Elucidation of the nature and function of the granular oyster amebocytes through histochemical studies of normal and traumatized oyster tissues

Summary A remarkable humoral component of the oyster inflammatory response was elucidated by employing the tools of the determinative histochemist. The humoral component, characterized by the release of copper and a diazotized p-nitroaniline-positive material from an acidophilic granular amebocyte, was associated with the oyster inflammatory reaction. Grossly, this humoral response was associated with the appearance of an avocado or pea green coloration in the traumatized area. A second amebocytic cell type, termed the basophilic granular amebocyte, was observed swelling in traumatized areas and may have released an additional humoral component into injured regions. Copper released in response to trauma was bound to the cells in and around the wound site and appeared to be most avidly bound by the granules of the basophilic granular amebocytes. Once incorporated into the granular matrix of these amebocytes, copper appeared to stabilize and prevent the granule from swelling.A portion of this work was excerpted from a Ph.D. thesis submitted to the Graduate School, University of Washington, Seattle.This work was supported by Public Health Service Contract No. 5 to 1 ES 00038-02. The costs of publication were defrayed in part by HSAA Award No. RR 06138 and Tumor Biology Training Grant, NIH CA 05245.     

Studies on fish scale formation and resorption

Summary Electron microscopic investigation of scales of the goldfish Carassius auratus revealed that the lamellae of fibrillary plates contain sheet-like structures composed of vertically oriented collagen fibers embedded in an organic matrix. The fibers (TC fibers) are smaller in diameter (35–45 nm) than those of the lamellae and the matrix is stained intensely with lead citrate.The sheet-like structures as well as the lamellae are formed by fibroblasts located beneath the lamellae. The orientation of the collagen fibers of the sheets and the lamellae seems to be controlled by the orientation of the ridges and invaginations of the surface of the fibroblasts.The fibrillary plate of C. auratus was found to be partially calcified. Calcification was initiated by the deposition of needle-like or flaky crystals of hydroxyapatite in the organic matrix of the sheet-like structure and proceeded into the TC fibers and the matrix region of the lamellae. The potassium pyroantimonate-osmium tetroxide method showed a heavy concentration of calcium in the osteoblasts, fibroblasts, and in the matrix regions of the fibrillary plate. Calcium-containing precipitates were also present in the hole zone of the collagen fibers in the lamellae, but the significance of this location in calcification remains to be elucidated.Contribution No. 285, Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina, 29208, USA     

Limulus amebocyte clotting cascade: Roles of endotoxin and adenylate cyclase

Endotoxin, the lipopolysaccharide from the cell wall of Gram-negative bacteria, causes blood clotting in the horseshoe crab,Limulus polyphemus. Minute amounts of endotoxin stimulate the amebocytes in the blood to undergo exocytosis, which release the contents of their secretory granules to form a clot. An endotoxin-binding protein that possesses calmodulin-like activity has been isolated from the amebocyte plasma membrane. This endotoxin-binding protein can activate adenylate cyclase fromBordetella pertussis to the same extent as rat testes calmodulin. The effect of endotoxin and the endotoxin-binding protein on cyclic AMP synthesis inLimulus amebocytes was examined. Amebocytes exposed to endotoxin have increased levels of intracellular cyclic AMP. Amebocyte membranes contain an adenylate cyclase which is stimulated by NaF, guanosine (,r-imido)triphosphate, and for skolin. This adenylate cyclase is also stimulated by the endotoxin-binding protein and calcium. Exposure of amebocytes to forskolin or dibutyryl cyclic AMP are stimulated to secrete clot components. Activation of adenylate cyclasein vivo by endotoxin via the endotoxin-binding protein may be one of the ways in which endotoxin stimulates secretion. It is suggested that endotoxin may have two actions in theLimulus system: (1) binding of endotoxin to the endotoxin-binding protein activates adenylate cyclase, promoting secretion by the amebocytes; and (2) endotoxin catalyzes a reaction on the secreted material to form a blood clot. This latter reaction is not elicited by forskolin or dibutyryl cyclic AMP.A preliminary report of this work has been presented elsewhere (Liu and Liang, 1984).     

