Development of respiratory trees in the holothurian <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> (Aspidochirotida: Holothuroidea)

The development of respiratory trees in the holothurian Apostichopus japonicus has been studied using light and electron microscopy. Primordial respiratory trees emerge in 2–3-mm-long animals (2 months after fertilization). They arise as two independent outgrowths from the dorsal wall of the anterior part of the cloaca. Upon first emerging and throughout the course of development, the left respiratory tree is longer than the right one. A common base develops in 4-mm-long animals (2–3 months after fertilization). In yearlings, the left respiratory tree grows into gaps between the loops of the intestinal tube interlaced with intestinal hemal vessels. The developing coelomic and luminal epithelia have ultrastructural peculiarities. The luminal epithelium of respiratory trees has been shown for the first time to comprise nerve cells and their processes. Characteristic structural features of the epithelia are shown to begin developing as early as in 4-mm-long animals (2–3 months after fertilization). In yearlings, the respiratory trees demonstrate definitive structural patterns and are entirely functional.     

Ultrastructure of coelomic lining in echinoderm podia: significance for concepts in the evolution of muscle and peritoneal cells

Summary Ultrastructural data are presented on the histological organization of coelomic lining in the podia of ten species of the five major groups of extant echinoderms. Further evidence of the incorporation of podial retractor muscle cells (myocytes) into a monociliated myoepithelial coelomic lining is provided. In the podia of the crinoid Nemaster rubinginosa and the ophiuroid Ophiophragmus wurdemani as well as in the feeding tentacles of the holothurian Leptosynapta tenuis, coelomic linings are organized as simple myoepithelia consisting of non-contractile peritoneal cells (peritoneocytes) and myocytes. Coelomic linings in the holothurian Thyonella gemmata, the echinoids Eucidaris cf. tribuloides and Lytechinus variegatus, and the asteroids Asterias forbesi and Astropecten sp. are pseudostratified or bipartite pseudostratified myoepithelia consisting of subapical myocytes and apically situated peritoneocytes. The ophiuroid podia of Ophioderma brevispinum and Ophiothrix angulata exhibit transitions from simple myoepithelia to partially pseudostratified epithelia. Intermediate forms between the extremes in myoepithelial organization also occur in the podial lining of single species (e.g. Eucidaris cf. tribuloides). These data supplement recent ultrastructural studies on the podial lining of echinoderms and, in conjunction with published ultrastructural data on the myoepithelial organization of other coelomic linings in echinoderms and in other coelomates, suggest myoepithelial organization of the coelomic lining is a plesiomorph feature in Bilateria.     

Structure of the Digestive Tube in the Holothurian Eupentacta fraudatrix (Holothuroidea: Dendrochirota)

In the holothurian Eupentacta fraudatrix,the gut wall exhibits trilaminar organization. It consists of an inner digestive epithelium, a middle layer of connective tissue, and an outer mesothelium (coelomic epithelium). The pharynx, esophagus, and stomach are lined with a cuticular epithelium composed of T-shaped cells. The lining epithelium of the intestine and cloaca lacks a cuticle and consists of columnar vesicular enterocytes. Mucocytes are also encountered in the digestive epithelium. The connective tissue layer is composed of a ground substance, which houses collagen fibers, amoebocytes, morula cells, and fibroblasts. The gut mesothelium is a pseudostratified epithelium, which is dominated by peritoneal and myoepithelial cells and also includes the perikarya and processes of the neurons of the hyponeural plexus and vacuolated cells.     

