Structure and function of the major ampullate spinning duct of the golden orb weaver, Nephila edulis.

Silks are fibres produced by spiders, some insects and even a crustacean, and are formed from protein solution by a pulltrusion process that is not well understood. Here we describe three aspects of the functional anatomy of the spinning apparatus in a spider: (i) changes in the diameter of the duct of the silk gland along its length for individuals at different stages of development, (ii) the correlation between the morphology of the duct and size and (iii) changes in the thickness of the wall of the duct. We conclude that in the distal part of the duct both the lumen's geometry and change in diameter with distance remains remarkably constant as the duct increases in length from moult to moult as the spider grows. This suggests constancy in the region where the nascent silk filament is drawn down within the lumen of the duct, which is likely to be fundamental for forming strong and tough fibres.

Regenerative potential of silk conduits in repair of peripheral nerve injury in adult rats.

Various attempts have been made to develop artificial conduits for nerve repair, but with limited success. We describe here conduits made from Bombyx mori regenerated silk protein, and containing luminal fibres of Spidrex(®), a silk-based biomaterial with properties similar to those of spider silk. Assessment in vitro demonstrated that Spidrex(®) fibres support neurite outgrowth. For evaluation in vivo, silk conduits 10 mm in length and containing 0, 100, 200 or 300 luminal Spidrex(®) fibres, were implanted to bridge an 8 mm gap in the rat sciatic nerve. At 4 weeks, conduits containing 200 luminal Spidrex(®) fibres (PN200) supported 62% and 59% as much axon growth as autologous nerve graft controls at mid-conduit and distal nerve respectively. Furthermore, Spidrex(®) conduits displayed similar Schwann cell support and macrophage response to controls. At 12 weeks, animals implanted with PN200 conduits showed similar numbers of myelinated axons (81%) to controls, similar gastrocnemius muscle innervation, and similar hindpaw stance assessed by Catwalk footprint analysis. Plantar skin innervation was 73% of that of controls. PN200 Spidrex(®) conduits were also effective at bridging longer (11 and 13 mm) gaps. Our results show that Spidrex(®) conduits promote excellent axonal regeneration and function recovery, and may have potential for clinical application.

[The molecular mobility of water in natural polymers: silk Bombyx mori with a low water content as studied by 1H DQF NMR]. / Molekuliarnaia podvizhnost' v prirodnykh polimerakh: shelk Bombyx mori s nizkim soderzhanien vody po dannym 1H DQF IAMR.

The molecular mobility of water in fibres of natural silk (Bombyx mori) was studied by the double-quantum-filtered (DQF) and single-pulse 1H NMR techniques. The results obtained showed a slow motion of water molecules and their strong interaction with silk macromolecules. At different model functions for resonance lineshape in 1H NMR spectra, the influence of signal linewidth on the estimation of relaxation times and cross-relaxation parameters was considered. The observed 1H DQF NMR signal in B. mori silk fibres (BC = 0.065) indicated a local order and anisotropic motion of water molecules, which leads to 1H-1H dipolar interactions in natural silk fibers due to the creation of the second-rank tensors (T(2,+1), T(2,-1)). DQF spectra were the difference of two Lorentzians with different linewidths and were analyzed using the theory of 1H DQF NMR and the data on residual dipolar interactions in systems with the anisotropic mobility of water molecules. The residual dipolar interactions was insignificant and, as the humidity increased (0.18), no DQF-signals and residual dipolar interactions were observed.

[Self-diffusion of water into silk fibers from magnetic field pulse gradient data]. / Samodiffuziia vody v volokonakh prirodnogo shelka po dannym impul'snogo gradienta magnitnogo polia.

