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Eicosanoids are a group of oxygenated arachidonic acid metabolites which include Prostaglandins, thromboxanes, leukotrienes, lipoxins, and various hydroperoxy and hydroxy fatty acids. Eicosanoids are implicated in diverse cellular functions such as Chemotaxis, proliferation, cell-cell signaling etc. The first committed step in the biosynthesis of each of the eicosanoids, the incorporation of molecular oxygen into polyunsaturated fatty acids, is catalyzed by one of a group of enzymes calles fatty acid oxygenases. This group includes the cyclooxygenase (COX) of Prostaglandin endoperoxide synthase (PGH) and various lipoxygenases (LOX).

We reported previously that, in cultured goldfish xanthophores, dispersion of aggregated carotenoid droplets (CDs) requires the specific phosphorylation of the CD protein p57 by a cAMP-dependent protein kinase and the presence of cytosol. We report here that, in permeabilized cells, the addition of the catalytic subunit of cAMP-dependent protein kinase and ATP phosphorylates p57 and converts the CDs from an immobile to a mobile state (first stage of CD dispersion). However, the CDs are restricted to the vicinity of the original site of the CD aggregate and do not actually disperse (second stage of CD dispersion) unless cytosol is also added. We propose that this process may be related to aspects of secretory processes.

In melanophores of Fundulus heteroclitus, hormone-stimulated melanosome aggregation is accompanied by cytoplasmic flow from the cellular processes to the perikaryon, and reversal of these events takes place upon hormone-induced melanosome dispersion. These cells contain parallel arrays of microtubules, the majority of which are located in the perikaryon and in cortical regions of the processes. Studies with heavy meromyosin binding demonstrated two types of actin filaments: 11 a decorated meshwork of filaments similar to those usually found in close association with plasma membranes, and 2) filaments decorated in a manner similar to that of stress fibers. There is an apparent increase in the amount of filaments during melanosome aggregation. These results are discussed in relation to intracellular movement.

Three calcium channel blockers of the dihydropyridine class were tested in vitro for their effects on tumor cell-platelet-endothelial cell interactions and in vivo for antimetastatic properties. Felodipine, nimodipine and nifedipine inhibited tumor cell-induced platelet aggregation in vitro in a dose-dependent manner. These compounds also inhibited platelet-enhanced tumor cell adhesion to endothelial cells in vitro . Lung colony formation (“experimental” metastasis) and spontaneous pulmonary metastasis were inhibited by felodipine, nimodipine and nifedipine. From the present studies on three calcium channel blockers of the dihydropyridine class we hypothesize that calcium channel blockers may represent a new generic class of antimetastatic agents.

Cells of the uncloned goldfish erythrophoroma lines, GEM-81 and GEM-218, have been induced to melanize by cultivation in autologous serum. The melanized cells continue to proliferate and exhibit clonal heterogeneity in terms of morphology, growth rates, contact behavior and pigment content, and distribution and translocation in response to hormones. Based on these characteristics and those of their normal counterparts, the melanized tumor cells have been categorized as type-I and type-I1 melanocytomas, and melanophoromas. The melanophoroma cells are capable of pigment translocation in response to epinephrine, melatonin, and/or MSH, whereas melanocytoma cells are not. The distinguishing characteristics of each type are apparent at the first appearance of melanized cells and appear to be stable except in some type-II melanocytoma clones which contain cells capable of differentiating into melanophoroma cells in long-term cultures. It appears that the parent erythrophoroma lines contain stem cells, melanoblastomas, which are capable of melanogenesis. These stem cells may themselves be a heterogeneous population with respect to the characteristics of the melanized cells to which they give rise.