Mitotic Activity in Tissues of the Regenerating Respiratory Tree of the Holothurian Apostichopus japonicus (Holothuroidea,Aspidochirota)

Mitotic activity in the regenerating respiratory tree was studied in the holothurian Apostichopus japonicus after evisceration. Significant proliferation was recorded in the regenerating tissues. The highest number of mitoses was revealed in the inner epithelium, the mitotic index (MI) of which reached 3.44 ± 0.01% on the 10th day of regeneration, which was comparable to MI values of blastema of a regenerating amphibian limb. In the coelomic epithelium the number of dividing cells varied from 0.49% in the upper part (20th day) up to 2.87% in the base (10th day). The proliferative activity gradually decreased in the further course of regeneration. In spite of high MI values, the distribution of mitoses in tissues was even at all stages of regeneration and blastema did not form.     

Transdifferentiation in holothurian gut regeneration

It has recently been shown that the whole spectrum of cell types constituting a multicellular organism can be generated from stem cells. Our study provides an example of an alternative mechanism of tissue repair. Injection of distilled water into the coelomic cavity of the holothurian Eupentacta fraudatrix results in the loss of the whole digestive tract, except the cloaca. The new gut reforms from two separate rudiments. One rudiment appears at the anterior end of the body and extends posteriorly. The second rudiment grows anteriorly from the cloaca. In the anterior rudiment, the luminal epithelium (normally derived from endoderm) develops de novo through direct transdifferentiation of the coelomic epithelial cells (mesodermal in origin). In the posterior rudiment, the luminal epithelium originates from the lining epithelium of the cloaca. After 27 days, the two rudiments come into contact and fuse to form a continuous digestive tube lined with a fully differentiated luminal epithelium. Thus in this species, the luminal epithelia of the anterior and posterior gut rudiments develop from two different cell sources-i.e., from the mesodermally derived mesothelium and the endodermally derived epithelium of the cloacal lining, respectively. Our data suggest that differentiated cells of echinoderms are capable of transdifferentiation into other cell types.     

Synthetic ACTH-Like Peptide GKVLKKRR,Corresponding to the Fragment 81–88 of Human Pro-Interleukin-1α, Acts as an Antagonist of ACTH Receptor

Synthetic adrenocorticotropic hormone (ACTH)-like octapeptide leukocorticotropin (GKVLKKRR), corresponding to the amino acid sequence 81–88 of pro-interleukin-1α, was labeled with tritium (specific activity of 22 Ci/mmol) and was found to bind to rat adrenal cortex membranes with high affinity and specificity (K _d = 2.2 ± 0.1 nM). Synthetic ¹²⁵I-labeled ACTH fragment 11–24 was also obtained (specific activity of 98 Ci/mmol) and shown to bind to ACTH receptor on rat adrenal cortex with high affinity (K _d = 1.8 ± 0.1 nM). Unlabeled leukocorticotropin was found to actively replace ¹²⁵I-labeled ACTH (11–24) in the receptor-ligand complex (K _i = 2.0 ± 0.1 nM). Leukocorticotropin at concentration range of 1–1000 nМ did not affect the adenylate cyclase activity in adrenocortical membranes. Thus, leukocorticotropin is an antagonist of ACTH receptor. ACTH-like peptide GKVLKKRR is an antagonist of ACTH This work financeed by the Russian Foundation for Basic Research (grant No. 05-04-48060), by the programs Leading Scientific Schools (grant No. 312.2003.4), Molecular and Cellular Biology (chairman V.M. Lipkin), and Naukogrady (grant No. 04-04-97200), and by the International Science and Technology Center (project No. 2615). V.V. Yurovsky is supported by the American Heart Association (grant No. 0555415U).     