Self-diffusion of water was studied in fibers of natural silk (Bombyx mori) with a water content of 0.18 g H2O/g dried material. Self-diffusion measurements were conducted by pulsed gradient of magnetic field (stimulated echo) at diffusion times from 10 to 200 mc. The dependence of experimental diffusion coefficients Dexp = f(delta) (observed decrease when delta increased) was determined to be responsible for the restricted diffusion. A model of planar and regularly spaced permeable barriers to diffusion of water molecules was applied to estimate the barrier spacing a and the permeability constant p. The maximal value of Dexp (at short diffusion time) in B. mori silk fibres was about 0.06 of the value of Dexp in bulk free water. The results obtained are compared to literature data on self-diffusion of water in hydrated biopolymer fibers and are discussed in connection with molecular mobility in natural macromolecular systems with low water content.

Comparison of the spinning of selachian egg case ply sheets and orb web spider dragline filaments.

Liquid crystal spinning appears to be widespread in the animal kingdom, utilizing protein dopes to give materials with a range of different secondary structures including beta-pleat, alpha-helix and collagen-fold. Here we seek to identify the essential design features used in natural liquid crystal spinning by comparing the spinning of two very different materials: the egg case wall of Selachians (dogfish, rays, and their allies) and the dragline silk of orb web spiders. The fish extrudes a "sea and island" composite in which the islands consist of flat ribbons of carefully orientated collagen and the sea, small quantities of an amorphous matrix. Dragline silk filaments are largely constructed from spidroin, a beta protein and have a skin and core structure together with two to three coats. The essential design features common to both systems appear to be the following: (i) intracellular co-storage of a hexagonal columnar liquid crystalline component and a peroxidase within the same secretory vesicles; (ii) luminal storage of a highly concentrated liquid crystalline dope; (iii) use of a dope containing immiscible droplets; (iv) hyperbolic extrusion dies; (v) control of pH and water content of the dope; (vi) preorientation of dope molecules before assembly into fibrils; (vii) combination of extrusion die, treatment/coating bath, and solvent recovery plant within a single microminiaturized device; (viii) slow natural spinning rates. The most important difference is that spiders produce a tough material by unfolding and hydrogen-bonding their silk dope molecules while Selachian fish do it by covalently cross-linking the molecules without unfolding them.

Changes in element composition along the spinning duct in a Nephila spider.

The silk gland of the golden orb spider Nephila edulis connects to the exit spigot through a long S-shaped duct that assists in the formation of the thread. Previous evidence suggests that the epithelium of the distal (last) part of the duct is specialized for ion transport and that a proton pump is involved in this process. Here, we present evidence from SEM (scanning electron microscope)-EDAX (energy dispersive X-ray) microanalysis of rapidly frozen material maintained at approximately -150 degrees C and from the use of pH indicators that the element composition and pH change progressively as the dragline silk dope (spinning solution) passes down the duct to form the thread. Na+ and Cl- composition decreased while K+ and P and S increased. Indicators suggested that the pH dropped from 6.9+/-0.1 to 6.3+/-0.1. These novel findings suggest that the absorption of Na+ and secretion of the more chaotropic K+ may help the silk protein molecules to refold while the secretion of H+ may assist in this process and reduce the repulsive charges on them. This in turn may allow the molecules to approach one another more closely to crystallize. Thus precise control of the ionic environment within the spider's spinning duct may be important in forming a tough insoluble thread and when devising mimetic processes to spin silk proteins industrially.

Liquid crystalline spinning of spider silk.

Spider silk has outstanding mechanical properties despite being spun at close to ambient temperatures and pressures using water as the solvent. The spider achieves this feat of benign fibre processing by judiciously controlling the folding and crystallization of the main protein constituents, and by adding auxiliary compounds, to create a composite material of defined hierarchical structure. Because the 'spinning dope' (the material from which silk is spun) is liquid crystalline, spiders can draw it during extrusion into a hardened fibre using minimal forces. This process involves an unusual internal drawdown within the spider's spinneret that is not seen in industrial fibre processing, followed by a conventional external drawdown after the dope has left the spinneret. Successful copying of the spider's internal processing and precise control over protein folding, combined with knowledge of the gene sequences of its spinning dopes, could permit industrial production of silk-based fibres with unique properties under benign conditions.