Stage 14 (Gallien and Durocher, 1957) Pleurodeles waltlii embryos were treated with α- or β-melanophore-stimulating hormone (MSH), ACTH, dbc-AMP, c-AMP plus theophylline, theophylline, 5′-AMP, or 2′,3′-AMP. The development of melanophores was accelerated (appearing two stages earlier than in control embryos) by α- or β-MSH, ACTH, dbc-AMP, c-AMP plus theophylline, or theophylline alone. By the time embryos developed to stage 28, the control and treated embryos were indistinguishable in the number, distribution, and general morphology of their melanophores, suggesting these agents do not induce melanophore formation or mitosis. Cyclic AMP alone, 5′-AMP or 2′,3′-AMP were ineffective. Accelerated cytodifferentiation apparently requires preinduction by the invaginating chordo-mesoderm during stages 13 and 14 as presumptive neural plate explants cultured before chordo-mesoderm induction (stage 8) failed to produce melanophores with or without α-MSH or dbc-AMP. Explants from stage 14 embryos treated with α-MSH or dbc-AMP developed the same number of melanophores at the approximate time that treated whole embryos developed melanophores. Whole embryo experiments involving the faster developing embryos of Xenopus laevis were similar to those described for P. waltlii.

This study examined gel filtered rat platelet activation by Walker 256 rat carcinoma cells and characterized fibronectin release. Using aggregometry measurements, a biphasic platelet response was characterized and the timing of alpha granule release was determined. The localization and association of released platelet fibronectin with tumor cell-platelet aggregates was determined by immunofluorescent and immunocytochemical methods. The immunofluorescent studies showed that the platelet fibronectin was released and became associated with the peripheries of the tumor cells following alpha granule discharge. The ultrastructural immunocytochemical data revealed that this fibronectin was associated with a fibrin-like material, enmeshing the tumor cell-platelet aggregates. The significance of the release and localization of platelet fibronectin to tumor cell metastasis is discussed.

Calcium channel blockers of 3 chemical classes and calmodulin antagonists have recently been shown to enhance the cytotoxicity of conventional cancer chemotherapeutic agents. We tested the ability of nifedipine, a dihydropyridine class calcium channel blocker, to enhance the cytotoxicity of cisplatin, the current chemotherapeutic of choice against human head and neck tumors and testicular carcinoma. Both in vitro and in vivo, combination therapy with nifedipine and cisplatin resulted in synergistic inhibition of tumor cell proliferation, primary tumor growth and appearance of spontaneous pulmonary metastases. This combination also significantly increased the survival of mice bearing cisplatin-resistant tumors. This is the first indication that calcium channel blockers can enhance the cytotoxicity of cisplatin as well as the first demonstration that calcium channel blockers can enhance cytotoxicity of chemo-therapeutic agent against a solid tumor and its metastases.

We have isolated from murine solid tumors (B16a) subpopulations of cells possessing high and low metastatic potential. Tumors were dispersed by collagenase treatment. The resulting heterogeneous population of cells (i.e., viable and non-viable tumor cells and host cells) were separated by centrifugal elutriation. Four of the fractions (100, 180, 260, 340) contained tumor cells of high viability (greater than 95%) and high purity (less than 1% host cell contamination). The four fractions were characterized by flow cytometry and found to differ in distribution of cells in G1, S and G2. The cell populations were also found to differ in metastatic potential as determined by their ability to form lung colonies following intravenous injection. The 340 fraction was approximately 5-fold more metastatic than the 100 fraction. We also observed that cells from the 100 fraction failed to induce platelet aggregation whereas cells from the 340 fraction induced significant platelet aggregation. These observations demonstrate that cells of B16a tumors are heterogeneous for phenotypic characteristics (i.e., metastatic potential; platelet aggregation, etc.) and that their ability to induce platelet aggregation is positively correlated with metastatic potential.

A panel of monoclonal and polyclonal antibodies raised against human platelet GpIb or the GpIIb/IIIa complex were used to detect immunologically related molecules on two cell lines derived from human solid tumors. Human cervical carcinoma (MS751) and human colon carcinoma (clone A) expressed molecules immunologically related to platelet GpIb and GpIIb/IIIa complex. These molecules were localized to their plasma membranes by immunofluorescence and immunocytochemistry. The immunologically related GpIb was evenly distributed on the tumor cell membrane with occasional areas of aggregates, whereas the immunologically related GpIIb/IIIa had a pronounced punctate distribution of aggregates in prefixed cells. When MS751 or clone A cells were pretreated with antibodies against platelet GpIb and/or the GpIIb/IIIa complex, their ability to induce platelet aggregation was significantly inhibited. In addition, when tumor cells were pretreated with antibodies against the platelet IIb/IIIa complex, adherence to fibronectin-coated plates was also significantly inhibited. These results suggest a role for these immunologically related tumor cell glycoproteins in tumor cell-host cell (i.e., platelet, endothelial cells) interactions, tumor cell interactions with components of the subendothelial matrix, and subsequent tumor metastasis.

