[The role of skin substitutes in the surgical treatment of extensive burns covering more than 60 % of total body surface area. A review of patients over a 10-year period at the Tours University Hospital]. / Place des substituts cutanés dans le traitement chirurgical des grands brûlés sur plus de 60 % de la Surface corporelle. Revue de patients sur 11ans dans le centre des brûlés adultes du CHRU de Tours.

INTRODUCTION: Progress in intensive care and surgery has made it possible to significantly improve the survival of victims with burns over 60% of total body surface area (TBSA). Coverage of the excised areas of these patients can be difficult when there is a shortage of skin donor sites; then the role of skin substitutes can be important. MATERIALS AND METHODS: This retrospective study included patients with burns covering more than 60% TBSA and treated at the Tours University Hospital over a period of 10 years. Patients who died during the first week or who presented superficial burns were excluded. The various substitutions means to temporarily or permanently replace the cutaneous barrier are presented. The biological dressings associated with grafts expanded by six according to the sandwich technique, allografts and xenografts, widely expanded postage stamp skin grafts using a modified Meek technique (Humeca(®)), temporary cutaneous substitutes such as Biobrane(®) and skin substitutes colonized by autologous cells (Integra(®)) are presented. RESULTS: Forty-four patients were admitted. Self-immolations represented 52% of the cases. Twenty-one patients were treated with Integra(®), 5 with Biobrane(®), 17 with sandwich grafts and 4 with postage stamp skin grafts. Integra(®) was widely used when donor sites were insufficient. The mean number of surgical procedures per patient was 8.4. The mean duration of hospitalization was 155 days. Twenty-four patients survived until the end of treatment. Eighteen patients died during the first week before any surgery could be performed. Two patients died at the end of treatment. The overall survival rate was 55%. It was 92% for patients who survived the first week. The principal sequel were functional (hand, cervical, thoracic and axillary contractures) and aesthetic (face and hands). Associated treatments were pressotherapy, physical therapy, ergotherapy and thermal water therapy. CONCLUSION: By temporarily replacing the cutaneous barrier in the absence of sufficient donor sites, skin substitutes make it possible to increase the survival of patients with very extensive burns and to optimize their treatment.

White matter volume is decreased in the brain of patients with neuromyelitis optica.

BACKGROUND: Neuromyelitis optica (NMO) is an inflammatory disease involving predominantly the spinal cord and optic nerves. Whether patients with NMO have a loss in white or grey matter (GM) volumes remains to be determined. METHODS: Thirty patients with NMO, 30 healthy subjects matched for age and gender, 21 patients with multiple sclerosis (MS) and 20 patients with a clinically isolated syndrome (CIS) were studied. We applied a SIENAX post-treatment software. We compared white matter (WM) and GM volumes between groups and explored correlations of changes in NMO patients with age, gender, duration, disease severity, visual acuity and T2 hyperintensities. We also performed a voxel-based morphometry (VBM) analysis to identify the regions affected by loss of volume. RESULTS: White matter volume was significantly reduced in patients with NMO (764.4 ± 58.3 cm(3) ) compared to healthy subjects (843.1 ± 49.3 cm(3) ) (P < 0.001), whereas no difference was observed for the GM. Patients with CIS also presented an elective atrophy of WM and MS an atrophy of both WM and GM. We did not find any predictive factors of brain atrophy. The decrease in WM volume in NMO was noted even in the absence of visible MRI hypersignals. The VBM analysis found a few regions of WM atrophy (corpus callosum and optic radiations, P < 0.005, uncorrected) and a few regions of GM atrophy (thalamus and prefrontal cortex, P < 0.001, uncorrected). CONCLUSION: These results suggest a significant brain involvement in NMO, especially an involvement of WM which appears not to be limited to secondary degeneration after spinal cord and optic nerve damage.

