A proposed solution to a fine-structural puzzle: the organization of gill cuticle in a crayfish (Panulirus)

Crayfish gill cuticle is approximately 2 micron thick and comprises an epicuticle and an endocuticle, which is subdivided into outer and inner layers. Sections demonstrate indistinct lamellae in the outer endocuticle and vertically striated lamellae in the inner endocuticle. Microfibrils cannot be seen in sections. Difficulties in interpretation of the fibrous architecture of the cuticle from thin sections have been overcome by examining tilted series of micrographs of sections and also by making freeze-fracture replicas of the cuticle, which reveal the microfibrils clearly. A model for the endocuticle based on a helicoidal configuration of microfibrillar laminae is proposed and the vertically striated structures seen in sections of the outer layer are accounted for by including regular rows of particles oriented perpendicular to microfibrils. The model is compared with cuticles and coverings reported from other invertebrates.     

Mode of action of lufenuron on larval cat fleas (Siphonaptera: Pulicidae)

Adult cat fleas, Ctenocephalides felis (Bouché), were fed suboptimal in vitro concentrations of lufenuron in blood to allow hatching of flea larvae for cytological study. At concentrations of 0.125, 0.25, and 0.5 ppm, larval hatch was 64, 15, and 4%, respectively. Larvae hatching from eggs laid by adults fed lufenuron at concentrations of 0.025, 0.08, or 0.125 ppm did not differ significantly from the control. However, many larvae from the 0.08-ppm group and higher concentrations died during the 1st instar. Examination of these larvae revealed that they were dying from desiccation caused by bleeding from microscopic lesions in the cuticle or the inability to complete the molt to the next instar. Electron micrographs showed that lufenuron often disrupted formation of the endocuticle resulting in the deposition of an amorphous mass of randomly oriented chitin microfibrils. Other larvae formed normal endocuticle but were unable to digest the old endocuticle or produce new procuticle after apolysis. Failure of larvae to digest old cuticle or form new cuticle was caused by degeneration of the epidermal cells needed for the synthesis of molting fluid and chitin.     

Purification and properties of two blue biliproteins from the larval hemolymph and integument of Rhodinia fugax (Lepidoptera: Saturniidae)

Blue biliproteins (BPs) are found in the hemolymph and integument of the fifth instar larvae of the saturniid silkworm, Rhodinia fugax. An efficient method of isolating BPs from the hemolymph, epidermis and cuticle using hydrophobic interaction chromatography and ion-exchange chromatography was devised. The BPs from the hemolymph, epidermis and cuticle have molecular weights of approximately 24,000, 48,000 and 23,000 Da by gel-filtration, respectively. Using matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS), the respective molecular masses were determined to be 22,641, 22,908 and 22,737 Da. Based on these results, BP molecules from the hemolymph and cuticle are assumed to be monomers, whereas the epidermal BP is a dimer. The amino acid composition and N-terminal amino acid sequences of the BPs from the hemolymph and cuticle (BP-I) are very similar, but the BP from the epidermis (BP-II) is quite different. The N-terminal amino acid sequences of these BPs share approximately 50% identity with the biliproteins from other lepidopteran insects. The blue color of BP is due to the presence of bile pigments, which are non-covalently bound to the apoprotein. The absorbance spectrum of BP-I from the hemolymph revealed maxima at 280 and 669 nm, while that of BP-II showed maxima at 280, 385 and 663 nm. The pigment dimethyl esters were extracted from BP-I and BP-II with acidic methanol and dichloromethane. The results of these analyses suggest that the blue pigments of BP-I and BP-II are different; BP-I contains a phorcabilin-like pigment while BP-II contains biliverdin IX gamma. In an immunoblot analysis, anti-BP-I antibodies, produced against hemolymph BP-I, reacted with immunoreactive proteins in the hemolymph and cuticle of R. fugax. These anti-BP-I antibodies did not react with BP-II and only cross-reacted weakly with Samia cynthia ricini biliverdin-binding protein (BBP)-II.     