Beta transition and stress-induced phase separation in the spinning of spider dragline silk.

Spider dragline silk is formed as the result of a remarkable transformation in which an aqueous dope solution is rapidly converted into an insoluble protein filament with outstanding mechanical properties. Microscopy on the spinning duct in Nephila edulis spiders suggests that this transformation involves a stress-induced formation of anti-parallel beta-sheets induced by extensional flow. Measurements of draw stress at different draw rates during silking confirm that a stress-induced phase transition occurs.

Partially systematic molecular packing in the hexagonal columnar phase of dogfish egg case collagen.

The collagen that forms the egg case of the dogfish Scyliorhinus canicula is stored in bulk in the female nidamental glands. Here the collagen molecules are thought to undergo a series of distinct pH-dependent liquid crystalline aggregation phase changes before assembling into the final arrangement encountered in the mature egg case. One liquid crystalline phase is hexagonal with the centres of two adjacent hexagons about 36 nm apart. We have collected tilt series of the hexagonal phase from plastic sections of the nidamental gland and have produced a three-dimensional reconstruction of the collagen arrangement of this phase. The reconstruction features axial columns of protein density lying regularly on the vertices of hexagonal cells of edge length 21 nm. Each column is connected to three nearest neighbours by irregular sheets of protein, but there appear to be preferred molecular directions at about 40 degrees to 50 degrees to the columns. The reconstruction has been interpreted in terms of known interactions of this collagen in other assemblies.

Structure and function of the silk production pathway in the spider Nephila edulis.

Our observations on the major ampullate gland of the spider Nephila edulis indicate that the exceptionally tough and strong core and coat composite structure of the dragline thread is formed by the co-drawing of two feedstocks through a single die. The cuticle that lines the gland's duct has the structure of an advanced hollow fibre dialysis membrane and is thought to facilitate a rapid removal of water and change in ionic composition involved in the spinning process. A structure previously termed the 'valve' is thought to advance the broken thread and act as a pump to restart spinning after the accidental internal rupture of a thread. Together, these observations indicate that the spider silk production pathway is highly optimised for the production of silk threads and shows considerable biomimetic potential.

The acupuncture system and the liquid crystalline collagen fibers of the connective tissues.

We propose that the acupuncture system and the DC body field detected by western scientists both in here in the continuum of liquid crystalline collagen fibers that make up the bulk of the connective tissues. Bound water layers on the collagen fibers provide proton conduction pathways for rapid intercommunication throughout the body, enabling the organism to function as a coherent whole. This liquid crystalline continuum mediates hyperreactivity to allergens and the body's responsiveness to different forms of subtle energy medicine. It constitutes a "body consciousness" working in tandem with the "brain consciousness" of the nervous system. We review supporting evidence from biochemistry, cell biology, biophysics and neurophysiology, and suggest experiments to test our hypothesis.

Characterization of selachian egg case collagen.

The egg case of the dogfish Scyliorhinus canicula is a remarkable collagenous structure that combines mechanical strength and toughness with high permeability to small molecules and ions. The collagenous lamellae that form over 80% of the thickness of the case wall are secreted by the D-zone of the nidamental (oviducal gland). An acid-soluble collagen extracted from this zone and partially purified ran as a single band on a native gel at pH 4.3. A single band of identical mobility was extracted from egg cases removed from the oviducal gland. SDS-PAGE of both extracts revealed a major component with an apparent molecular weight of 35 kDa and a minor component at 34 kDa. Neither of these components appeared to be glycosylated. Amino acid analysis of the partially purified collagen extracted from the oviducal gland revealed a composition similar to that of the collagenous lamellae of the egg case with glycine accounting for 16% and imino acids for 10% of the total residues. Partial N-terminal and internal sequences were obtained by Edman degradation for peptides extracted from the D-zone of the nidamental gland. Four of the internal sequence fragments showed repeated G-X-Y triplets showing them to be collagenous. These four fragments were novel but showed similarity to the triple-helical domains of mammalian type IV, X, and VI collagens. The noncollagenous N-terminal and pepsin-resistant sequences were unique, showing no significant similarity to known proteins in the database. Several possible N-myristoylation and phosphorylation sites were identified in the noncollagenous sequences.

