Generation of a nonequlibrium plasma in heterophase atmospheric-pressure gas-liquid media and demonstration of its sterilization ability

Results are presented from experiments on the generation of a low-temperature nonequilibrium plasma in atmospheric-pressure heterophase gas-liquid media of different compositions: (i) a liquid with air bubbles and (ii) air with liquid aerosol. To illustrate possible application of a low-temperature plasma in a heterophase medium, experiments on the inactivation of some microorganisms by a low-temperature plasma have been performed.     

Studies on the host/parasite interface during the development of a pentastomid arthropod parasite in rodent intermediate hosts, with observations on protective surface membranes

The changing structure of the cuticle of the arthropod pentastomid parasite Porocephalus crotali, during growth to the infective stage in mouse and rattlesnake hosts, is described. The outermost cuticulin layer of the cuticle in instars II-VI is elevated to form a dense mat of epicuticular hairs. Since the VI larval cuticle is retained by the infective (VII) nymph as a protective sheath, effectively all stages in mice present a hairy surface to the host and this may constitute a physical barrier to inflammatory cells. The entire surface is overlain by a triple-track 'unit' membrane whose biophysical properties resemble those of a conventional plasma membrane, and there is evidence to suggest that this membrane is susceptible to immune attack. Under natural circumstances, epicuticular hairs entrap secretion, delivered to the cuticle via innumerable minute ducts which communicate with tegumental secretory cells termed subparietal cells (SPC). SPC synthesize lamellate droplets which unfold on the cuticle to constitute a layer of protective polymorphic vesicles. By contrast, infective nymphs in snakes possess a smooth cuticle and SPC membranous secretion is stacked over the entire surface, in sheets up to 20 deep. The function of the lipid and protein components of SPC secretion is discussed.     

Assembly of the Drosophila larval exoskeleton requires controlled secretion and shaping of the apical plasma membrane.

The apical plasma membrane of epithelial cells plays a central role in producing and shaping the apical extracellular matrix (aECM) that eventually adopts a stereotypic architecture required for the physical and physiological needs of the epithelium. To assess the implication of the apical plasma membrane on aECM differentiation, we have studied the function of the apical plasma membrane t-SNARE Syntaxin 1A in the embryo of the fruit fly Drosophila melanogaster during differentiation of the stratified exoskeleton, the cuticle, which is composed of proteins and the polysaccharide chitin. The cuticle layers of syntaxin1A deficient larvae are rudimentary. Consistently, Syntaxin 1A is required for the secretion of O-glycosylated proteins and components involved in pigmentation and protein cross-linking. By contrast, localization of chitin synthesis and organising proteins to the apical plasma membrane or to the extracellular space does not depend on Syntaxin 1A activity. However, chitin microfibrils have a random orientation instead of being arranged in parallel. This correlates with the lack of corrugations at the apical plasma membrane of epidermal cells, the apical undulae that have been proposed to be crucial for chitin microfibril orientation. Hence, Syntaxin 1A contributes to cuticle differentiation by controlling correct apical plasma membrane topology as well as mediating secretion of a subset of extracellular proteins required for layer organisation. Our data also indicate that yet another unidentified t-SNARE is needed in parallel to Syntaxin 1A to deliver extracellular material for complete cuticle assembly. Evidently, coordination of apical membrane modelling and two secretion routes are essential for stereotypic aECM organisation.     

Dusky-like functions as a Rab11 effector for the deposition of cuticle during Drosophila bristle development

The morphogenesis of Drosophila sensory bristles is dependent on the function of their actin and microtubule cytoskeleton. Actin filaments are important for bristle shape and elongation, while microtubules are thought to mediate protein and membrane trafficking to promote growth. We have identified an essential role for the bristle cuticle in the maintenance of bristle structure and shape at late stages of bristle development. We show that the small GTPase Rab11 mediates the organized deposition of chitin, a major cuticle component in bristles, and disrupting Rab11 function leads to phenotypes that result from bristle collapse rather than a failure to elongate. We further establish that Rab11 is required for the plasma membrane localization of the ZP domain-containing Dusky-like (Dyl) protein and that Dyl is also required for cuticle formation in bristles. Our data argue that Dyl functions as a Rab11 effector for mediating the attachment of the bristle cell membrane to chitin to establish a stable cuticle. Our studies also implicate the exocyst as a Rab11 effector in this process and that Rab11 trafficking along the bristle shaft is mediated by microtubules.     