Platelet involvement during tumor cell adhesion to subendothelial matrix was examined in vitro. Platelets were subjected to thrombin stimulation and mechanical lysis and examined for their effects on tumor cell adhesion. These treatments altered the platelet ultrastructure and cytoskeletal integrity. Untreated washed rat platelets (WRP) exhibited extensive adhesion to and spreading on substrates and substantially enhanced tumor cell adhesion to the same substrates (i.e., 250% greater than tumor cells without platelets). Thrombin prestimulation of platelets limited platelet adhesion and spreading and platelet facilitation of tumor cell adhesion. Complete mechanical lysis disrupted both the platelet membrane and the cytoskeleton and eliminated the ability of platelets to adhere or to enhance tumor cell adhesion. Partially lysed platelets resembled membrane ghosts and facilitated tumor cell adhesion by a mechanism independent of spreading and cytoskeletal rearrangement. Fractionation studies indicated that platelet cytoskeletal components played a role in the adhesion process. Pretreatment of WRP with cytochalasin A or B dose dependently inhibited microfilament-mediated platelet spreading and platelet-enhanced tumor cell adhesion. Colchicine and vinblastine induced microtubule depolymerization, but they had no observable effect on platelet spreading or platelet-enhanced tumor cell adhesion. It was concluded that platelet-enhanced tumor cell adhesion to subendothelial matrix depends on an intact platelet cytoskeleton and on a platelet membrane component(s) and is mediated by surface contact between platelets and tumor cells. Furthermore, platelet-mediated tumor cell adhesion to subendothelial matrix may involve two mechanisms: one dependent on, and one independent of, platelet spreading and cytoskeletal rearrangement.

The cytoskeleton of goldfish xanthophores contains an abundance of unique dense structures (400 nm in diameter) that are absent in goldfish nonpigment cells and are probably remnants of pterinosomes. No major difference in protein composition between xanthophores and nonpigment cells (without these structures) was found that could account for these structures. In xanthophores, these structures are foci of radiating filaments. The addition or withdrawal of ACTH causes a radical rearrangement of the xanthophore cytoskeleton accompanying redistribution of carotenoid droplets, namely, the virtual exclusion of these dense bodies with associated filaments from the space occupied by the carotenoid droplet aggregate vs. a relatively even cytoplasmic distribution of these structures when the carotenoid droplets are dispersed. These changes in cytoskeletal morphology are not accompanied by any major changes in the protein or phosphoprotein composition of the cytoskeleton.