[Use of Matriderm® 1mm in reconstructive surgery. Series of 31 cases]. / Utilisation de Matriderm® 1mm en chirurgie reconstructrice. Série de 31 cas.

INTRODUCTION: Dermal substitute are used for soft-tissue defect for their functional and aesthetic advantages. Matriderm® 1mm, single layer dermal matrix, composed of collagen and elastin covered by a split thickness skin graft simultaneously to its application, has been used most often in burned surgery. This prospective series evaluates the interest of this recent dermal matrix in reconstructive surgery. PATIENTS AND METHOD: Twenty-eight patients have been treated with the substitute in our department for reconstructive surgery indication between November 2008 and May 2012. Indications were tissue losses treatment after limb or trunk sarcoma resection, melanoma, extended baso- or spinocellular carcinoma, palmoplantar keratodermy, burn sequels, or traumatic tissue losses. Indications were preferentially deep tissue losses, functional areas and the face. RESULTS: Mean treated area has been 82.4 cm(2) (10 to 600 cm(2)). Mean taken rate has been 87±19% of the area and mean day of discharge has been 4.8 days and the mean cost per patient 906.5 euros. Negative wound therapy, until D3, was used 6 times. Three patients treated for limb sarcoma had radiotherapy performed on the grafted area. Aesthetic and functional results have been encouraging. CONCLUSIONS: Matriderm® 1mm, dermal substitute commonly used in acute burn treatment can be indicated in reconstructive surgery. This series show that it permits to obtain a good graft taken rate, a quick healing with a satisfying aesthetic and functional results and permit an early discharge. However, its indications are limited by its cost.

Plasmodesmatal pores in the torus of bordered pit membranes affect cavitation resistance of conifer xylem.

The pit membrane in bordered pits of conifer tracheids is characterized by a porous margo and central thickening (torus), which is traditionally considered to function as an impermeable safety valve against air-seeding. However, electron microscopy based on 33 conifer species, including five families and 19 genera, reveals that pores occur in the torus of 13 of the species studied. The pores have a plasmodesmatal origin with an average diameter of 51 nm and grouped arrangement. Evidence for embolism spreading via pores in tori is supported by the pore sizes, which correspond relatively well with the pressure inducing cavitation. Predictions based on earlier correlations between pit structure and cavitation resistance were only weakly supported for species with punctured tori. Moreover, species with punctured tori are significantly less resistant to cavitation than species with non-punctured tori. Nevertheless, absolute pore diameters must be treated with caution and correlations between theoretical and measured air-seeding pressures are weak. Because most pores appear not to traverse the torus but are limited to one torus pad, only complete pores would trigger air-seeding. Embolism spreading through a leaky torus is not universal across gymnosperms and unlikely to represent the only air-seeding mechanism.

Adaptive responses for seed and leaf phenology in natural populations of sessile oak along an altitudinal gradient.

We assessed the adaptive potential of seed and leaf phenology in 10 natural populations of sessile oak (Quercus petraea) sampled along two altitudinal transects using common garden experiments. Population differentiation for both phenological traits was observed with high-altitude populations germinating and flushing later than low altitude ones. However, high genetic variation and heritability values were also maintained within populations, despite slightly decreasing for dates of leaf unfolding with increasing altitude. We suggest that biotic and abiotic fluctuating selection pressures within populations and high gene flow are the main mechanisms maintaining high genetic variation for these fitness related traits. Moreover, changes in selection intensity and/or selection pressures along the altitudinal gradient can explain the reduction in genetic variation observed for leaf phenology. We anticipate that the maintenance of high genetic variation will be a valuable resource for future adaptation of sessile oak populations undergoing an upslope shift caused by climate change.

Effects of galvanic mastoid stimulation in seated human subjects.