Fine structure of the prothoracic mycangium, a chamber for the culture of symbiotic fungi, in the southern pine beetle, Dendroctonus frontalis

The ultrastructure of the prothoracic mycangium of female Dendroctonus frontalis is examined. The mycangium consists of a cuticular invagination within which symbiotic fungi are cultured by the pine beetle and transported to new host trees. Secretions from two types of gland cells pass into the mycangial lumen. The plasma membrane of type-1 cells is invaginated to form an enclosed extracellular cavity. The secretory product passes into the cavity, then through fine cylindrical channels into an end apparatus and finally via an efferent cuticular ductule to the lumen of the mycangium. Secretion of the type-2 cells is released into a cavity just beneath the mycangial cuticle. The cuticle over this cavity is quite thin (1-2mu), consisting mostly of inner epicuticle riddled with irregular canals through which the secretion reaches the lumen. Beneath the patches of porous cuticle are ribs (up to 1Omu in thickness) which flank the cavities and presumably provide structural support for the porous secretory zones.     

Apolysis and the turnover of plasma membrane plaques during cuticle formation in an insect.

The apical plasma membranes of Calpodes epidermal cells have small fattened areas or plaques with an extra density upon their cytoplasmic face. The plaques are typically at the tips of microvilli. The are present during the deposition of fibrous cuticle and the cuticulin layer. Since the plaques are close (less than 15nm) to the sites where these kinds of cuticle first appear, they are presumed to have a role in their synthesis and/or deposition and orientation. When fifth stage larval cuticle deposition ceases prior to pupation, the plaques are lost as the area of the apical plasma membrane is reduced. The plaques pass from the surface into pinocytosis vesicles and multivesicular bodies where they are presumably digested. The loss of plaques occurs as the blood level of moulting hormone reaches a peak at the critical period after which the prothoracic glands are no longer needed for pupation. Apolysis or separation of the epidermis from the old cuticle is the stage when plaques are absent, the old ones have been lost but the new ones have yet to form. After the critical period, the epidermis prepared for pupation with a phase of elevated RNA synthesis at the end of which plaques and microvilli reform in time to secrete the new cuticulin layer and later the fibrous cuticle of the pharate pupa. There is a new generation of plaques for each moult and succeeding intermoult and each generation is involved in two kinds of cuticle deposition before involution and redifferentiation.     

The localization of a peroxidase associated with hard cuticle formation in an insect, Calpodes ethlius stoll, Lepidoptera, Hesperiidae

The distribution of a peroxidase associated with the formation of hard cuticle has been studied in developing larvae of Calpodes ethlius. It occurs in granules in several cell types but is most easily observed in the cells making the proleg spines at the 4th to 5th molt. Light microscopy shows peroxidase in numerous granules about 0.5mu in diameter at the time the cuticle of the spine shaft is being deposited. Electron microscopy shows these granules to be multivesicular bodies with peroxidase in the matrix. Peroxidase is also found in cisternae of the rough ER near Golgi complexes, in vesicles of Golgi complexes and in the secretory vesicles which discharge to make cuticle at the apical surface. The cuticle above the plasma membrane where peroxidase is being deposited reacts with DAB in the absence of hydrogen peroxide. Presumably this cuticle has been 'peroxidized' as a first stage in stabilization by cross-linking. Some of the peroxidase secreted at the apical surface is pinocytosed and transported to the multivesicular bodies, suggesting that there may be a precise control of the cuticular environment through the turnover of its soluble components.     