There has been considerable controversy surrounding the ability of inhibitors of arachidonic acid metabolism to concomitantly inhibit tumor cell induced platelet aggregation (TCIPA). Reconciliation of this controversy has been difficult due to the wide variability of experimental conditions (e.g., inhibitor concentration, strength of the inducing agonist).In the present study, we examined the effects of several cyclooxygenaseand lipoxygenase inhibitors on the induction of platelet aggregation by Walker 256 carcinosarcoma (W256) cells. We have previously demonstrated that aggregation of platelet rich plasma (PRP), induced by W256 cells, was initiated via a thrombin dependent mechanism. Platelet aggregation was induced by the addition of W256 cells (75,000-J500,000 cells/cuvette) to rat PRP preincubated with inhibitor(s) or diluent. The strength of the inducing stimulus affected both the degree of aggregation and the production of thromboxane A2 (TXA2) in a dose dependent manner. A weak stimulus (low concentration of W256 cells) produced only a low level of aggregation and low TXA2 production, whereas aggregation induced by a strong stimulus (high concentration of W256 cells) resulted in significant aggregation and increased TXA2 production. Preincubation (5 min., 37°C) of rat PRP with cyclooxygenase inhibitors (e.g., aspirin, indomethacin, ibuprofen) resulted in complete inhibition of platelet aggregation at low agonist concentration (75,000 W256 cells), whereas when a high agonist concentration (500,000 W256 cells) was used to induce aggregation, the inhibitors failed to inhibit TCIPA. The addition of fewer than 50,000W256 cells failed to induce any measurable platelet aggregation in the presence or absence of inhibitors. TCIPA was not affected by lipoxygenaseinhibitors (e.g.,quercetin) alone regardless of agonist concentration. Both cyclooxygenase and lipoxygenase inhibitors, however, were required to significantly inhibit TCIPA induced by high agonist concentration. Compounds which inhibited both the cycloogygenase and lipoxygenase pathways (e.g.,hydroquinone, BW755c) inhibited TCIPA at all agonist concentrations. Nafazatrom failed to inhibit TCIPA consistant with a lack of effect on platelet cyclooxygenase and lipoxygenase. Therefore, we conclude cyclooxygenase (e.g., TXA2) and lipoxygenase (e.g., 12-HETE) products of platelet arachidonic acid metabolism and the strength of the inducingagonist are important criteria in TCIPA. This study may help to clarify the current controversy regarding the inhibition of TCIPA by inhibitors of arachidonic acid metabolism.

Ultrastructural studies, and cytochemical and biochemical determinations of tyrosinase activity were conducted on the pigment epithelium of albino and xanthic goldfish eyes. In eyes of xanthic goldfish, two types of melanosomes are present, spherical and elongated. Melanized melanosomes are absent in the eyes of the albino goldfish, but elongated lamellar premelanosomes are observed. Internal vesicles are present in both melanosome types in the pigment epithelium of the xanthic goldfish but are absent in premelanosomes of the albino. There are also differences in the distribution of lipid droplets, smooth endoplasmic reticulum and Golgi complexes with the latter two being more abundant in the albino. Tyrosinase was not identified cytochemically; however, the enzyme was demonstrated biochemically in the pigment epithelia of both albino and xanthic goldfish. The enzyme is associated with the particulate and soluble fractions of both types of eyes. Particulate albino tyrosinase may be solubilized by triton X-100 treatment. Tyrosinase inhibitors are present in the particulate fractions of both albino and xanthic goldfish eyes. Thus, in the goldfish, ocular albinism appears to be a multiple defect at the molecular and ultrastructural levels.

The ability to metastasize is a characteristic which distinguishes benign from malignant neoplasms. Metastasis can be simply defined as a loss of contiguity between a tumor cell or a clump of tumor cells and the primary tumor with successful transfer to, and growth at, a spatially separate site. The overall process can be regarded as a series of sequential events representing complex interactions between the tumor cell and the host (Figure 1).

Metastases represent perhaps the single most important hindrance to improved cancer patient survival. The formation of metastases is the culmination of a complex series of tumor cell-host interactions called the metastatic cascade (Figure 1; 1,2). During tumor progression, tumor cells become separated from the primary tumor mass and invade into the surrounding tissue. Occasionally, tumor cells will invade blood vessels or lymphatics (intravasation) and are readily disseminated throughout the host. A relatively small percentage of the circulating tumor cells will arrest at the vessel wall and invade into the surrounding tissue (extravasation) to establish a secondary tumor site (3–5). The interaction of the circulating tumor cells with host immune cells may result in destruction of the tumor cells, whereas interaction with host platelets and/or blood coagulation components may enhance the metastatic process. Evidence for the involvement of platelets in metastasis has been demonstrated by several investigators (for review see 6,7). Enhancement of tumor cell arrest and adhesion to the vessel wall by platelets has been postulated as the mechanism for this phenomenon, but its exact nature is undetermined. Because of the potent effects of arachidonic acid metabolites (i.e., prostacyclin and thromboxane A2) on platelet function, we have hypothesized that tumor cells shift the balance between these metabolites in favor of platelet aggregation (8). We have previously proposed the use of modifiers of arachidonic acid metabolism as antimetastatic agents, although the efficacy of antimetastatic therapy with these modifiers has been inconsistent. Prostacyclin (PGI2) has been recently shown to reduce the incidence of pulmonary.