Muscle regeneration in the holothurian Stichopus japonicus

The regeneration of longitudinal muscle bands (LMBs) in the sea cucumber Stichopus japonicus was studied using light and electron microscopic and immunocytochemical methods. Previous investigations of holothurian organs showed the presence of some cytoskeletal proteins which were specific for LMBs only. One of them, the 98 KDa protein, was isolated by means of SDS-electrophoresis and used as an antigen to obtain polyclonal antibodies. When tested on paraffin sections of sea cucumber organs, the antibodies were shown to interact only with coelomic epithelial cells covering the LMBs. The antibodies were used to study LMB regeneration after transverse cutting. During regeneration no signs of myocyte dedifferentiation or mitotic division were observed. In the wound region, damaged myocytes degenerated and muscle bundles desintegrated. However, the coelomic epithelial cells dedifferentiated and began to invade the LMB. Just beneath the surface these cells formed clusters (muscle bundle rudiments). The number and size of the clusters gradually increased, the cells lengthened and developed contractile filaments. These observations suggest that new muscle bundles arise from coelomic epithelial cells covering the LMBs. The migration of coelomic epithelial cells into the damaged LMBs and their myogenic transformation are the basic mechanism of holothurian muscle regeneration.     

Regeneration of the Digestive Tube in the Holothurian Apostichopus japonicusafter Evisceration

We performed a TEM study of regeneration of the intestine in the Far Eastern trepang, the holothurian Apostichopus japonicus, after evisceration. The following stages were distinguished in the restoration process of the digestive tube: the growth of connective tissue along the margin of the mesenterium, in the place of rupture; dedifferentiation of cells and their migration and proliferation; the rooting of the esophagus lining into the connective tissue anlage; and the transformation of esophagus cells into cells of the middle part of the intestine. The migration of epithelium into the area of regeneration takes place through a solid cellular layer, without breaking of the cell contacts. The mitotic activity was registered in all stages of restoration; the dividing cells were located chaotically, without the development of a blastema.     

The distribution of the Wnt5 protein in the tissues of the holothurian Eupentacta fraudatrix (Djakonov et Baranova, 1958) (Holothuroidea: Dendrochirotida) in the norm and during regeneration

The Wnt5 protein localization in holothurian Eupentacta fraudatrix tissues was examined in the norm and during regeneration. In healthy E. fraudatrix, Wnt5 was found in solitary cells of the hypodermis and in the radial nerve cords. During regeneration, the number of Wnt5 positive cells increased. They were observed in the connective tissue of the body wall and the pharyngeal bulb, in nervous system tissues, the coelomic epithelium, and amoebocytes. The Wnt5 protein may participate in regulating the regeneration in the holothurian E. fraudatrix; it probably modulates cell migration, extracellular matrix reorganization, and neurogenesis.     

Participation of the coelomic epithelium of the body wall in muscle regeneration in <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> (Holothuroidea: Aspidochirota)

The cellular sources of regeneration of longitudinal muscles were studied in the holothurian Apostichopus japonicus. An autoradiographic method tracing the distribution of cells labeled with tritiated thymidine (³HT) revealed that the majority of ³HT-cells, which were initially localized in the coelomic epithelium of muscles and the body wall at the beginning of active morphogenesis, were then found in the structure of new muscular bundles during subsequent terms of restoration. Thus, the coelomic epithelium of the body wall participated in the regeneration of muscle tissue concurrently with the coelomic epithelium of muscle, contributing to the recruitment of a pool of myogenic cells.     

Immunoreactivity to molluskan neuropeptides in the central and stomatogastric nervous systems of the earthworm, Lumbricus terrestris L.

Polyclonal antisera against two related command neuropeptides (CNP2 and CNP4) described in neurons of the terrestrial snail Helix were used in a study of the nervous system of the earthworm Lumbricus. The CNP-like peptides belong to the same neuropeptide subfamily and bear a C-terminal signature sequence Tyr-Pro-Arg-X. The distribution patterns of immunoreactive (IR) neurons were studied in the central nervous system (CNS), skin, and stomatogastric nervous system of the earthworm. IR neurons were found in all CNS ganglia, the patterns being similar for both antibodies used. Several clusters of IR cells were observed in the cerebral and subesophageal ganglia. In the ventral cord ganglia, the number of IR cells decreased in the rostro-caudal direction, and the IR cells sent their fibers mostly into the median fiber bundle. Segmental nerves contained no IR fibers. After injury of the worm body, the number of IR neurons in the CNS significantly increased. In the skin, IR sensory neurons were present in sensory buds. The stomatogastric ganglia only contained IR fibers. Numerous scattered IR neurons were found in the inner subepithelial layer of the esophagus and formed the enteric plexus in which the cell bodies displayed a segmentally repeated pattern. Possible involvement of CNP-like-IR neurons in central integratory processes, sensory processes, and the regulation of feeding is discussed.This work was supported by INTAS (grant 01-2117), CRDF (grant RB1-2321-MO-02), and the Russian Foundation for Basic Research (grants 05-04-48724 and 03-04-48179).     