In vitro formation by reverse dialysis of collagen gels containing highly oriented arrays of fibrils.

Acid extracts of rat tail tendon were subjected to reverse dialysis against 0.5% PEG at 4 degrees C in an attempt to induce liquid crystallization. After 48 h, gel and fibril formation were initiated by continuing dialysis at 20 degrees C against the same PEG solution adjusted to pH 7.4. The inclusion of calcium- or magnesium chloride (final concentration 0.3-33 mM) in the collagen solution before dialysis resulted in strongly birefringent gels that showed a progressive rotation of the slow axis of birefringence with increasing distance from the lateral margin of the gel. The gels contained fibers running predominantly in the plane of the flattened gel and crossing at angles of between 55 degrees and 90 degrees. We suggest that liquid crystallization is responsible for this phenomenon and that it might be possible to exploit this to produce materials for tissue engineering.

The effect of non-polar liquids and non-ionic detergents on the ultrastructure and assembly of rat tail tendon collagen fibrils in vitro.

Non-ionic detergents or emulsions of non-polar liquids when added to solutions of rat tail tendon collagen (RTTC) or to the dispersed fibrils produced similar conspicuous ultrastructural modifications in the form of a D-periodic lesion between bands c2 and d in the 'gap region' of the fibril close to the start of the overlap region. The size and extent of the lesion in some fibrils indicates that at least some of the collagen molecules rupture. In an attempt to detect peptide fragments produced in this way we ran SDS-PAGE gels of collagen fibrils treated with the non-ionic detergent Triton X-100. These contained two peptides (44 and 32 kDa) not seen in controls. The lesions are thought to result from interactions between the hydrophobic part of non-polar liquids or detergents with an anomalous part of the fibril's D-period. The anomalous region has a high concentration of hydrophobic and alanyl residues but exceptionally few charged and hydroxyproline ones. We suggest that the anomalous region may play a part in storing and dissipating strain energy and permitting cross-link formation. Similar collagen-lipid interactions may occur under pathological conditions.

The effect of deamination and/or blocking arginine residues on the molecular assembly of acid-extracted rat tail tendon collagen.

We describe the effect of deamination of lysine and blocking of arginine residues on the assembly of collagen into native fibrils and SLS aggregates. Treatment of collagen solutions with one or both of these procedures does not prevent the formation of fibrils or SLS aggregates but reduces their ability to form assemblies with accurate longitudinal registration. These observations provide direct confirmation that hydrophobic interactions are important in collagen assembly. Unbanded fibrils were formed within the first 24 h at 4 degrees C from both acidic and neutralized deaminated and from neutralized control collagen solutions, transversely banded fibrils appearing later. This is compatible with the suggestion that initially, collagen fibrils are assembled by lyotropic liquid crystallization and with other observations which suggest that collagen molecules are initially free to move laterally within the fibril before being locked into place. Fibrils assembled from deaminated collagen solution show two variant longitudinal registration patterns which grade into one another. This suggests that, with a reduction in positively charged side chains, the thermodynamic energy minima responsible for longitudinal registration are less sharp compared with control collagen solutions. Reduction of positive charge by chemical modification helps to explain why the chemical modifications reduce swelling of collagen fibres. It also helps to explain why fibrils form spontaneously at 4 degrees C in both arginine-blocked and deaminated collagen solutions. Thus chemical modifications of rat tail tendon provides new insight into the mechanisms in collagen assembly.