Chitons, common on tropical limestone shores, use magnetite-capped radula teeth to scrape algae from upon and within the substrate, and at the same time remove quantities of rock debris. A quantitative estimate of the erosive activities of intertidal chitons at Aldabra Atoll shows that 0.023 g/day of rock debris are defecated by each chiton, which amounts to 40.20 g/m2/yr/chiton. This accounts for a 0.017 mm/m2/yr rate oF erosion of the rock surface, which is about 3.4% of the total erosion. Chitons, although effective agents at rock removal, are insufficiently abundant to be a major cause of intertidal erosion at Aldabra.

Previous work using primary cell cultures of xanthic goldfish skin in heterologous (fetal calf) serum failed to produce significant numbers of melanocytes by ACTH treatment. This report shows that by the use of autologous (goldfish or red carp) serum, and cell cultures enriched in xanthophores and deep dermal non-pigment cells, melanization is readily induced with ACTH. The major melanization process is the formation of colonies of actively dividing melanocytes. A rarer melanization process is the transdifferentiation of xanthophores into xanthomelanophores containing both orange and dark pigments.

We reported previously the isolation of a melanized cell line that can undergo reversible dedifferentiation and redifferentiation. A heavily pigmented cell line, designated as P15, originally isolated by fish serum-induced melanization of some GEM 81 cells, cloned and serially passaged in fish serum medium, became noticeably less pigmented after several months in fetal calf serum medium and completely unpigmented after another year in the same medium. Addition of fish serum to the medium of this dedifferentiated cell line, designated P15D, induced pigmentation within a week. This re-induced pigmented cell line, designated as P15DI, became unpigmented when cultured in fetal calf serum medium for one month. We report here that the dedifferentiation of P15 occurs in two stages. One week after withdrawal of fish serum, the specific activity of tyrosinase of the culture dropped by approximately 70% and remained at this reduced level for at least one month. After one year, the specific activity of tyrosinase had dropped to a barely detectable level and the culture became completely unpigmented (P15D). Electron microscopic studies showed that the P15D cells have no melanosomes, probably no large vesicles for melanosome formation, but some dopa-positive trans-Golgi network (TGN). Addition of fish serum to the growth medium of P15 cultures led to a steady increase in the specific activity of tyrosinase, detectable after one day. There was also an increase in the amount of dopa-positive TGN within one day. Melanosomes first appeared after three days and became numerous after one week. Upon removal of fish serum, these re-induced cells (P15D1) underwent a rapid decrease in the specific activity of tyrosinase, reaching, after eight days, the basal level seen in P15D cells. We also report that a protein designated as p75 (Mr approximately 75,000), previously shown to be associated with melanosomes in two melanized cell types of goldfish origin, is present in all melanized cell lines, including P15 and P15DI but absent in unmelanized cell lines, including P15D.

Epinephrine-induced melanosome aggregation in cultured terminally differentiated Black Moor goldfish melanophores can be over-ridden by ACTH but not by cAMP.

Treatment of cultured goldfish xanthophores by hormone (ACTH) or c-AMP induces not only pigment dispersion, but subsequent outgrowth of processes, and pigment translocation into these processes. These latter effects are shown to proceed as follows: First the edge of the cytoplasmic lamellae takes on a scalloped contour with numerous protrusions. These presumably serve as nucleation centers where short microfilament bundles are assembled. Later, the microfilament bundles elongate ("grow"), often resulting in an extension of the protrusions to become filopodia while the proximal end of the microfilaments penetrates into the thicker portion of the cellular process which now houses the pigment, i.e., the carotenoid droplets. Carotenoid droplets appear to migrate along the microfilament bundles, or cytoplasmic channels associated with them, into the filopodia. Finally, some of the filopodia become broader, thicker and laden with carotenoid droplets and are then recognized by light microscopy as pigmented cellular processes. The microfilaments have been shown to be actin filaments by their thickness, the size of their subunits, and decoration by heavy meromyosin. Evidence is presented which suggests that the growth of these actin filaments may come about by recruitment from short F-actin strands found in random orientation in adjacent areas.