Initiation of acellular extrinsic fiber cementum on human teeth

Summary The development of acellular extrinsic fiber cementum (AEFC) has never before been studied in human teeth. We have therefore examined the initiation of AEFC in the form of a collagenous fiber fringe and its attachment to the underlying dentinal matrix, in precisely selected, erupting human premolars with roots developed to 50%–60% of their final length. Freshly extracted teeth were prefixed in Karnovsky's fixative, decalcified in EDTA and subdivided into about 10 blocks each, cut from the mesial and distal root surfaces, vertical to and along the root axis. The blocks were postfixed in osmium tetroxide, embedded in Epon and cut for light- and electron-microscopic investigation. Starting at the advancing edge of the root, within a region extending about 1 mm coronal to this edge, fibroblast-like cells were seen closely covering the external root surface. Along the first 100 m from the root edge, these cells extended cytoplasmic processes and contacted the dentinal collagen fibrils. Between these cells and the dentinal matrix, new collagen fibrils and very short collagen fibers gradually developed. Within the second 100 m from the root edge, this resulted in the formation of a cell-fiber fringe network. Newly formed fibers of the fringe were directly attached to the non-mineralized matrix containing dentinal collagen fibrils and could be distinguished from the latter by differences in fibril orientation. During the process of dentin mineralization, the transitional zone between the fiber-fringe base and the dentinal matrix, i.e., the future dentino-cemental junction, also mineralized. It is suggested that this fiber fringe is the base of AEFC, which later increases in thickness by fiber extension and subsequent mineralization.Abbreviations AEFC acellular extrinsic fiber cementum - AIFC acellular intrinsic fiber cementum - CIFC cellular intrinsic fiber cementum - CMSC cellular mixed stratified cementum - ARE advancing root edge - CP cytoplasmic process - D dentin - DCJ dentinocemental junction - E enamel - EBL external basal lamina - EC epithelial cell - EDTA ethylene diaminetetraacetic acid - ERM epithelial rests of Malassez - FF fiber fringe - HRS Hertwig's epithelial root sheath - IBL internal basal lamina - MD mineralized dentin - NMD non-mineralized dentin - OB odontoblast - PD predentin - PL periodontal ligament     

Outer root sheath (ORS) cells organize into epidermoid cyst-like spheroids when cultured inside Matrigel: a light-microscopic and immunohistological comparison between human ORS cells and interfollicular keratinocytes

In organotypic cultures, outer root sheath (ORS) cells of the human hair follicle develop into a stratified epithelium largely reminiscent of the epidermis; this apparently reflects their importance during wound healing. In the present study, ORS cells were grown inside a three-dimensional network of extracellular matrix proteins (Matrigel), together with different mesenchymal cells, in an attempt to mimic their follicular environment. Thus, inside Matrigel, ORS cells formed spheroids differentiating toward the center and showing all the markers of epidermal keratinization. Under identical conditions, normal epidermal keratinocytes developed similar spheroids, but of a significantly smaller size. Human dermal fibroblasts and dermal papilla cells, cocultured in the matrix, had a positive influence on both the proliferation and differentiation within both types of spheroids. Epidermal differentiation markers, such as suprabasal keratins, involucrin, filaggrin, gp80 and pemphigoid antigen, were readily expressed in ORS spheroids, whereas hard (hair) keratins were not detectable by immunostaining. Cells positive for an epithelial membrane antigen, strongly expressed in sebaceous glands, were seen in numerous spheroids. In contrast to organotypic surface epithelia, the expression and location of different integrin chains was normalized in ORS spheroids, indicating an enhanced mesenchymal influence in this in vitro system.     

Observations on the biology ofThiothrix

Thiothrix nivea grows profusely in tufts of greyish white material on pebbles in a local sulfide spring. The spring remained a constant 10.8° C during the course of the study. Chemical analyses indicated that the spring water contained 0.27 mg/l sulfide, 0.37 mg/l oxygen, and 0.77 mg/l dissolved organic carbon. Tufts ofThiothrix growth were removed from the pebbles and examined by phase and electron microscopy. The cell filaments contained numerous sulfur granules which disappeared upon incubation in the refrigerator. The average cell diameter was 1.5 m and the length 4.0 m. When cells were lysed by hyperplasmolysis a sheath was readily discernible. The presence of a sheath, 60 nm thick, was confirmed in thin sections. Fine structure analysis also revealed that the organism was a gram negative bacterium. Sulfur granules were bound by a unit membrane extending from the cytoplasmic membrane.Thiothrix nivea was grown for short periods of time in slide culture. In some cases the filaments fragmented into short segments ca 15m long. These may represent gonidia as initially reported by Winogradsky.     

Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes

Summary In vitro experiments were conducted to clarify the involvement of the epithelium-amebocyte interaction in epithelial regeneration of bivalves. The outer epithelia of the pallial mantle of the pearl oyster, Pinctada fucata martensii, were separated in cell sheets from the inner connective tissue layers by digestion with Dispase. Clumps of the separated mantle epithelia were inoculated onto the amebocyte layers prepared on the bottom of culture dishes and maintained at 20° C in 5% CO₂:95% air for 1 wk. Balanced salt solution with 0.03% (wt/vol) glucose was used as a culture medium. The epithelial cells adhered to the amebocyte layers within 24 h, changed their shape from cuboidal to squamous, and migrated and formed monolayer sheets within 3 d. Electron microscopy confirmed maintenance of epithelial polarity and cell to cell junction in the sheets; 6 d after the inoculation, 5-bromo-2′-deoxyuridine was added to the culture at 30 μM. After labeling for 24 h, the cultures were fixed and stained with anti 5-bromo-2′-deoxyuridine antibody. Cells with immunoreactive nuclei were clearly observed in the epithelial cell sheets, indicating active DNA synthesis in the epithelial sheets. Thus, cocultured with amebocytes, the outer epithelial cells from pallial mantle tissue formed a monolayer sheet and started DNA synthesis. The morphological features of the mantle outer epithelial cells are analogous to those described for the in vivo cutaneous wound healing process, suggesting that the epithelium-amebocyte interaction is important in the regeneration of epithelium in bivalves.     

Spicule formation in the calcareous sponge Sycon ciliatum

Summary The spicule primordium is formed in an intercellular cavity within a group of sclerocytes. This cavity contains organic material which ensheaths the growing spicule but does not appear to determine the nature of the mineral morph (magnesian calcite) or the crystallographic orientation of the spicule. The tip of each growing spicule ray is seated in a dense cup in the cytoplasm of the sclerocyte concerned. Both ends of monaxons are initially inserted each into a dense cup. As rays elongate the sclerocyte membrane around the tip becomes invaginated and forms a system of converging spaces that possibly indicate high secretory activity in that region. Spicule growth involves the displacement and expansion of the organic sheath by the enlarging spicule. Fully formed spicules which are exposed to the mesohyl become surrounded by collagen fibrils. However, these fibrils are in no way concerned with the process of mineral deposition and are never found within the spicule calcite.     

Analysis of head and foot formation inHydra by means of an endogenous inhibitor

Summary In tissue regenerating the head, the ability to initiate head formation in a host increases with the time allowed for regeneration before grafting, while the foot-initiating ability decreases concomitantly. The reverse was found for tissue about to regenerate a foot. The early divergent changes thus indicated are counteracted in both head and foot regeneration by treatment with an inhibitor (Berking, 1977) in low concentrations.The inhibitor also interferes with processes which determine wether or not hypostome and tentacles are formed, and how many tentacles (if any) appear. The circumferential spacing of the tentacles was regular whether their number was normal or below normal.Secondary axes caused by implanted tissue either detach after having formed a head and a foot (i.e. behave like buds) or do not detach, having only formed a head. This alternative depends on the origin and amount of the implanted tissue and on the position of the implant within the host.The following model based on these findings is proposed: Head and foot formation start with pre-patterns which cause a continuously increasing change of the tissue's ability to initiate a head or a foot. Along the body axis this ability is determined by a graded distribution of sources. As development progresses, the high source density which accumulates in the head region causes the formation of a hypostome and tentacles; the angular spacing of tentacles is also dependent on source density. At a certain low source density foot-formation is initiated. The inhibitor counteracts the increase of source density in head-forming tissue as well as the decrease of source density in foot-forming tissue. It thus appears to be part of the mechanism which controls morphogenesis in hydra.     

