Stephen W. Downing

Keratin-like components of gland thread cells modulate the properties of mucus from hagfish (Eptatretus stouti)

SummaryThe hagfishes (cyclostomes) are known to secrete copious amounts of mucus mainly by the holocrine mode from the slime glands. Stressed animals release two types of cells (gland thread cells, GTCs; gland mucous cells, GMCs) which rupture on contact with water and rapidly form a mass of viscous mucus. Herein we report some key sequential events of this process and document a novel role for cytoskeletal polymers. After electrostimulation of Pacific hagfish (Eptatretus stouti), the exudate was collected in a stabilization buffer and GTCs segregated from GMC vesicles. Water was added progressively to mixtures of known quantities of these entities. The changing mucous composition and properties were monitored by light- and electron microscopy, viscometry and immunogold assay. Sequentially, the threads uncoil from GTCs, aggregate with the vesicles, the vesicles rupture and release mucin-like substances, at least some of which adhere to the thread. It was found that the intermediate filament (IF)-rich threads markedly facilitate hydration and modulate the viscoelastic and cohesive properties of the resultant mucus. It was speculated that the thread abets localization of mucus in an aqueous environment and promotes adhesion of mucus to surfaces such as the fish integument. As judged by immunostaining in situ, GTCs, as well as several cell-types in the epidermis, contain keratin-like components. The role of biopolymers on the properties of teleost and mammalian mucus is discussed.

Fractionation of Hagfish Slime Gland Secretions: Partial Characterization of the Mucous Vesicle Fraction

This paper deals with the collection, fractionation and partial characterization of the slime gland secretion of the Pacific hagfish (Eptatretus stouti) with emphasis on the mucous fraction. Secretions were collected by electrical stimulation of the glands of anesthetized hagfish and, using three different methods, separated into three fractions: 1) the thread cells, 2) the mucous vesicles of the mucous cells, and 3) the soluble fraction. The methods take advantage of the stabilization of the thread cells and mucous vesicles by ammonium sulfate and sodium citrate.

Metabolic-morphologic events in the integument of the Pacific hagfish (Eptatretus stoutii).

SummaryLight- and electron-microscopic autoradiography were used to obtain a coordinated metabolic-morphologic view of some of the events of cellular differentiation that occur across the epidermis of the Pacific hagfish (Eptatretus stoutii) and which enable this animal to secrete copious amounts of mucus. As judged by epidermal incorporation of [3H]-thymidine in vivo, about 98 % of DNA replication is confined to the basal three layers of the total of 6–8 layers of cells. Small mucous cells (SMC), the most numerous of the three major cell types involved in mucigenesis, show in vitro and in vivo radioincorporation profiles of [3H]-L-lysine and [3H]-D-glucosamine which differ markedly from those of [3H]-L-fucose and [3H]-D-galactose. Time-course incorporation profiles (mean silver grains/cell and percentage of cells with at least one cluster of silver grains) of [3H]-L-lysine and [3H]-D-glucosamine not only reflected the metabolic activities of cell renewal and differentiation in basally-located cells but also the high mucigenic activity in cells near the epidermal surface. By contrast, [3H]-L-fucose and [3H]-D-galactose were mainly incorporated by the more mature SMC in juxtanuclear regions near Golgi complexes and newly formed secretory vesicles. The intensity of [3H]-fucose labeling appeared proportional to the intensity of histochemical staining of the apical cytoplasm. The prominent capsule, within SMC in basal and lateral regions, which arises from a tight intermingling of tonofilaments, appears to restrict secretory vesicles to apical regions while the cell progressively differentiates and migrates to the epidermal surface. The other mucigenic cell types, large mucous cells and thread cells, each show distinctive differentiation and radioincorporation patterns.

Adriamycin-induced inhibition of melanoma cell invasion is correlated with decreases in tumor cell motility and increases in focal contact formation