The vestibular responses evoked by transmastoid galvanic stimulation (GS) in the rectified soleus electromyogram (EMG) in freely standing human subjects disappear when seated. However, a GS-induced facilitation of the soleus monosynaptic (H and tendon jerk) reflex has been described in few experiments in subjects lying prone or seated. This study addresses the issue of whether this reflex facilitation while seated is of vestibulospinal origin. GS-induced responses in the soleus (modulation of the rectified ongoing EMG and of the monosynaptic reflexes) were compared in the same normal subjects while freely standing and sitting with back and head support. The polarity-dependent biphasic responses in the free-standing position were replaced by a non-polarity-dependent twofold facilitation while seated. The effects of GS were hardly detectable in the rectified ongoing voluntary EMG activity, weak for the H reflex, but large and constant for the tendon jerk. They were subject to habituation. Anesthesia of the skin beneath the GS electrodes markedly reduced the reflex facilitation, while a similar, although weaker, facilitation of the tendon jerk was observed when GS was replaced with purely cutaneous stimulation, a tap to the tendon of the sternomastoid muscle, or an auditory click. The stimulation polarity independence of the GS-induced reflex facilitation argues strongly against a vestibular response. However, the vestibular afferent volley, insufficient to produce a vestibular reflex response while seated, could summate with the GS-induced tactile or proprioceptive volley to produce a startle-like response responsible for the reflex facilitation.

The reliability of commonly used electrophysiology measures.

BACKGROUND: Electrophysiological measures can help understand brain function both in healthy individuals and in the context of a disease. Given the amount of information that can be extracted from these measures and their frequent use, it is essential to know more about their inherent reliability. OBJECTIVE/HYPOTHESIS: To understand the reliability of electrophysiology measures in healthy individuals. We hypothesized that measures of threshold and latency would be the most reliable and least susceptible to methodological differences between study sites. METHODS: Somatosensory evoked potentials from 112 control participants; long-latency reflexes, transcranial magnetic stimulation with resting and active motor thresholds, motor evoked potential latencies, input/output curves, and short-latency sensory afferent inhibition and facilitation from 84 controls were collected at 3 visits over 24 months at 4 Track-On HD study sites. Reliability was assessed using intra-class correlation coefficients for absolute agreement, and the effects of reliability on statistical power are demonstrated for different sample sizes and study designs. RESULTS: Measures quantifying latencies, thresholds, and evoked responses at high stimulator intensities had the highest reliability, and required the smallest sample sizes to adequately power a study. Very few between-site differences were detected. CONCLUSIONS: Reliability and susceptibility to between-site differences should be evaluated for electrophysiological measures before including them in study designs. Levels of reliability vary substantially across electrophysiological measures, though there are few between-site differences. To address this, reliability should be used in conjunction with theoretical calculations to inform sample size and ensure studies are adequately powered to detect true change in measures of interest.

Three-dimensional reconstruction of Limulus polyphemus hemocyanin from cryoelectron microscopy.

Hemocyanin (Hc) the respiratory pigment of the horseshoe crab Limulus polyphemus (Lp) is composed of 48 approximately 75 kDa copper-containing subunits arranged in eight hexameric groups. In this study, we used the random conical tilt series method to do a three-dimensional (3D) reconstruction of Lp Hc observed in vitreous ice. This approach allowed the unambiguous determination of the handedness of the molecule. Lp Hc contains two superimposed 4 x 6mer structures possessing the same structural features as the other 4 x 6meric Hcs, namely flip and flop views and a rocking effect. Moreover, 3D fitting of the X-ray structure of subunit LpII with the reconstruction volume shows that the intra4 x 6meric contacts described in arthropod Hcs also occur within Limulus Hc. The two half-molecules composing the 8 x 6mer have their flop faces in contact (flop/flop association), the main links being formed by subunits LpIV. Model building shows that the flop/flop association is the only possible arrangement which allows the assembly of the whole particle. The two alternate constructions (flip/flop and flip/flip) are forbidden because of steric hindrance.

Quaternary structure of Octopus vulgaris hemocyanin. Three-dimensional reconstruction from frozen-hydrated specimens and intramolecular location of functional units Ove and Ovb.