The effect of PH on fibrillogenesis of collagen in the egg capsule of the dogfish, Scyliorhinus canicula.

The collagen of the egg capsule of the dogfish, Scyliorhinus canicula is stored and secreted by the secretory cells of the D-zone of the nidamental gland (Rusaouën-Innocent, 1990b). The collagen appears to pass through several morphologically distinct textures during storage, secretion and fibril formation which may represent different lyotropic liquid crystalline phases (Knight et al., 1993). In the present communication we report evidence that a fall in hydrogen ion concentration induces fibrillogenesis during the secretion of the dogfish egg capsule. In an attempt to understand the factors involved in collagen assembly, we investigated the effects of subjecting isolated collagen storage granules in vitro to solutions ranging in pH from 2-11 and Na(+), K(+), Ca(++), Mg(++), Zn(++) and Cu(++) ions at concentrations varying from 0.01-0.5 M. From pH 2 to pH 4 most granules appeared completely amorphous; from pH 5 to pH 7 granules showed the following previously reported liquid crystalline textures: isotropic, lamellar, micellar, hexagonal columnar, transversely banded twisted nematic, and unbanded twisted nematic. At pH 8 granules showed both the hexagonal columnar phase (phase IV) and small quantities of the final fibrillar phase together with a previously undescribed texture. The latter texture, which we refer to as phase VII, had a D period (17.5 nm) half that of the lamellar texture (phase II) and the final egg capsule fibrils (phase VI). From pH 9 to pH 11, only the final fibrillar texture (phase VI) together with small quantities of the new texture (phase VII) were present. Na(+), K(+), Ca(++), Mg(++), Zn(++) and Cu(++) ions did not appear to have an observable effect on the phases found in isolated granules at pH 7.0. The role of pH in collagen storage and fibrillogenesis was confirmed by direct estimation of the pH in vivo using vital staining with neutral red, a range of pH indicators applied to unfixed cryostat sections and direct measurements of the pH of the jelly within the egg capsule. The implications of these findings for the mechanism of collagen storage and fibrillogenesis in the dogfish egg capsule and other collagenous systems are discussed.

Some observations on the collagen fibrils of the egg capsule of the dogfish, Scyliorhinus canicula.

The egg capsule of the dogfish Scyliorhinus canicula is a collagenous material with a laminated, plywood (orthogonal) construction. The collagen fibrils which constitute the bulk of the egg capsule wall have a unique, highly ordered structure (Knight and Hunt, 1974; 1976, 1986; Gathercole et al., 1993) which is thought to represent a smectic A liquid crystalline phase (Knight et al., 1993). The egg capsule is extremely strong and chemically inert (Hunt, 1985). It is stored, secreted and formed by the nidamental gland (Rusaouën 1976, 1990 a, b; Knight and Feng, 1992). During intracellular storage, secretion and fibrillogenesis, the dogfish egg capsule collagen appears to pass through a remarkable series of textures within a lyotropic liquid crystalline phase diagram (Knight et al., 1993). In the present communication, further observations on the ultrastructure of the collagen fibrils and their arrangement within the laminae of the fully-formed egg capsule are reported. The effect of tilting ultrathin sections of fibrils in the goniometer stage of a transmission electron microscope are described, demonstrating that the crystalline lattice within the fibril appeared twisted more or less regularly into a long pitch helix. Other observations indicated that some of the fibrils were in turn twisted round one another to form fibres which therefore had a coiled-coil structure. The fibres are arranged parallel to one another in the laminae which are stacked to give an orthogonal plywood construction. The effects of staining fibrils with cuprolinic blue and with tannic acid are reported. Reduction in the water content of the fibrils before fixation appeared to move some of the fibrils through the part of the lyotropic phase transition diagram converting them from smectic A to smectic C. Finally, evidence is presented that the fibrils shrank, but remarkably, still retained a longitudinally-ordered but modified, molecular arrangement even after boiling in water for periods of up to 10 min. These observations are discussed in relation to other collagens.