Number of nucleoli and nucleolar organizer regions per nucleus and nucleolus--prognostic value in canine mammary tumors

The carbohydrate residues of the surface coat of 20 axenic cultures of Blastocystis hominis were studied using FITC-labelled lectins (ConA, WGA, DBA, HPA, SBA, PNA, UEAI and LPA). The specific affinity of reactive lectins was determinated by competitive inhibition assay with specific carbohydrates or by enzymatic pre-treatment of cells. All stocks strongly bound ConA and HPA; WGA, UEAI and LPA were partially reactive, and the remaining lectins were nonreactive. Inhibition assays showed abolition (WGA, LPA, UEAI and HPA) or partial reduction (ConA) of lectin affinity, which demonstrated the specificity of binding assay. These results indicate that B. hominis has surface components containing alpha-D-mannose, alpha-D-glucose, N-acetyl-alpha-D-glucosamine, alpha-L-fucose, chitin and sialic acid.     

Porous channels in the cuticle of the head-arrester system in dragon/damselflies (Insecta:Odonata)

The ultrastructure of the porous channels (PC) of the postcervical sclerite (SPC), which provides additional head fixation to the neck in adult odonates, was studied using TEM and high resolution SEM microscopy. Single chitin-protein microfibrils, about 0.14 micron thick, are arranged into channels with cylinder-like shapes. The axial rod of the chitin fiber (0.04 micron thick) is located in the center of the cylinder. The orientation of the axial rods was three-dimensionally demonstrated after dissolving the protein cover with NaOH. The PCs are arranged vertically to the surface and pass from the epidermal cells through all the cuticular layers to the surface of the cuticle. In the exo- and endocuticle, the PCs are usually oval in cross-section and about 0.3 micron thick. In the endocuticle, the cross-sectional area of the PCs varies widely, from 0.01-0.15 micron2. The shape of the PC is determined by the macromolecular organization of the chitin-protein microfibrils: the long axis of the channel is orientated parallel to the axis of the preferred orientation of the cuticular microfibrils. The microfibrils tend to follow the line of the channel very closely. In fractures orientated perpendicular to the surface, the PC resembles a ribbon-like construction, which was clearly demonstrated by casts. The strongly parallel orientation of PCs in the deep layers of the cuticle changes within the microtrichia (MT), and they begin to be curved. Numerous PCs pass through the microtrichium, and most of them end on its side wall. PCs usually contain channel filaments about 0.09 micron thick. Usually, a single channel contained one filament, but channels located in the deep layers of the endocuticle have from one to five single filaments. The filaments were observed in the intact cuticle and in the cuticle enzymatically treated with chitinase, while in the cuticle treated with NaOH filaments were absent. The porous channel system of the odonate arrester is interpreted as a device transporting adhesive excretions from the epidermal cells to the cuticular surface.     

In vitro chiral nematic ordering of chitin crystallites

Microfibrillar fragments of purified crab and shrimp chitin were prepared by hydrolysis in 3 M HCl at its boiling point (104 degrees C). After removal of the acid by centrifugal washing and dialysis, an ultrasound treatment converts the residual product to a colloidal suspension stabilized by NH3+ charges. When dewatered to a critical concentration, spontaneous formation of a two-phase equilibrium system system occurs. The upper phase (lower concentration) is isotropic and the lower phase is anisotropic. The latter displays chiral nematic order and dries to a solid film which mimics the helicoid organization characteristic of the chitin microfibrils in the cuticle of arthropods.     