A frozen-hydrated sample of Octopus vulgaris hemocyanin was imaged at 0 degree and 40 degrees tilt angle under low dose conditions by transmission electron microscopy. A three-dimensional reconstruction by the method of random conical tilt series produced a three-dimensional volume to which a D5 symmetry was applied. Examination of serial sections in the volume and surface representation at various thresholds allowed the five arches containing functional unit Ovg to be localized at the interdimeric subunit groove. In another set of experiments specific polyclonal antibodies were used to label functional units Ovb and Ove in the cylinder wall. The observation of the negatively stained immunocomplexes showed that Ovb is located in the external tiers of functional units and Ove in the internal tier. These results suggest that the direction of the polypeptide chains in the cylinder wall may be only partially antiparallel. A model of the quaternary structure is proposed with the following features: (1) the external tiers of functional units comprise four units each (Ova-d) coming from a single polypeptide chain; (2) the internal tier comprises two functional units from each polypeptide chain (Ove-f); (3) the interdimeric subunit arches connect the two copies of a single functional unit (Ovg) located in each polypeptide chain.

Reassembly of Lumbricus terrestris hemoglobin: a study by matrix-assisted laser desorption/ionization mass spectrometry and 3D reconstruction from frozen-hydrated specimens.

Dodecamers and four types of linker chains (L1-L4) were purified from dissociated hemoglobin of the earthworm Lumbricus terrestris. Various preparations comprising dodecamer of globin chains and linker chains were allowed to reassemble at neutral pH. They produced various oligomers that were purified by gel filtration, analyzed in matrix-assisted laser desorption/ionization mass spectrometry and submitted to 3D reconstruction from isolated particles observed in cryoelectron microscopy. Despite the impossibility to completely free the L2, L3, and L4 preparations from L1, the following conclusions were obtained. First, hemoglobin molecules indistinguishable from native hemoglobin at 25 A resolution were obtained in the absence of linker chains L2, L3, or L4. Second, the 3D reconstruction volumes of reassembled hemoglobins containing dodecamers and L1+L3 or dodecamers and L1+L4 demonstrate that reassembly of native-like structures can be obtained from at most two linker chains and dodecamers. Third, the 3D reconstruction volumes of native and reassembled hemoglobins containing dodecamers and (1) L1, L2, and L4, (2) L1, L3, and L4, (3) L1 and L4, and (4) L1 and L3 were highly similar. Since these structures comprise two types of substructures (one involved in the c3a, c3b, and c4 linking units of the hollow globular substructure and the other in the c5 connection and the toroid), it seems highly probable that the minimal number of linker chains required to reassemble native-like hemoglobin is at most two.

Intramolecular localization of the functional units of Sepia officinalis hemocyanin by immunoelectron microscopy.

The quaternary structure of Sepia officinalis hemocyanin (Hc) as studied in immunoelectron microscopy with rabbit IgGs and Fab fragments raised against functional units (FU) Soc, Sod, Soe, Sof, Sog, and Soh and fragment Soab. The architecture of immunocomplexes shows that (i) epitopes characteristic of FUs Soc and Sog and of fragment Soab are located in the two external tiers of FUs, (ii) FUs Soh and Soe or Sod are located in arches. These results were confirmed using immunocomplexes made up of Sepia Hc and IgGs or Fab fragments purified from antisera raised against FUs of Octopus vulgaris and Octopus dofleini. Frozen-hydrated immunocomplexes containing one Hc molecule and at least one FU-specific Fab fragment were observed in the electron microscope and submitted to image processing. When the Hc molecule is viewed along its 5-fold axis (i) anti-Soc Fab fragments project on a radius passing through the arch's pillar, (ii) anti-Sof Fabs project slightly out of the arches, and (iii) anti-Soh Fabs project between neighboring arches. When applied to a recent three-dimensional (3D) reconstruction volume, these results allow us to deduce the intramolecular location of five of the eight FUs. For the last three FUs limited uncertainties remain: (i) Soc can be located in two positions in the external tier of FUs; (ii) Soa and Sob can both occupy three positions in the external tiers; and (iii) because of an immunological cross-reactivity Sod may be located in the wall and Soe in the arch, or vice versa. An analysis of the quaternary structure considering the possible locations of the 80 FUs and postulating a single type of subunit shows that 80 possibilities of paths still exist for the polypeptide chain. To solve definitely these 80 possibilities only five questions remain to be answered.