A member of the PLEIOTROPIC DRUG RESISTANCE family of ATP binding cassette transporters is required for the formation of a functional cuticle in Arabidopsis

Although the multilayered structure of the plant cuticle was discovered many years ago, the molecular basis of its formation and the functional relevance of the layers are not understood. Here, we present the permeable cuticle1 (pec1) mutant of Arabidopsis thaliana, which displays features associated with a highly permeable cuticle in several organs. In pec1 flowers, typical cutin monomers, such as ω-hydroxylated fatty acids and 10,16-dihydroxypalmitate, are reduced to 40% of wild-type levels and are accompanied by the appearance of lipidic inclusions within the epidermal cell. The cuticular layer of the cell wall, rather than the cuticle proper, is structurally altered in pec1 petals. Therefore, a significant role for the formation of the diffusion barrier in petals can be attributed to this layer. Thus, pec1 defines a new class of mutants. The phenotypes of the pec1 mutant are caused by the knockout of ATP BINDING CASSETTEG32 (ABCG32), an ABC transporter from the PLEIOTROPIC DRUG RESISTANCE family that is localized at the plasma membrane of epidermal cells in a polar manner toward the surface of the organs. Our results suggest that ABCG32 is involved in the formation of the cuticular layer of the cell wall, most likely by exporting particular cutin precursors from the epidermal cell.     

The Ultracytochemical Localization of ATPase Activity in the Ovules of Sunflower

The ultracytochemical localization of ATPase activity was determined employing the method of lead precipitation in the ovules of sunflower (Helianthus annuus L.). No ATPase activity is observed in the egg and synergids except some at the filiform apparatus. Much ATPase activity is localized on the plasma membrane and wall of the central cell. In the antipodal cells, ATPase activity is also found on the plasma membranes, but only a little in their walls. In the integumentary tapetum, besides the plasma membranes, most of the nuclei are rich in ATPase. Between the integumentary tapetum and uncontinuous cuticle surrounding the embryo sac, there is a gap where a lot of ATPase are found. These ATPases are continuously linked with those in the central cell wall throuth the intervals of the cuticle. At the sites of the wall ingrowths of the central celT, abundant vesicles and other structures with high ATPase activity aggregate noticeably in the gap region. According to the ATPase distribution in the ovules, we propose that the whole surface of embryo sac functions in absorbing nutrients directly from the apoplast outside the cuticle, especially via the wall-membrane apparatus of 'he central cell.     

Fine structure of the spermatheca of the mealworm beetle (Tenebrio molitor L.)

The spermatheca of the female mealworm beetle is an inflorescence of branching cuticular ducts which is connected to the bursa copulatrix via a cuticular neck surrounded by a muscular coat. The infolded bursal cuticle consists of a distinct outer epicuticle, inner epicuticle, procuticle, and a subcuticular zone; the latter is rich in mucopolysaccharides. The cuticle of the neck lacks a distinct procuticle. The cuticle of the spermatheca itself is mostly inner epicuticle with two thin underlying lamellae of procuticle. The cells of the bursa are loosely coupled to the procuticle, whereas cuticular projections bind the epithelia of the "neck" and the spermatheca proper to the underlying epithelia. The apical plasma membranes of the spermathecal epithelium are sinuous and much infolded; we believe that this epithelium controls the micro-environment within the cuticular ducts.     

Kelvin-Helmholtz instability for a bounded plasma flow in a longitudinal magnetic field

Kelvin-Helmholtz MHD instability in a plane three-layer plasma is investigated. A general dispersion relation for the case of arbitrarily orientated magnetic fields and flow velocities in the layers is derived, and its solutions for a bounded plasma flow in a longitudinal magnetic field are studied numerically. Analysis of Kelvin-Helmholtz instability for different ion acoustic velocities shows that perturbations with wavelengths on the order of or longer than the flow thickness can grow in an arbitrary direction even at a zero temperature. Oscillations excited at small angles with respect to the magnetic field exist in a limited range of wavenumbers even without allowance for the finite width of the transition region between the flow and the ambient plasma. It is shown that, in a low-temperature plasma, solutions resulting in kink-like deformations of the plasma flow grow at a higher rate than those resulting in quasi-symmetric (sausage-like) deformations. The transverse structure of oscillatory-damped eigenmodes in a low-temperature plasma is analyzed. The results obtained are used to explain mechanisms for the excitation of ultra-low-frequency long-wavelength oscillations propagating along the magnetic field in the plasma sheet boundary layer of the Earth’s magnetotail penetrated by fast plasma flows.     