Ozone-induced microscopical changes and quantitative carbohydrate contents of hybrid poplar (Populus × euramericana)

Summary Cuttings of hybrid poplar (Populus × euramericana var. Dorskamp) were exposed to ozone (80 g/m³ from 2100 hours to 0700 hours, 180 g/m³ from 0700 hours to 2100 hours) for 3 months. Ozone reduced the starch content in leaves and stem bark, whereas starch granules accumulated in bundle sheath cells along small leaf veins. At the same time, sucrose and inositol content increased in the leaves. Mesophyll cells in the vicinity of the stomata were injured first, and droplet-like material appeared on their walls. In the sieve plates of fumigated trees, the pores showed a higher degree of narrowing than those of the control treatment. Cell collapse in the leaves was accompanied by water loss and an increase in air space. In the stems, the ozone treatment led to a reduced radial width, particularly in the xylem tissue. These results are discussed in relation to reduced or inhibited phloem loading and ozone-induced drought stress. The plants injured by ozone showed quite distinct patterns of metabolite responses as well as enzyme activities (PEP- and RubP-carboxylase) in the leaves from the top to the bottom. There were also remarkable differences in the reaction of sucrose and inositol between leaves and stem bark. Future research should therefore increasingly follow a whole-plant approach for a better understanding of complex plant reactions.     

Studies on resistance in snails. 4. Induction of ventricular capsules and changes in the amebocyte-producing organ during sensitization of Biomphalaria glabrata snails.

Formation of an amebocyte aggregate in the ventricular cavity of Biomphalaria glabrata can be induced 30 hr or more after a single infection by irradiated or (less frequently) by normal Echinostoma lindoense miracidia. The resulting amebocyte mass frequently encapsulated and destroyed the developing E. lindoense sporocysts within the ventricle. The constituent amebocytes of the capsule correspond in vitro and by staining characteristics to circulating amebocytes of uninfected snails, but with additional inclusion bodies, increased mucopolysaccharide, acid phosphatases, and lipid-positive staining reactions. Mitotic activity, rapid growth, and later regression of the amebocyte-producing organ (located between the posterior mantle epithelium and anterior pericardial endothelium) follow the growth and regression sequence of the ventricular capsule. Though peripheral foci of secondary amebocyte production have been found and were previously known, the amebocyte-producing organ appears to be the primary amebocyte source responsible for the snails' rapid intraventricular sporocyst encapsulation.     

Endotoxin-induced degranulation of the Limulus amebocyte

Exocytosis and gelation of the granule contents of the amebocyte of Limulus polyphemus have been studied in a perfusion chamber observed with Nomarski differential interference contrast microscopy. Degranulation in response to bacterial endotoxin or the ionophore A23187 was significantly inhibited by the anion channel blocking agents suramin, SITS, DNDS and sodium isethionate. db-cAMP, PGI₂ and theophylline also succeeded in imparing degranulation of the amebocytes. All of the agents tested produced inhibition of degranulation which was readily reversed by washing the system free of the inhibitor and rechallenging the amebocytes with either endotoxin or the ionophore. After isolation in vitro, amebocytes underwent spontaneous degranulation in the absence of endotoxin at a rate 1–2 orders of magnitude slower than in the presence of endotoxin. Gelation of the clottable protein released from the amebocyte granules could occur in the absence of endotoxin. This is the first demonstration of gelation under endotoxin-free conditions.     

Collagen and proteoglycan in a sea urchin ligament with mutable mechanical properties

Summary The problematic ligament of sea urchins is a connective tissue which crosses the ball-and-socket joint between spine and body wall. The problem of this ligament is that it is composed of parallel collagen fibrils, yet normally undergoes rapid and dramatic alterations in mechanical properties and in length. Previous work has suggested that the collagen fibrils of the ligament are able to slide past one another during length changes but are inhibited from sliding when the ligament is in catch. In this model of the ligament both the collagen fibrils and the interfibrillar matrix are mechanically important. We have found that the collagen fibrils of the spine ligament of the pencil urchin Eucidaris tribuloides are discontinuous and end by tapering within the body of the ligament. Intact fibrils that have been isolated from the ligament vary by more than an order of magnitude in length and in radius but have a constant length/radius (aspect) ratio of about 5300. This is the first determination of the aspect ratio of collagen fibrils from any source. The constant aspect ratio of the fibrils is consistent with their functioning as the discontinuous fiber phase in a fiber-reinforced composite material, while the high value of the aspect ratio indicates that the nonfibrillar matrix, which must act to transfer stress between fibrils, can produce a stiff and strong ligament even if it is several orders of magnitude weaker and more compliant than the fibrils. Moreover, the tensile properties of the ligament may be determined by the properties of the matrix. A prominent component of the interfibrillar matrix is a proteoglycan which associates with specific bands at the surface of the collagen fibrils through noncovalent binding of its core protein. The glycosaminoglycan moiety of this proteoglycan is partly comprised of chondroitin sulfate/dermatan sulfate polymers. These results are consistent with the sliding fibril hypothesis and suggest that the proteoglycan may be an important component of the stress-transfer matrix.     