Tumor cell adhesion to the extracellular matrix (ECM) is closely linked with tumor cell invasion and metastasis. In this study, we demonstrate that low levels of adriamycin, a widely used anticancer drug, can inhibit the invasion of highly metastatic K1735-M2 mouse melanoma cellsin vitro through a reconstituted basement membrane extract. Adriamycin-induced inhibition of melanoma cell invasion occurred at levels of the drug (i.e. 1 ng/ml) that did not inhibit tumor cell growth, suggesting that the observed inhibition in tumor cell invasion was not due to the well-documented ability of adriamycin to interfere with DNA and/or RNA synthesis. Rather, these studies indicated that adriamycin-induced inhibition of melanoma cell invasion was accompanied by a corresponding decrease in the ability of adriamycin-treated tumor cells to migrate in response to several isolated ECM components including fibronectin, laminin and basement membrane (type IV) collagen. The decreased migration of adriamycin-treated tumor cells was not accompanied by a decrease in the adhesion or spreading of the adriamycin-treated cells on substrata coated with these ECM components. Instead, adriamycin-treated cells actually exhibited a slightly increased propensity (compared to untreated control cells) to adhere on fibronectin-, laminin-, and type IV collagen-coated substrata. Additionally, adriamycin treatment caused a dramatic increase in focal contact formation by these melanoma cells, as assessed by fluorescent microscopy of actin and vinculin. In addition to providing a useful model for which to study the molecular and cellular basis for focal contact formation, these results further emphasize the results of several other investigators that have suggested an important role for focal contacts in modulating tumor cell motility, invasion and metastasis.

Entamoeba histolytica: Autoradiographic analysis of nuclear sites of RNA synthesis

Abstract Autoradiographs of sectioned Entamoeba histolytica labeled with [5-3H]uridine for time periods ranging from 15 sec to 24 hr revealed (1) some evenly distributed nuclear label and (2) extensive labeling of the peripheral nuclear chromatin. This nuclear area is the probable site of synthesis (or accumulation) of ribosomal RNA and/or ribosomal precursor RNA. Also seen was (3) evenly dispersed cytoplasmic label with peaks of labeling after ca. 48 hr of exposure to isotope. Pulses of 5 min, 15 min, 1 hr, and 2 hr followed by a 5-min to 3-hr chase in unlabeled medium with carrier uridine demonstrated that most of the pulse-labeled nuclear RNA was lost from the nucleus; however, a small amount of stable nuclear RNA was detected after a 2- to 3-hr chase. Further, an instability of cytoplasmic RNA was also detected.

The Preparation of Poly(A)±mRNA from the Hagfish Slime Gland

The isolation of translatable poly(A)+mRNA from the slime glands of the Pacific hagfish, Eptatretus stouti, is not possible by the commonly used procedures because of the viscous slime that is formed when the contents of the glands are hydrated. This paper reports on a procedure developed to overcome this problem. Briefly, the tissue was powdered in liquid nitrogen, mixed with sodium lauroylsarcosine and proteinase K and lyophilized. The lyophilized powder was then mixed with 0.3 mm diameter glass beads, thoroughly ground and wetted with buffer and digested at 37 degrees C. The RNA from the digest was recovered by ultracentrifugation through a CsCl cushion. Further purification of the RNA was accomplished by the usual methods with slight modifications.

Metabolic-morphologic characteristics of the integument of teleost fish with mature lymphocystis nodules

SummaryNormal and virus-infected (lymphocystis disease) integument from five species of teleosts was examined by light and TEM autoradiography and SEM to establish metabolic-morphologic characteristics of integument with mature lymphocystis cells (LCs). LCs with numerous morphologic attributes of a late developmental stage showed highest incorporation of [3H]-thymidine in vivo (1–91 h) above the intracytoplasmic inclusion body (ci) with little radiolabel in nuclei, cytoplasmic icosahedral deoxyriboviruses (ICDVs) or capsule. Analysis by quantitative autoradiography revealed that the % total cell label in ci and cytoplasm did not vary appreciably from 1–91 h and was corroborative with morphologic criteria of maturity. A possible phylogenetic difference was noted between teleosts, wherein normal integument showed uptake of [3H]-thymidine in vivo (1 h) by cells at all levels of the epidermis, and cyclostomes (Spitzer et al. 1979) wherein labeling was confined to the basal third of the epidermis. Among four infected teleost species, the mean diameters of the ICDVs measured under the same conditions, ranged from 259.5 nm to 290.0 nm with the mean for each species differing significantly (p < 0.01) from each of the other means. Ruptured LCs were shown by TEM and SEM to have released ICDVs onto the lesions and integument. Various stages of LC degeneration, host response, and integumental repair processes were documented. An evaluation of labeling in vivo of the capsular matrix was compatible ([3H]-D-galactose> [3H]-L-lysine ≫ [3H]-L-fucose) with a glycosaminoglycan-protein structure.