Three-dimensional architecture of human alpha 2-macroglobulin transformed with methylamine.

A frozen-hydrated sample embedded in vitreous ice of human alpha 2-macroglobulin transformed by methylamine was imaged by cryoelectron microscopy and reconstructed in three dimensions. In the reconstruction, the cage-like architecture of this protease inhibitor is fully revealed with a clear visualization of two lozenge-shaped lateral walls connected by thin bridges. The shape and dimensions of the internal cavity normally containing the trapped protease(s) is described. The possible locations of the thiol ester sites and inter-subunit connections are also discussed.

Three-dimensional reconstruction from a frozen-hydrated specimen of the chiton Lepidochiton sp. hemocyanin.

A frozen-hydrated specimen of the hemocyanin of the chiton Lepidochiton sp. has been subjected to a three-dimensional reconstruction by the random conical tilt- series m wall and a collar complex. The wall is composed of five oblique wall units, disposed as a five-stranded, right-handed helix, separated by five clefts. The oblique wall unit is composed of two strings of functional units separated by a groove parallel with the cleft. The collar complex is a crown-like structure composed of five collar complex units, located at one end of the molecule and slightly protruding outside the cylinder wall. The collar complex unit comprises a collar unit probably composed of two functional units, one of which is connected to the wall, and an arch composed of two additional functional units, each connected to the wall by a narrow bridge. Each arch crosses a cleft between adjacent oblique wall units. The indentations present on both circular faces of the molecule and the dispositions of the masses resemble those of cephalopod hemocyanins.

Concerning the axial rotational flexibility of the Fab regions of immunoglobulin G.

By electron microscopy, we have observed immunocomplexes with both negative stain and in amorphous ice using monoclonal antibodies directed against one of the 24 subunits of scorpion haemocyanin. A copy of this subunit occurs at each of the corners of the square-shaped haemocyanin molecule. Three distinct orientations of adjacent haemocyanin molecules may be observed in immunocomplex pairs or chains using both the above-mentioned methods. These observations, coupled with low-resolution computer simulations of immunocomplex formation, argue strongly in favour of the existence of a considerable degree of rotational flexibility within the IgG molecule and around the long axis of the Fab arms, as was suggested by previous observations with negative stain. We find that the arms can rotate by up to 180 degrees with respect to the Fc region.

Three-dimensional reconstruction of Lumbricus terrestris hemoglobin at 22 A resolution: intramolecular localization of the globin and linker chains.