Stimulation (in vivo and in vitro) of dermal melanophores of the leaf frog, Agalychnis dacnicolor, by melanophore stimulating hormone (MSH) elicits two responses in addition to the dispersion of melanosomes: (1) dispersion of heterochromatin; and (2) blebbing of the outer membrane of the nuclear envelope. The latter leads to the formation of dilated rough endoplasmic reticulum with the involvement of 80–100 A microfilaments. N6,O2-dibutyryl adenosine 3′,5′-monophosphate (db-cAMP) elicits a similar response. Actinomycin D prevents both heterochromatin dispersion and membrane blebbing while cytochalasin B (CB) prevents only the latter.

Melanosomes of phyllomedusid frogs are unusually large and are composed of an amorphous matrix of thick fibers. Their hitherto undescribed dark red pigment is neither phaeomelanin nor eumelanin, but seems to be related to melanins. Melanophores of at least one of these species, Agalychnis dacnicolor, exhibit color change in direct response to illumination, and it is suggested that these chromatophores are innervated.

The skin of the lizard, Anolis carolinensis, changes rapidly from bright green to a dark brown color in response to melanophore stimulating hormone (MSH). Chromatophores responsible for color changes of the skin are xanthophores which lie just beneath the basal lamina containing pterinosomes and carotenoid vesicles. Iridophores lying immediately below the xanthophores contain regularly arranged rows of reflecting platelets. Melanophores containing melanosomes are present immediately below the iridophores. The ultrastructural features of these chromatophores and their pigmentary organelles are described. The color of Anolis skin is determined by the position of the melanosomes within the melanophores which is regulated by MSH and other hormones such as norepinephrine. Skins are green when melanosomes are located in a perinuclear position within melanophores. In response to MSH, they migrate into the terminal processes of the melanophores which overlie the xanthophores above, thus effectively preventing light penetration to the iridophores below, resulting in skins becoming brown. The structural and functional characteristics of Anolis chromatophores are compared to the dermal chromatophore unit of the frog.

Chromatophores of two color forms of the salamander, Plethodon cinereus were studied by electron microscopy. Erythrophores found in the red-backed form possess pterinosomes, some with regions of high electron density. In addition, a few melanosome-like organelles are present. On the other hand, the lead-backed form displays no visible erythrophores but instead melanophores with melanosomes being the most prevalent organelle and a few pterinosomes. The possibility that this represents a kind of hybrid chromatophore with intermediate stages in melanosome-pterinosome interconversion is discussed.

Summary: A series of 55 cases of acute appendicitis in pregnancy and the puerperium over the 6-year period, 1960–1965 is presented. Pregnancy modifies the classical presentation of the disease, partly because of the altered position of the appendix, and partly because of less efficient localization of the infection. Accordingly, the site of maximum tenderness may be above McBurney's point and peritonitis is a frequent complication and this may be associated with abortion and perinatal death. Immediate appendicectomy is the correct treatment; combination with Caesarean section should be avoided whenever possible.

Publisher Summary This chapter discusses that the melanin-containing pigment cells, melanocytes, and melanophores have been favorite targets of study because of the fact that the pigment melanin serves as a built-in cytochemical marker and facilitates microscopic examinations. It has been found through embryological studies that these cells are derived from specific unpigmented cells of the neural crest that migrate to specific locations during a specific phase of embryonic development and give rise, at a much later stage, to pigmented cells. The melanocyte-stimulating hormone (MSH), and other structurally related polypeptides, including adrenocorticotropic hormone (ACTH), cause dispersion of the pigment granules, melanosomes, and therefore, produce visible darkening of the cell or of skin containing these cells. This effect is mediated via second messenger, cyclic adenosine monophosphate (cAMP), and, as in the case of most other polypeptide hormones, probably involves membrane-bound complexes of adenyl cyclase and hormone receptor molecules. Results discussed in the chapter indicate that the mythical inductor for melanocytogenesis is the well-known hormone MSH acting via the second messenger, cAMP. The chapter also discusses some future avenues of experimental approaches.