An important developmental role for oligosaccharides during early embryogenesis of cyprinid fish

Derivatives of chitin oligosaccharides have been shown to play a role in plant organogenesis at nanomolar concentrations. Here we present data which indicate that chitin oligosaccharides are important for embryogenesis in vertebrates. We characterize chitin oligosaccharides synthesized in vitro by zebrafish and carp embryos in the late gastrulation stage by incorporation of radiolabeled N-acetyl-D-[U14C]glucosamine and by HPLC in combination with enzymatic conversion using the Bradyrhizobium NodZ alpha-1, 6-fucosyltransferase and chitinases. A rapid and sensitive bioassay for chitin oligosaccharides was also used employing suspension-cultured plant cells of Catharanthus roseus. We show that chitin oligosaccharide synthase activity is apparent only during late gastrulation and can be inhibited by antiserum raised against the Xenopus DG42 protein. The DG42 protein, a glycosyltransferase, is transiently expressed between midblastula and neurulation in Xenopus and zebrafish embryogenesis. Microinjection of the DG42 antiserum or the Bradyrhizobium NodZ enzyme in fertilized eggs of zebrafish led to severe defects in trunk and tail development.     

The chitin crystallite in arthropod cuticle

Electron microscopy has revealed that chitin from a representative selection of insect orders (plus one crustacean and one arachnid) is localized in crystallites about 2.8 nm across. Furthermore, these crystallites are arranged on an hexagonal or pseudo-hexagonal lattice, the lateral order of which varies considerably. The lattice becomes secondarily reoriented during cuticle expansion following an ecdysis. The size of the 'unit cell' has been measured both by optical diffraction and direct measurements of the micrographs, permitting an estimate of the chitin and resilin content for locust rubberlike cuticle. The number of poly-N-acetyl-glucosamine chains per sheet and sheets per crystallite can be estimated from the physical dimensions of the crystallite. Each crystallite is unlikely to comprise more than 3 sheets and 6 chains per sheet. The calculated and measured density of alpha-chitin can be shown to be in close agreement.     

A cuticular protein from the moulting stages of an insect

A 22 kDa peptide was purified from prepupal cuticles of 5th instar Calpodes ethlius caterpillars. It was absent earlier in the stadium and from the egg and adult, i.e. it is related to cuticle turnover rather than cuticle structure. It was present at larval and metamorphic moults, showing that it is related to moulting not just metamorphosis. The cDNA corresponding to the 22 kDa peptide was isolated by antibody screening of an epidermal cDNA expression library. Hybridization to Calpodes genomic DNA showed that the gene was present as a single copy. The deduced amino acid sequence is not like any of the sequences of cuticular structural proteins that have been published, but has a 47 amino acid sequence similar to bacteriophage T7 N-acetylmuramoyl-L-alanine amidase (34% identical, 51% similar). The amino acid sequence, the timing of expression in development, and the similarity between the substrate of the bacteriophage amidase and components of insect cuticle, all suggest that the 22 kDa protein may have a role in cleaving chitin-peptide bonds as a prerequisite for digestion of the cuticle by chitinases and proteases.     

Recognition of B-cell epitopes of the 65 kDa HSP in Beh?et's disease

B-cell epitopes of the mycobacterial 65 kDa heat shock protein (HSP) were mapped in sera from patients with Beh?et's Disease (BD). A series of 47 overlapping synthetic peptides (15ers) derived from the sequence of the Mycobacterium tuberculosis 65 kDa HSP was used in ELISA. Significant increases in IgA and IgG antibody levels were observed with peptides 111-125, 154-172 and 311-326 in sera from BD, compared with those from controls. Homologous peptides derived from the sequence of the human mitochondrial 60 kDa HSP were then examined. Peptides 136-150 and 336-351 showed comparable results to the homologous mycobacterial peptides 111-125 and 311-326, respectively. The B-cell epitopes defined in this investigation overlap with the T-cell epitopes the authors have previously reported in BD. Inhibition studies are consistent with the view that antibodies to each of the three B-cell epitope peptides represent a small proportion of the total B-cell epitope repertoire elicited by the 65 or 60 kD HSP. Sequential antibody studies suggest that IgA and IgG antibody titres to one or all three peptides tested may increase during exacerbation of ocular disease. The functional role of these antibodies needs to be determined, but the peptides may be involved in the immunopathogenesis of BD as they can induce experimental uveitis in Lewis rats, which is a principal manifestation of BD.     