A structural model of the chitin-binding domain of cuticle proteins

The nature of the interaction of insect cuticular proteins and chitin is unknown even though about half of the cuticular proteins sequenced thus far share a consensus region that has been predicted to be the site of chitin binding. We previously predicted the preponderance of beta-pleated sheet in the consensus region and proposed its responsibility for the formation of helicoidal cuticle (Iconomidou et al., Insect Biochem. Mol. Biol. 29 (1999) 285). Consequently, we have also verified experimentally the abundance of antiparallel beta-pleated sheet in the structure of cuticle proteins (Iconomidou et al., Insect Biochem. Mol. Biol. 31 (2001) 877). In this work, based on sequence and secondary structure similarity of cuticle proteins, and especially that of the consensus motif, to that of bovine plasma retinol binding protein (RBP), we propose by homology modelling an antiparallel beta-sheet half-barrel structure as the basic folding motif of cuticle proteins. This folding motif may provide the template for elucidating cuticle protein-chitin interactions in detail and reveal the precise geometrical formation of cuticle's helicoidal architecture. This predicted motif is another example where nature utilizes an almost flat protein surface covered by aromatic side chains to interact with the polysaccharide chains of chitin.     

The formation of the ecdysial droplets and the ecdysial membrane in an insect

Insect cuticle forms as a result of overlapping sequences of two kinds of process, those involving vesicles of the Golgi complex, and those related to transport through and/or assembly at the apical plasma membrane. The ecdysial droplets are the last layer of old cuticle to be deposited before ecdysis and form from the contents of secretory vesicles from Golgi complexes. Ecdysial droplets and secretory vesicles both stain with PTA and react with silver hexamine after oxidation with periodic acid. The vesicles discharge in localized apical areas devoid of microvilli where they accumulate as droplets measuring about 3 [ x 1 [. The. droplets span the last few lamellae of the endocuticle which becomes the ecdysial membrane. They dissolve to leave the ecdysial membrane full of holes at the time that the rest of the old cuticle is digested.     

Deconstructing the design of a biological material

By identifying the functional conflicts in its design, the cuticle of arthropods can be shown to cope with IR and UV irradiation in the same manner as our technology-by controlling spectral properties (transmission and reflection). However, the skeletal properties of cuticle are integrated with demands for sensory transmission, movement, etc, by controlling the local properties of the material rather than by changing global parameters (which would be the technical solution). On the basis of this study, the biomimetic similarity of cuticle with technology is only about 20%, suggesting that we can learn from the design of arthropod cuticle.     

Arabidopsis LTPG Is a Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Required for Export of Lipids to the Plant Surface

Plant epidermal cells dedicate more than half of their lipid metabolism to the synthesis of cuticular lipids, which seal and protect the plant shoot. The cuticle is made up of a cutin polymer and waxes, diverse hydrophobic compounds including very-long-chain fatty acids and their derivatives. How such hydrophobic compounds are exported to the cuticle, especially through the hydrophilic plant cell wall, is not known. By performing a reverse genetic screen, we have identified LTPG, a glycosylphosphatidylinositol-anchored lipid transfer protein that is highly expressed in the epidermis during cuticle biosynthesis in Arabidopsis thaliana inflorescence stems. Mutant plant lines with decreased LTPG expression had reduced wax load on the stem surface, showing that LTPG is involved either directly or indirectly in cuticular lipid deposition. In vitro 2-p-toluidinonaphthalene-6-sulfonate assays showed that recombinant LTPG has the capacity to bind to this lipid probe. LTPG was primarily localized to the plasma membrane on all faces of stem epidermal cells in the growing regions of inflorescence stems where wax is actively secreted. These data suggest that LTPG may function as a component of the cuticular lipid export machinery.     

Coagulation of dust grains in the plasma of an RF discharge in argon

Results are presented from experimental studies of coagulation of dust grains of different sizes injected into a low-temperature plasma of an RF discharge in argon. A theoretical model describing the formation of dust clusters in a low-temperature plasma is developed and applied to interpret the results of experiments on the coagulation of dust grains having large negative charges. The grain size at which coagulation under the given plasma conditions is possible is estimated using the developed theory. The theoretical results are compared with the experimental data.     