Biological behavior of myoepithelial cells in the regeneration of rat atrophied sublingual glands following release from duct ligation

Summary The present study aimed to clarify how myoepithelial cells behave during regeneration of an atrophied sublingual gland by investigating cell proliferation and ultrastructure. Atrophy of rat sublingual glands was induced by unilateral ligation of the excretory duct near the hilum with metal clips, which were then removed after one week of ligation for regeneration. The sublingual glands 0–14 days after unligation were examined with single immunohistochemistry for actin as a marker of myoepithelial cells, double immunohistochemistry for actin and proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells, and transmission electron microscopy (TEM). The single immunohistochemistry and TEM showed that myoepithelial cells surrounded residual ducts in the atrophied glands and immature and mature acini in the regenerating glands. Although PCNA-positive myoepithelial cells were identified during regeneration, PCNA labeling indices of myoepithelial cells were low at all time points except at day 7. Ultrastructurally, myoepithelial cells showing bizarre shaped structures in the atrophy changed with maturation of differentiating acinar cells and appeared normal in the regenerated glands. There was no differentiation of the remaining duct cells to myoepithelial cells. These observations suggest that proliferation of myoepithelial cells and differentiation to myoepithelial cells do not commonly participate in the regeneration of atrophied sublingual glands and that the bizarre shaped myoepithelial cells in the atrophied sublingual glands recover the original shapes with acinar cell regeneration.     

The syndrome of sea cucumber (Apostichopus japonicus) infected by virus and bacteria

A outbreak of disease with symptoms of evisceration and skin ulteration led to mass mortality in sea cucumber Apostichopus japonicus cultivated in indoor ponds near the Dalian coast from December 2004 to April 2005. Spherical virus particles with a diameter of 75–200 nm were found in the cytoplasm of cells in the water-system, the alimentary canal and in the respiratory trees of the diseased and dying sea cucumber individuals by electron microscopic observation of ultrathin sections. Examination by negative stained samples revealed that all the diseased sea cucumbers were infected by the virus, while the healthly ones cultivated outside the contagious area were not. Two bacterial strains were also isolated from the diseased animals. When exposed to a medium containing the virus particles, regardless of whether the bacterial suspension was added, healthy sea cucumbers exhibited identical disease symptoms as the ones in the indoor ponds, and had a mortality of 90%–100%. However, when exposed to a medium in which there was only one of the two bacterial strains, 30%–80% of the sea cucumbers were infected and nearly 20% died. Negative staining showed that the viral particles were detected only in the bodies of the tested animals that were exposed to the viral medium. Histopathologically, the diseased sea cucumbers are characterized by karyopycnosis, and disintegration of the endoplasmic reticula and mitochondria in the epithelial cells in the water-system, the respiratory tree and the alimentary canal. Fundation items: Supported by commonweal program of State Oceanic Administration of China (200705007), Science and Technology Project of Liaoning Province (2004203001) and Science and Technology Project of Dlian (2004 BINS030).     