Cigarette smoking as a determinant of high-grade carotid artery stenosis in Hispanic, black, and white patients with stroke or transient ischemic attack

The effect of chitin, poly-beta-(1 --> 4)-N-acetyl-glucosamine, and chitosan, a polymer of glucosamine obtained by the deacetylation of chitin, on growth and nutrient digestibility was studied in grass shrimp, Penaeus monodon. Shrimp were fed for 8 wk diets containing no supplement (control) or 2, 5 or 10 g/100 g chitin or chitosan. Each diet was fed to triplicate groups of shrimp with a mean initial body weight of 0.45 +/- 0.05 g. Significantly higher body weight gains were observed in shrimp fed the 5% chitin diet than in those fed the 10% chitin or the control diet. The weight gain of shrimp decreased as dietary chitosan supplementation level increased (r = 0. 87, P < 0.05). Feed efficiencies (FE) and protein efficiency ratios (PER) followed the same pattern. Lower protein and lipid digestibilities and lower body protein and lipid contents were observed in shrimp fed all chitosan-containing diets than in controls (P < 0.05). Carbohydrate digestibility was lower in shrimp fed the 10% chitosan diet than in those fed the control diet. Lower protein and lipid digestibilities, body lipid content and blood cholesterol concentration were observed in shrimp fed the 10% chitin diet compared with controls (P < 0.05). Higher weight gains, body lipid contents and blood cholesterol concentrations were observed in shrimp fed the 2 and 5% chitin diets than in those fed the chitosan diets. Shrimp fed the 5% chitin diet had higher protein and lipid digestibilities and higher body protein content than those fed the 5% chitosan diet (P < 0.05). These data suggest that dietary chitin, supplemented at 5%, enhances P. monodon growth, whereas chitosan depresses shrimp growth, regardless of the supplementation level.     

The chitin catabolic cascade in the marine bacterium Vibrio furnissii. Molecular cloning, isolation, and characterization of a periplasmic chitodextrinase

Chitin catabolism in Vibrio furnissii comprises several signal transducing systems and many proteins. Two of these enzymes are periplasmic and convert chitin oligosaccharides to GlcNAc and (GlcNAc)2. One of these unique enzymes, a chitodextrinase, designated EndoI, is described here. The protein, isolated from a recombinant Escherichia coli clone, exhibited (via SDS-polyacrylamide gel electrophoresis) two enzymatically active, close running bands ( approximately mass of 120 kDa) with identical N-terminal sequences. The chitodextrinase rapidly cleaved chitin oligosaccharides, (GlcNAc)4 to (GlcNAc)2, and (GlcNAc)5,6 to (GlcNAc)2 and (GlcNAc)3. EndoI was substrate inhibited in the millimolar range and was inactive with chitin, glucosamine oligosaccharides, glycoproteins, and glycopeptides containing (GlcNAc)2. The sequence of the cloned gene indicates that it encodes a 112,690-kDa protein (1046 amino acids). Both proteins lacked the predicted N-terminal 31 amino acids, corresponding to a consensus prokaryotic signal peptide. Thus, E. coli recognizes and processes this V. furnissii signal sequence. Although inactive with chitin, the predicted amino acid sequence of EndoI displayed similarities to many chitinases, with 8 amino acids completely conserved in 10 or more of the homologous proteins. There was, however, no "consensus" chitin-binding domain in EndoI.     

Cancer conferences. Can they be improved?