Interaction of collagen with hydrophobic protein granules in the egg capsule of the dogfish scyliorhinus canicula.

The egg capsule of the dogfish is a composite material containing collagenous fibrils and 2 mum spherical hydrophobic protein granules. The latter appear to owe much of their hydrophobicity to an exceptionally high tyrosine content (approximately 20% of total amino acid residues). The hydrophobic component appears to form as an emulsion in the secretory granules of the D and E zone gland cells of the nidamental gland. Droplets of the hydrophobic material appear to become coated with remarkably regular layers of radially-arranged collagen molecules which form a series of concentric, evenly spaced layers around each hydrophobic granule. Numerous disclinations were seen where the layers around adjacent granules interfered with one another. The layers are thought to represent a lamellar liquid crystalline phase previously described for this collagen (Knight et al., 1993). The fine structural appearance of the concentric layers and evidence for radial arrangement of collagen molecules within them is compatible with the suggestion that the layers are built from a dumbbell-shaped unit approximately 35 nm long with hydrophobic groups concentrated at the ends. This unit may represent a dumbbell-shaped molecule or an oligomer of two or more molecules lying parallel with one another in a head-to-tail arrangement. Such a unit can be readily incorporated into models for the micellar, hexagonal columnar and final fibrillar phases previously described for this collagen (Knight et al., 1993). Evidence from the TEM study of stretched egg capsule wall suggests that there is a mechanical interaction between the hydrophobic granules and the collagen fibrils in the fully formed material. We suggest that the radial, concentric layered arrangement of collagen molecules is established by hydrophobic interactions within the liquid crystalline material and locked into place by oxidative covalent cross-linking to give a 3-dimensional cross-linked meshwork of collagen fibrils and hydrophobic granules. The latter arrangement helps to account for the high tensilestrength and toughness of this material.

Secretion and stabilization of the layers of the egg capsule of the dogfish Scyuorhinus canicula.

The egg capsule of the dogfish is a unique, collagcnous, layered structure secreted by the nidamental gland which has nine, remarkably discrete, transverse zones of tubular glands. The present paper traces the origin of the four layers of the capsule to particular zones within the gland. Evidence is presented for the existence of DOPA, DOPA oxidase. protein(s) rich in tyrosyl residues and a peroxidase within the same storage granules within the secretory cells of the C and E zones. It is suggested that these interact when secreted to cross-link the inner and outer surfaces of the egg capsule. Evidence is presented that the middle layer which forms the bulk of the thickness of the egg capsule and has the highest collagen content may be partly stabilized by the peroxidation of tyrosyl residues. The mechanical significance of crosslinking in this system and the possible cytological mechanisms involved in the secretion of the tanning agents and enzymes are discussed.

Improved specificity of the CA 125 enzyme immunoassay for ovarian carcinomas by use of the ratio of CA 125 to carcinoembryonic antigen.

We found that ovarian cyst fluids contained carcinoembryonic antigen (CEA) and CA 19-9 and CA 125 tumor markers. However, only the ratio of CA 125 to CEA concentrations provided sufficient specificity to differentiate serous from mucinous cysts. For CEA measurement, our results suggested the use of a monoclonal CEA kit. When CEA was determined with a Hybritech monoclonal CEA kit, all ratios in mucinous ovarian cysts were less than 10 and most of the ratios were greater than 1000 in serous ovarian cysts. We also found that the ratio of CA 125 to CEA in serum could be used to differentiate ovarian from nonovarian malignant diseases when both sera contain increased CA 125 concentrations. The nonovarian malignancies consisted of colorectal, breast, lung, and pancreatic carcinomas. The mean ratio for serum from patients with nonovarian cancers was 0.94 (n = 19); for ovarian-cancer patients (n = 45), 916. Therefore, determining this ratio will greatly improve the specificity of the CA 125 test for ovarian cancer.