A 3D reconstruction of the hemoglobin (Hb) of the earthworm Lumbricus terrestris was carried out by the 3D projection alignment method from electron microscopy images of a frozen-hydrated specimen at 22 A resolution. The results were analyzed by a new approach taking into account the evolution of the 210 densities forming the 3D volume as a function of the threshold of surface representation. The whole oligomer with D6point-group symmetry is comprised of 12 hollow globular substructures (HGS) with local 3-fold symmetry tethered to a complex network of linking subunits (linker complex). The 12 globin subunits of each HGS are distributed around local 3-fold axis in four layers of three subunits. The first layer, the most external, contains monomeric globin chains 2A, 3A, and 5A. The three trimers corresponding to the nine remaining subunits have one subunit in each of the second (2B, 3B, 5B), third (1A, 4A, 6A), and fourth (1B, 4B, 6B) layer. The distances between the centers of the globin chains forming the trimers are in the ranges 20-32 A and 45-52 A. The linker complex is made up of two types of linking units. The first type forms three loops connecting globin chains of the second, third and fourth layers. The average molecular mass (Mm) of these subunits was 25 kDa. The second type forms the central structure, termed hexagonal toroid, and its 12 connections to the HGS. This structure corresponds to a hexamer of a single linking unit with a Mm (31.2 kDa), size and a shape different from those of the HGS loops. A careful study of 3D volume architecture shows that each toroid linking unit is bound to the three loops of a HGS pair located in the upper and lower hexagonal layers, respectively. As shown in a model of architecture, hexagonal bilayered (HBL) Hbs can be built very simply from 144 globin chains and 42 linker chains belonging to two different types. We also propose a simple assembly sequence for the construction of HBL Hbs based on the architecture model.

Crystals of the carboxyl-terminal functional unit from Octopus dofleini hemocyanin.

The carboxyl-terminal oxygen-binding unit of the polypeptide from Octopus dofleini hemocyanin has been crystallized in a form suitable for three-dimensional X-ray analysis. This proteolytic fragment has a molecular weight of 47 kDa and reversibly binds O2 while exhibiting a slight Bohr effect. Two types of crystals have been grown. Type I crystals, currently under analysis, belong to the orthorhombic space group P2(1)2(1)2(1) and have unit cell dimensions of 92.6 A x 167.4 A x 59.2 A. A composition of two protein molecules per asymmetric unit and 50% solvent content is consistent with a self-rotation function that identifies a non-crystallographic 2-fold axis of symmetry relating these molecules. Diffraction extending beyond 1.9 A Bragg spacings can be detected with synchrotron X-radiation.

Three-dimensional reconstruction of native Androctonus australis hemocyanin.

A sample of native 4 x 6-meric hemocyanin of Androctonus australis was negatively stained with the double-layer technique, and was observed by transmission electron microscopy under low-dose conditions with a 50 degree and 0 degree tilt. The three-dimensional reconstruction method from "Single-exposure, random conical tilt series" was then applied. Independent three-dimensional reconstructions were obtained from the top, side and 45 degree views. Despite a pronounced flattening effect, presumably due to the specimen preparation technique, the positions of the 24 subunits composing the oligomer were unequivocally determined. This experiment definitely solves the problem of the architectural organization of the subunits in the cheliceratan 4 x 6-meric hemocyanins. Moreover, distinction between the flip and flop faces and an attenuated rocking effect were observed.

Occurrence of two architectural types of hexagonal bilayer hemoglobin in annelids: comparison of 3D reconstruction volumes of Arenicola marina and Lumbricus terrestris hemoglobins.

A 3D reconstruction at 25 A resolution of native hemoglobin of the polychaete worm Arenicola marina was carried out from frozen-hydrated specimens examined in the electron microscope. The reconstruction volume of this large extracellular multimeric respiratory pigment appears as a hexagonal bilayer structure with eclipsed vertices in its upper and lower hexagonal layers. Conversely, in hemoglobins of oligochaetes, achaetes, and vestimentiferans and in chlorocruorins of the Sabellidae (polychaete) family, the vertices of the upper layer are 16 degrees clockwise rotated with respect to those of the lower layer. The fact that two other polychaete hemoglobins (Alvinella pompejana and Tylorrhynchus heterochaetus) have the same architecture as Arenicola led us to define two types of hexagonal bilayer hemoglobins/chlorocruorins: (i) type-I present in oligochaete, achaete, and vestimentiferan hemoglobins and in Sabellidae chlorocruorins; and (ii) type-II present in polychaete hemoglobins. A comparative study of the hemoglobins of Lumbricus terrestris (type-I) and Arenicola marina (type-II) showed that only two small differences located in the c4 and c5 linking units are responsible of the important architectural difference present in oligomers. A likely scheme proposed to explain the phylogenic distribution of the two types suggests that Clitellata, Sabellida (polychaete), and vestimentiferan hemoglobins and chlorocruorins derive from a type-I ancestral molecule, while Terebellida (Alvinella), Phyllodocida (Tylorrhynchus), and Scolecida (Arenicola) and possibly other polychaetes derive from an ancestor molecule with type-II hemoglobin. The architectures of the hollow globular substructures are highly similar in Arenicola and Lumbricus hemoglobins, with 12 globin chains and three linking units (c3a, c3b, and c4). The central piece of Arenicola hemoglobin is an ellipsoid while that of Lumbricus is a toroid. No phylogenic correlation could be found between the structure of the central pieces and the architecture type.