In a previous study, we showed that overexpression of cyclin D, a G1 cyclin, is frequently associated with keratinocyte carcinogenesis as an early event. Another G1 cyclin, cyclin E, was recently suggested to be a prognostic marker for breast cancer. In order to evaluate the role of cyclin E in human keratinocyte carcinogenesis, we analysed the expression of cyclin E by immunohistochemistry in normal skin, seborrheic keratosis (SK), keratoacanthoma (KA), actinic keratosis (AK), Bowen's disease (BD), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Positive cells were seen rarely in normal epidermis, in 9 of 20 cases of SK, in 5 of 6 cases of KA, in 9 of 13 cases of AK and in all 27 cases of BD. Some of the cases of AK and BD had positive cells in the superficial epidermis, where atypicality is less obvious. In contrast, positive cells were seen in 4 of 25 cases of SCC and none of 15 cases of BCC. These results suggest that expression of cyclin E plays a role in the formation of benign and premalignant keratinocytic tumors, whereas down-regulation of cyclin E expression may be involved in carcinogenesis in human keratinocytes.     

Effects of alpha-D- and beta-L-glucose pentaacetate on effluent radioactivity from pancreatic islets labelled with [(32)P]phosphate and myo-[2-(3)H]inositol

The anomers of both D-glucose pentaacetate and L-glucose pentaacetate were recently found to display insulinotropic potential. In order to progress in understanding the mode of action of these esters in islet cells, we have now investigated whether they mimic the effect of nutrient secretagogues to cause a phosphate flush and activation of phospholipase C in isolated islets. For this purpose, rat pancreatic islets were prelabelled with either [(32)P]orthophosphate or myo-[2-(3)H]inositol and placed in a perifusion chamber. In the absence of any other exogenous nutrient, the administration of alpha-D-glucose pentaacetate (1.7 mM) from 46 to 70 min of perifusion increased, after an initial transient fall, both 32P and 3H fractional outflow rates and stimulated insulin release from the perifused islets. No secondary rise in either (32)P or (3)H outflow and no sizeable stimulation of insulin release was observed, however, in response to Beta-L-glucose pentaacetate (also 1.7 mM). These findings are consistent with the view that the insulinotropic action of alpha-D-glucose pentaacetate entails a nutrient-like component leading to the occurrence of both a phosphate flush and hydrolysis of phosphoinositides. This is not the case, however, for Beta-L-glucose pentaacetate. The latter ester might act directly on a yet unidentified receptor, the early secretory response to alpha-D-glucose pentaacetate also apparently involving such a direct effect of the ester itself.     

Atrial natriuretic peptides in the heart and hemolymph of the oyster, Crassostrea virginica: a comparison with vertebrates

1. The content of atrial natriuretic peptides (ANPs) in the auricles of oysters, Crassostrea virginica, was significantly (P < 0.01) greater than in their ventricles. 2. High-performance gel permeation chromatography (HP-GPC) followed by ANF radioimmunoassay revealed two peaks in both oyster and vertebrate (rat) hearts--a major peak where the 12.6-14 kDa ANF prohormone elutes and a smaller peak where the pure human form of ANF elutes. 3. HP-GPC evaluation followed by proANF 31-67 radioimmunoassay revealed only an ANF-like prohormone while HP-GPC followed by proANF 1-30 radioimmunoassay revealed the ANF prohormone and a proANF 1-30-like peptide in oyster and rat hearts. 4. ANPs concentrations in hemolymph were 940 +/- 129, 225 +/- 25, and 100 +/- 10 pg/ml by the proANF 1-30, proANF 31-67, and ANF radioimmunoassays, respectively. 5. Atrial natriuretic-like peptides are present in the oyster heart in molecular species similar to vertebrate species and these peptides are also present in hemolymph.     