Isolation and studies of the granules of the amebocytes of Limulus polyphemus,the horseshoe crab

Granules were isolated from the cytoplasm of the amebocytes of Limulus polyphemus, the horseshoe crab, by disruption of cells obtained from blood which had been drawn into 2 mM propranolol. The granules subsequently were purified by centrifugation through a sucrose gradient that contained heparin. Extracts of the granules were prepared by freezing and thawing the granule preparations in distilled water. Transmission and scanning electron microscopy of the granules revealed round or ovoid particles. However, only one type of granule appeared to be present. The ultraviolet spectrum of the extract of amebocyte granules demonstrated a peak at 277 nm at pH 7.4, and a shift into two peaks of 281 nm and 290 nm at alkaline pH. Analytical ultracentrifugation revealed a pattern similar to that observed with lysates prepared from intact amebocytes. Polyacrylamide gel electrophoresis, in the presence of urea at pH 4.5, demonstrated patterns similar to those observed with amebocyte lysate. Extracts of the granules were gelled by bacterial endotoxin. The blood of the horseshoe crab contains only one type of cell, the amebocyte. Previous studies have shown that the blood coagulation mechanism of Limulus is contained entirely within amebocytes. The current studies suggest that the granules, which pack the cytoplasm of these cells, contain all of the factors required for the coagulation of blood, including the clottable protein. The intracellularly localized coagulation system is released from amebocytes when their granules rupture during cell aggregation.     

The endomembrane system of differentiating carposporangia in the red algaChondria tenuissima: Occurrence and participation in secretion of polysaccharidic and proteinaceous substances

Summary The endomembrane system during carposporogenesis inChondria tenuissima was studied using electron microscopy and histochemistry. Profiles of the nucleus are convoluted, resulting in a highly increased surface area. Stacked cisternae are found within the peripheral part of the nucleus. Vesicles, tubules and membrane bound fibrillar bodies occur within the nucleoplasm. The endoplasmic reticulum surrounds the nuclear envelope.The endoplasmic reticulum and the Golgi apparatus, together with small transition vesicles, represent a functional unit. They form two different secretory substances during carposporogenesis. In young stages, carbohydrates are produced by normal dictyosomes within large, normal exocytotic Golgi vesicles. They do not react positively with PAS or Thiéry method and are believed to represent cell wall material. In later stages, the central area of the Golgi cisternae becomes filled with electron dense material. The individual cisternae are transformed into cored vesicles at the trans-face of the dictyosomes. The dense core of the vesicles is proteinaceous and stains with coomassie brilliant blue R. The peripheral fibrillar material is polysaccharidic and reacts positively using the Thiéry method. The contents of the cored vesicles are believed to participate in carpospore attachment. The ER gives rise to cytolysosomes in which starch grains are sequestrated and digested. Mucilaginous sacs seem to be similarly formed.     

Studies on resistance in snails. 6. Escape of Echinostoma lindoense sporocysts from encapsulation in the snail heart and subsequent loss of the host's ability to resist infection by the same parasite.

Formation of amebocyte aggregates in the ventricular cavity of Biomphalaria glabrata, induced by developing sporocysts of Echinostoma lindoense, does not always result in destruction of the parasites, as the sporocysts occasionally escape encapsulation in the heart. When this occurs, a remarkable loss of protective capacity follows and the host snails become highly susceptible to reinfection with the same species--even more so than in control susceptible snails exposed for the first time. Although the amebocyte-producing organ is considerably enlarged after a first infection and shows numerous mitoses, the amebocytes produced by snails harboring an "escaped" infection in the heart appear unable to attack the parasites of the first or of the second exposure. Instead, the amebocytes produced accumulate in the loose connective tissues between the liver lobuli, where early developmental stages of the parasites do not occur. These amebocytes apparently have lost their ability to recognize the parasites as foreign.     