Three-dimensional reconstruction by cryoelectron microscopy of the giant hemoglobin of the polychaete worm Alvinella pompejana.

A frozen-hydrated specimen of the hexagonal bilayer hemoglobin (HBL Hb) from the deep-sea hydrothermal vent polychaete worm Alvinella pompejana, the most thermophilic metazoan known to date, was observed in the electron microscope and subjected to three-dimensional (3D) reconstruction by the method of random conical tilt series. At a resolution of 34.6 A by the differential phase residual method and 27.7 A by the Fourier shell correlation method, the 3D volume possesses a D6 point-group symmetry. While in previous 3D reconstructions of annelid and vestimentiferan Hbs the vertices of the upper layer were 16 degrees rotated compared with those of the lower layer, in Alvinella Hb the vertices of the two hexagonal layers are almost perfectly eclipsed when viewed along the 6-fold axis. As in the HBL Hbs of Riftia pachyptila and Macrobdella decora, a central linker complex is decorated by 12 hollow globular substructures (HGS). The linker complex comprises (1) a central hexagonal toroid, (2) two internal bracelets onto which the HGSs are built, and (3) six connections between the two hexagonal layers. Each HGS is composed of six masses, which are separated when the volume is displayed at high threshold, plus one additional mass involved in the bracelet connecting the six HGSs in both hexagonal layers. The HGSs have a local pseudo 3-fold symmetry and a disposition of the high-density masses different from those of Riftia V1 Hb.

Three-dimensional reconstruction of the alpha D and beta C-hemocyanins of Helix pomatia from frozen-hydrated specimens.

The three-dimensional (3D) reconstructions of the di-decameric forms of alpha D and beta C-hemocyanins of the Roman snail Helix pomatia and of the decameric half molecules of alpha D-hemocyanin were carried out on frozen-hydrated specimens observed in the electron microscope by using the random conical tilt series method. The three 3D volumes were examined by computing solid-body surface representations and slices through the volume and by eroding the structure progressively through raising of the threshold. The di-decameric molecule of alpha D and beta C-hemocyanins, reconstructed from side views, are very similar and are composed of a cylindrical wall, comprising ten oblique wall units, and of two collar complexes located at both ends of the cylinder, comprising each five arches and an annular collar made up of five collar units. Erosion of the structure reveals that the wall looks like a segment of a five-stranded right-handed helix and that each oblique wall unit resembles a figure 8 inclined to the right. The decameric half molecule of alpha D-hemocyanin, reconstructed from end-on views, resembles the whole molecule, except that the collar is thinner and appears composed of five independent collar complex units. It is suggested that the difference in structural appearance of the collar complex between the whole and the half alpha D-hemocyanin may be due to the missing cone artifact, induced by the angular limitations imposed by the goniometer of the electron microscope. The comparison between the alpha D-hemocyanin and the beta C-di-decameric hemocyanin at high thresholds suggests that in the beta C-hemocyanin the oblique wall units of each half molecule may be linked by two connections, whereas in alpha D-hemocyanin there may be only one. This difference in the number of connections may be responsible for the lower stability of the alpha D molecule at high salt concentration.