轧制变形对U75V重轨钢珠光体晶粒度的影响

U75V钢重轨由280 mm×380 mm铸坯经14道次粗轧(BD1)-中轧(BD2)和CCS 6道次精轧(UR₁、ER、UR₂、UR₃、EF、UF)而成。用金相显微镜观察U75V重轨钢不同道次轧制变形试样的组织。结果表明,随重轨钢BD1E、BD2B、UR1和UF道次压缩比的增加,重轨头部、腰部和底部的珠光体晶粒度等级分别由6.0～6.5、6.0～6.5、6.0～6.5升至8.0～8.5、7.5～8.0、8.0～8.5,珠光体片层间距(μm)分别由0.51、0.41、0.49降至0.29、0.27、0.25,从而有利于提高重轨的力学性能。     

Xenon and krypton as radiographic inhalation contrast media with computerized tomography: preliminary note

Uptake of D-glucose anomers by isolated rat retina was studied. After 3 min incubation at 37 degrees C in the presence of alpha or beta anomer (750 mug/ml), a significantly greater uptake (1.32 mg/g wet tissue) of beta-anomer was observed compared with that of alpha-D-glucose (1.11 mg/g wet tissue). This result and other data suggest that the carrier for D-glucose transport in the retina prefers the beta-anomer stereospecifically.     

Effect of diflubenzuron on the incorporation of UDP-N-acetyl-[3H]glucosamine (UDP-[3H]NAGA) to chitin in permeabilized, and isolated integuments from the newly molted American cockroach Periplaneta americana

1. Diflubenzuron (DFB) was found to inhibit the incorporation of UDP-N-acetyl-[3H]glucosamine (UDP-[3H]NAGA) to chitin in permeabilized and isolated integument from newly molted American cockroach. 2. The favorable experimental conditions demonstrating the effect of diflubenzuron were: 10 mM phosphate, low calcium concentration (10(-6) M-10(-8) M), high potassium concentration (> 100 mM), and high pH (> or = 7). 3. The action of diflubenzuron was completely erased by preincubating the isolated integument with valinomycin, FCCP, or A23187. 4. By lowering the external pH to 5.2, it was also possible to reduce the rate of UDP-[3H]NAGA incorporation to the extent that DFB's effect was no longer recognizable. 5. Both Cs+ and Rb+ could replace K+ in maintaining a high level of chitin synthesis and the inhibitory action of DFB under the optimum conditions.     

Specificity of the glucose transport system in Leishmania tropica promastigotes

The glucose transport system in Leishmania tropica promastigotes was characterized by the use of labeled 2-deoxy-D-glucose (2-DOG), a nonmetabolizable glucose analog. The uptake system has a Q10 of 2 and a heat of activation of 10.2 kcal/mole. The glucose transport system is subject to competitive inhibition by 2-DOG, glucosamine, N-acetyl glucosamine, mannose, galactose, and fructose which suggests that substitutions in the hexose chain at carbons 2 and 4 do not affect carrier specificity. In contrast, changes at carbon 1 (alpha-methyl-D-glucoside, 1,5-anhydroglucitol) and carbon 3 (3-0-methyl glucose) lead to loss of carrier affinity since these sugars do not compete for the glucose carrier. Sugars that compete with the glucose carrier have one common feature--they all exist in the pyranose form in solution. The carrier for D-glucose does not interact with L-glucose or any of the pentose sugars tested. Uptake of 2-DOG is inhibited by glycerol. This inhibition, however, is noncompetitive; it is evident; therefore, that glucose and glycerol do not compete for the same carrier. Glycerol does not repress the glucose carrier since cells grown in presence of glycerol transport the sugar normally.     

Determination of peptide phi angles in solids by relayed anisotropy correlation NMR

A solid state NMR method is presented for determination of a backbone dihedral angle phi in peptides, being based on the previously reported method, relayed anisotropy correlation (RACO) NMR [Y. Ishii et al., Chem. Phys. Lett. 256 (1996) 133]. In the present method, the 15N-1H and the 13C-1H dipolar tensors in the 1H-15N-13C-1H system are two-dimensionally (2D) correlated via polarization transfer from 15N to 13C under magic angle spinning (MAS). This method was applied to N-acetyl[1,2-13C,15N]D,L-valine, and the H-C-N-H dihedral angle was determined to be 154.0 +/- 1.4 degrees or 206.0 +/- 1.4 degrees, the former agreeing with the X-ray value of 154 +/- 5 degrees.