The innervation and fine structure of paraneuronic cells in an amphibian pulmonary artery

Summary The pulmonary artery of Bufo marinus contains large numbers of bipolar cells situated in the tunica adventitia and in the outer layers of the media. These cells show a bright green-yellow fluorescence (emission spectra 485 nm) after formaldehyde pre-treatment suggesting that they contain a primary monoamine. The most characteristic fine-structural feature of these cells is the presence of numerous dense-cored vesicles (80—300 nm diameter) in their cytoplasm. The cells are in close contact (20 nm gap) with both agranular and granular nerve fibres. Both EM-cytochemical and formaldehyde-induced fluorescence tests indicate that the granule-containing nerve fibres are adrenergic. The agranular nerve fibres form discrete synaptic contacts with pre-and post-synaptic membrane thickenings on the cells. This was never observed with respect to the adrenergic fibres. Each process of the cells is about 45 m long. The processes do not bear any special relationship to either vessels of the arterial vasa vasorum or medial smooth muscle cells. Their location in the wall of the artery suggests that they are functionally significant with respect to activity of the arterial media.     

Selection of a standard culture medium for primary culture of <Emphasis Type="Italic">Limulus polyphemus</Emphasis> Amebocytes

Summary This study provides information relevant to future research aimed at producing Limulus Amebocyte Lysate (LAL) in vitro, which would potentially reduce the need to harvest and bleed horseshoe crabs as in the current methods of LAL production. To address the need for primary culture of horseshoe crab amebocytes, this study tested the effects of a variety of standard insect cell culture media on amebocyte morphology and viability after 7 d of maintenance. Amerbocyte morphology was least altered from in vivo form in Grace’s Modified Insect Medium, with no observed degranulation of cells, as compared to the other media tested. There were significant differences in amebocyte viability among the six insect cell culture media tested. Grace’s Modified Insect Medium sustained viability of 77.2±5.1% (mean ± standard deviation) of amebocytes, followed distantly by Grace’s Insect Medium with 35.1±8.7% amebocyte viability. Results indicate that Grace’s Modified Insect Medium with horseshoe crab serum supplementation was the best candidate of the six media tested for future medium optimization for Limulus amebocyte requirements.     

Cytoskeletal organization of limulus amebocytes pre- and post-activation: comparative aspects

One of the major functions of circulating Limulus amebocytes is to effect blood coagulation upon receipt of appropriate signals. However, the hypothesis that Limulus amebocytes are fundamentally similar to vertebrate thrombocytes and platelets has not been tested sufficiently in previous studies of their cytoskeletal organization. Whereas the earlier data were derived from transmission electron microscopy (TEM) of thin sections of a limited number of cells, improved fluorescence labeling methods that retain cell morphology have now enabled us to survey F-actin and microtubule organization in intact individual amebocytes and in large amebocyte populations pre- and post-activation. Anti-tubulin immunofluorescence showed the marginal band (MB) of microtubules to be ellipsoidal in most unactivated cells, with essentially no other microtubules present. However, minor subpopulations of cells with discoidal or pointed shape, containing corresponding arrangements of microtubules suggestive of morphogenetic intermediates, were also observed. Texas-red phalloidin labeled an F-actin-rich cortex in unactivated amebocytes, accounting for MB and granule separation from the plasma membrane as visualized in TEM thin sections, and supporting earlier models for MB maintenance of flattened amebocyte morphology by pressure against a cortical layer. Shape transformation after activation by bacterial lipopolysaccharide was attributable principally to spiky and spreading F-actin in outer cell regions, with the MB changing to twisted, nuclei-associated forms and eventually becoming unrecognizable. These major pre- and post-activation cytoskeletal features resemble those of platelets and non-mammalian vertebrate thrombocytes, supporting recognition of the Limulus amebocyte as a representative evolutionary precursor of more specialized clotting cell types.