The electron microscopy of the human hair follicle. II. The hair cuticle

1. During the early differentiation of the cuticle the cell membranes smooth out and the cells become closely attached over most of their surface. The change seems to be due to a layer of cement which forms between them. The plasma membranes also increase in density. 2. The decreased membrane activity of the cuticle cells may prevent a phagocytosis of the melanocyte processes and thus account for the non-pigmentation of the cuticle. 3. The flattening and imbrication of the cuticle may possibly be explained by a zipper-like spread of cell contacts. 4. Keratinisation of the cuticle occurs at a late stage in its development; the keratin formed is an amorphous type, similar to the gamma-fraction of the cortex which is produced at a similar level. 5. Keratinisation is accompanied by the formation of complex intercellular layers similar to structures observed in the inner root sheath (see Part 3). 6. In the final stage of keratinisation the remaining cytoplasm condenses with the result that the cell is divided into a laminated structure with an outer keratinised layer and an inner layer, which is insoluble in keratinolytic solvents.     

Unique features of the structural model of 'hard' cuticle proteins: implications for chitin-protein interactions and cross-linking in cuticle

Cuticular proteins are one of the determinants of the physical properties of cuticle. A common consensus region (extended R&R Consensus) in these proteins binds to chitin, the other major component of cuticle. We previously predicted the preponderance of beta-pleated sheet in the consensus region and proposed its responsibility for the formation of helicoidal cuticle (Iconomidou et al., Insect Biochem. Mol. Biol. 29 (1999) 285). Subsequently, we verified experimentally the abundance of antiparallel beta-pleated sheet in the structure of cuticle proteins (Iconomidou et al., Insect Biochem. Mol. Biol. 31 (2001) 877). Homology modelling of soft (RR-1) cuticular proteins using bovine plasma retinol binding protein (RBP) as a template revealed an antiparallel beta-sheet half-barrel structure as the basic folding motif (Hamodrakas et al., Insect Biochem. Molec. Biol. 32 (2002) 1577). The RR-2 proteins characteristic of hard cuticle, have a far more conserved consensus and frequently more histidine residues. Extension of modelling to this class of consensus, in this work, reveals in detail several unique features of the proposed structural model to serve as a chitin binding structural motif, thus providing the basis for elucidating cuticle's overall architecture and chitin-protein interactions in cuticle.     

An ultrastructural study of the larval integument of the midge,Chironomus riparius meigen (Diptera: Chironomidae)

Summary The larval integument of the midge, Chironomus riparius Mg., is unusually thin although it conforms with the normal insect pattern. The cuticle of the post-cephalic segments is about 3 m thick and overlies an epidermis which has an irregular basal plasma membrane resulting in spaces occurring between it and the basement membrane. The ventral tubuli have a similar epidermis but the cuticle is somewhat thinner. The anal papillae have the thinnest cuticular covering with a uniquely folded epicuticle of variable thickness, and their epidermis has the characteristics of a transporting epithelium. No evidence of pore canals could be found in the cuticle of any part except the head capsule which has a remarkably smooth epicuticle and a distinct layer which may represent the exocuticle. There are no spaces between the basement membrane and basal plasma membrane of the epidermis in the head. Ultrastructural evidence would suggest that gaseous exchange can occur across most of the post-cephalic integument.The author is indebted to Mrs. L. Rolph and Mr. R.L. Jones for their technical assistance     

The transfer of lipid in insects from the epidermal cells to the cuticle

The structure of the pore canals and the tubular filaments they contain are described in a series of insects and types of cuticle. In all these cuticles the tubular filaments arise from the plasma membrane of the epidermal cells and they contain argentaffin material, regarded as sclerotin precursors, and lipid-staining material, regarded as wax precursors. These materials are transferred to the inner epicuticle and are exuded over the surface of the outer epicuticle to form the waterproofing layer as described in the preceding paper. They are also transported to those parts of the endocuticle destined to form hard exocuticle. There are no terminations of tubular filaments in the soft cuticle of Manduca larva, in the soft expanding cuticle of Rhodnius, and in the non-sclerotized post-ecdysial endocuticle of Tenebrio. Apis. etc. In the puparium of Calliphora lipid appears to be added by the epidermal cells directly and not by way of tubular filaments. It is confirmed that lipid is a component of sclerotized cuticle.