Activated omentum becomes rich in factors that promote healing and tissue regeneration

In order to study the mechanism by which an omental pedicle promotes healing when applied to an injured site, we injected a foreign body into the abdominal cavity to activate the omentum. One week after the injection, we isolated the omentum and measured blood vessel density, blood content, growth and angiogenesis factors (VEGF and others), chemotactic factors (SDF-1α), and progenitor cells (CXCR-4, WT-1). We found that the native omentum, which consisted mostly of adipose tissue, expanded the mass of its non-adipose part (milky spots) 15– to 20-fold. VEGF and other growth factors increased by two– to four-fold, blood vessel density by three-fold, and blood content by two-fold. The activated omentum also showed increases in SDF-1α, CXCR-4, and WT-1 cells (factors and cells positively associated with tissue regeneration). Thus, we propose that an omentum activated by a foreign body (or by injury) greatly expands its milky-spot tissue and becomes rich in growth factors and progenitor cells that facilitate the healing and regeneration of injured tissue. This work was partly supported by a grant (no. 2000–241 to A.K.S.) from the Juvenile Diabetes Foundation International.     

Genetic Conservation of Insular Populations of Monterey Pine (Pinus Radiata D. Don)

Monterey pine (Pinus radiata D. Don) has only five extant native populations: three disjunct populations along the coast of California, USA and two on Mexican islands. All populations have been influenced by human activity, but the island populations in particular have been affected by introduced biota. On Guadalupe Island, the pine population has suffered drastically from overgrazing by introduced goats. We visited both island populations and described their status, took measurements, and made seed collections. We counted approximately 200 mature pine trees and virtually no seedlings on Guadalupe Island: a reduction of approximately half the population in the last 50 years. The trees are all large (mean diameter of 144 cm) –considerably larger than trees from the other four populations – and arguably near the end of their natural lifespan. The population on Cedros Island is much more robust, with thousands of trees. None sampled were as large as those on Guadalupe Island (mean diameter of 20 cm) and many groves were young and even-aged – presumably the consequence of natural regeneration after a recent fire. Tissue samples from trees on both islands did not show evidence of infection from the pitch canker pathogen, Fusarium circinatum, that has caused significant mortality in the three mainland populations. Caution is recommended in any restoration activity for the Guadalupe Island pines. Inbreeding levels could indicate the need for some planting or seeding intervention but there are also risks associated with this. Natural regeneration – after goat removal – is preferred.     

Distinction between vascular smooth muscle cells and myoepithelial cells in primary monolayer cultures of human breast tissue

Summary We report on the discrimination of vascular smooth muscle cells and myoepithelial cells in primary cultures of human breast tissue. Breast tissue was disaggregated enzymatically and the resulting organoids seeded in monolayer culture on collagen-coated plastic in serum-free medium CDM3a. Two main types of organoids were present after enzymatic digestion. One resembled small blood vessels and the other interlobular ducts or acini of the breast gland epithelium. Within 3 to 8 d after plating the organoids migrated into typical monolayer islets. These monolayer islets were evaluated using phase contrast microscopy and further tagged with monoclonal antibodies for immunocytochemical demonstration of Factor VIII-related antigen, muscle iso-forms of actin, type IV collagen, vimentin, desmin, and keratins. It is concluded that vascular smooth muscle cells resembled myoepithelial cells by expressing vimentin filaments, depositing type IV collagen, and showing immunoreactivity to muscle iso-forms of actin. However, whereas vascular smooth muscle cells were associated with endothelial cells and sometimes expressed desmin, myoepithelial cells appeared together with luminal epithelial cells and expressed cytokeratins. This work was supported by the Danish Medical Research Council, the Danish Cancer Society, the NOVO Foundation, and the Thaysen Foundation.     

Immunohistochemical studies of S-100 protein α and β subunits in adenoid cystic carcinoma of salivary glands

Immunohistochemical studies were performed to explore the distribution of S-100 protein and its α,β subunits in 76 adenoid cystic carcinomas (ACC) of the salivary glands. Histopathologically, ACC was divided into cribriform, tubular, basaloid and trabecular types which could be mixed in the same tumor. S-100 protein was usually positive in tumor cells forming cribriform structures; foci of strongly positive tumor cells were also distributed in the luminal layer of tubular structures, and in areas transitional between cribriform and tubular patterns. S-100 α staining was confined to some tumor cells in cribriform areas, to luminal tumor cells in tubular structures and to few tumor cells in basaloid structures. S-100β reaction was usually localized to luminal surfaces in a fine granular pattern in tubular and microtubular structures in a distribution somewhat similar to that in the normal salivary gland. Great heterogeneity in the immunohistochemical distribution of S-100, S-100 a and S-100β proteins was found in the various histologic types of ACC and the pattern was different from that seen in pleomorphic adenomas. It is possible that the ACC tumor cells positive for S-100 protein may be closely related to true or modified myoepithelial cells.     

Ammonium sulphate induced stress related alterations in the respiratory epithelium of the airbreathing organ of the catfishHeteropneustes fossilis (Bloch)

In this paper, histopathological changes in the inner lining of the accessory respiratory organ ofHeteropneustes fossilis following exposure to sublethal concentration (0.2 g I^–1) of ammonium sulphate (3 mg I^–1 total ammonia-N) has been described. The goblet cells show periodic increased followed by decreased secretory activities. Necrosis and shedding of the epithelial cells over the secondary lamellae cause periodic haemorrhages which lead to degeneration and decreased number of secondary lamellae. Subsequently regeneration takes place each time as evidenced by the appearance of inflammatory tissue. Fusion of more than one secondary lamellae is also common. Regeneration also leads to uncontrolled hyperplasia of haphazardly arranged epithelial cells. This hyperplasia causes increased distance of respiratory blood-air barrier in the secondary lamellae, leading to impaired normal aerial respiration     

Morphology of the Respiratory Trees in the Holothurians Apostichopus japonicus and Cucumaria japonica

The morphology of the respiratory trees in the holothurians Apostichopus japonicus and Cucumaria japonica was studied using histochemical and electron microscopic techniques. The epithelium of the respiratory tree cavity in A. japonicus consists of columnar cells about 17–20 m high. In C. japonica, this epithelium is composed of two cell types: bulbous cells embedded in the connective tissue layer and secretory cells; cells are 1–12 m high. A characteristic feature of cells of the respiratory tree cavity epithelium in these species is the presence of numerous phagosomes and coated vesicles in the apical cytoplasm. The cell surface has many microvilli and a single cilium. Cells of the coelomic epithelium contain vacuoles in the apical part and myofibrils in the basal region; the thickness of this epithelium in A. japonicus and C. japonica is 9–16 and 10–30 m, respectively. Based on the different structure of the respiratory trees and the absence of hemocytes in A. japonicus, it is suggested that the holothurian species studied have different routes of oxygen transport from the environment to the internal organs.     

Regeneration in subalpine coniferous forests

The regeneration process of a subalpine coniferous forest, a mixed forest ofTsuga diversifolia (dominant species),Abies veitchii, Abies mariessi, andPicea jezoensis var.hondoensis, was studied on the basis of annual ring data. The age class distribution was discontinuous and four age groups occurred in the study plot (30m×30m). The canopy layer was a mosaic of patches (83.8–133.7 m² patch area), which had different mean ages. The recruitment of canopy trees was carried out only by advance regeneration in the plot. The diameter growth ofAbies andPicea exceeded diameter growth ofTsuga in the gap.Abies lived for 200–300 years and their trunks were susceptible to heart rot.Picea lived for 300–400 years andTsuga for more than 400 years. The regeneration process derived from the analysis of the plot consisted of three phases leading to the development of a even-aged patch; (1) the establishment of saplings before a gap opening, (2) the opening of a gap in the canopy and repair of the canopy by advance regenerated saplings dominated by rapid growth species,Abies andPicea, and (3) the dying off of canopy trees as each species reached the end of its life-span, resulting in pure patches of long-livedTsuga.