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Animal venoms are considered sterile sources of antimicrobial compoundswith strong membrane-disrupting activity against multidrug-resistant bacteria. However,venomous bite wound infections are common in developing nations. Investigating theenvenomation organ and venom microbiota offive snake and two spider species, weobserved venom community structures that depend on the host venomous animal spe-cies and evidenced recovery of viable microorganisms from black-necked spitting cobra(Naja nigricollis) and Indian ornamental tarantula (Poecilotheria regalis) venoms. Amongthe bacterial isolates recovered fromN. nigricollis,weidentified two venom-resistant,novel sequence types ofEnterococcus faecaliswhose genomes feature 16 virulencegenes, indicating infectious potential, and 45 additional genes, nearly half of whichimprove bacterial membrane integrity. Ourfindings challenge the dogma of venom ste-rility and indicate an increased primary infection risk in the clinical management of ven-omous animal bite wounds.

We have modified the periplasmic Escherichia coli glucose/galactose binding protein (GBP) and labelled with environmentally sensitive fluorophores to further explore its potential as a sensor for the evaluation of glucose concentration in airway surface liquid (ASL). We identified E149C/A213R GBP labelled with N,N’-Dimethyl-N-(iodoacetyl)-N’-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD, emission wavelength maximum 536nm) with a Kd for D-glucose of 1.02mM and a fluorescence dynamic range of 5.8. This sensor was specific for D-glucose and exhibited fluorescence stability in experiments for several hours. The use of E149C/A213R GBP-IANBD in the ASL of airway cells grown at air-liquid-interface (ALI) detected an increase in glucose concentration 10 minutes after raising basolateral glucose from 5 to 15mM. This sensor also reported a greater change in ASL glucose concentration in response to increased basolateral glucose in H441 airway cells compared to human bronchial epithelial cells (HBEC) and there was less variability with HBEC data than that of H441 indicating that HBEC more effectively regulate glucose movement into the ASL. The sensor detected glucose in bronchoalveolar lavage fluid (BALf) from diabetic db/db mice but not normoglycaemic wildtype mice, indicating limited sensitivity of the sensor at glucose concentrations

Fractal or focal dimension (FD) analysis is a valuable tool to identify physiologic stimuli at the cellular and tissue levels that allows for quantification of cell perimeter complexity. The FD analysis was determined on fluorescence images of caffeine- or epinephrine-treated (or untreated control) killifish Fundulus heteroclitus (Linneaus) melanophores in culture. Cell perimeters were indicated by rhodamine-phalloidin labeling of cortical microfilaments using box-counting FD analysis. Caffeine-treated melanophores displayed dispersed melanosomes in cells with less serrated edges and reduced FD and complexity. Complexity in epinephrine-treated cells was significantly higher than the caffeine-treated cells or in the control. Cytoarchitectural variability of the cell perimeter is expected because cells change shape when cued with agents. Epinephrine-treated melanophores demonstrated aggregated melanosomes in cells with more serrated edges, significantly higher FD and thus complexity. Melanophores not treated with caffeine or epinephrine produced variable distributions of melanosomes and resulted in cells with variably serrated edges and intermediate FD with a larger SE of the regression and greater range of complexity. Dispersion of melanosomes occurs with rearrangements of the cytoskeleton to accommodate centrifugal distribution of melanosomes throughout the cell and to the periphery. The loading of melanosomes onto cortical microfilaments may provide a less complex cell contour, with the even distribution of the cytoskeleton and melanosomes. Aggregation of melanosomes occurs with rearrangements of the cytoskeleton to accommodate centripetal distribution of melanosomes. The aggregation of melanosomes may contribute to centripetal retraction of the cytoskeleton and plasma membrane. The FD analysis is, therefore, a convenient method to measure contrasting morphologic changes within stimulated cells.

Pigmentary organelle translocations within fish chromatophores undergo physiological color changes when exposed to external signals. Chromatophores can be isolated in high yields, and their pigmentary organelles can be tracked readily by microscopy. The combined efforts of morphology and biomolecular chemistry have led to the identification of and determination of the interrelationships between cytoskeletal elements and accessory proteins, motor molecules, cytomatrix, and pigmentary organelles of various sizes. Fish chromatophores have been classified as fast, intermediate, and slow translocators, based on the relative numbers of microtubules. Studies on cultured goldfish (Carassius auratus L.) xanthophores for over 20 years have demonstrated that in this slow translocator, tubulovesicular structures of the smooth endoplasmic reticular (SER) cisternae are involved in the disperson and aggregation of associated carotenoid droplets (CD) with some involvement of cytoskeletal elements. Killifish (Fundulus heteroclitus L.) melanophore, a fast translocator, was also examined. Recent work demonstrates a bright fluorescent "starburst"-like spot that we call an actin filament-organizing center (AFOC) with radiating microfilaments, akin to the microtubule-organizing center (MTOC) with radiating microtubules. Melanosomes translocate single-file on microtubules and are not associated with SER cisternae. Slower CD dispersion or aggregation in goldfish xanthophores seems to be predominantly microfilament-based transport, or microfilament- and microtubule-based transport, respectively. Faster melanosome translocations in killifish melanophores are based on microtubules, with our evidence indicating microfilament involvement. Neural crest-derived chromatophores are models for vesicular transport in axons, and immunocytochemical and imaging technologies may help to elucidate the cellular transport mechanisms.

Two veneroidean bivalves Granicorium indutum from Australia and Samarangia quadrangularis from the tropical Indo-Pacific region, cement a thick, hard layer of sand over most of their shells. In Granicorium this layer forms low commarginal ribs while in Samarangia it forms more prominent radial features. Sand grains are cemented to the shell and to each other with growths of a crystalline aragonitic cement similar in morphology to inorganic marine cements. Both species secrete mucus layers at the growing shell margin which initially hold the sediment grains together and form a substrate for the nucleation and growth of calcium carbonate crystals. The ribs of Samarangia are formed by the accretion of successive sheets of spherulitic growths. In G. indutum, the middle and outermost of two inner mantle folds are large, glandular and capable of considerable extension beyond the shell margin. Mucus secreted by the folds contains abundant bacteria and small calcium carbonate crystals. It is proposed that initial nucleation of the calcium carbonate cement takes place within this biofilm possibly mediated by the bacteria. The function of the sand layers is unknown but predation resistance and protection of the shells from endobionts are the most likely possibilities.

12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], a lipoxygenase metabolite of arachidonic acid, has been shown to be involved in a wide variety of cellular activities (i.e., adhesion, spreading, motility, invasion) which promote metastasis to occur in tumor cells. In this study, several techniques (Western blotting, flow cytometry and DNase I assay) were performed to examine the alterations in the distribution of G- and F-actin expressed in B16a melanoma cells. Each of these methods independently revealed that 12(S)-HETE treatment (0.1 mM, 15 min) resulted in an increase in the F-actin content in the cytoskeletal preparations. Since the integrity of cytoskeletal networks (i.e., actin filaments) can be dynamically regulated through protein phosphorylation, we investigated the potential role of several protein kinases in the 12(S)-HETE-induced actin polymerization. By flow cytometric analysis, 12(S).HETE was found to increase the actin filament contents. This effect could be inhibited by protein kinase C (PKC) inhibitors (calphostin C and staurosporine) as well as by protein tyrosine kinase (PTK) inhibitor (genistein) but not by protein kinase A inhibitor (H8), suggesting that the 12(S).HETE effect involves PKC and PTK. This conclusion is consistent with the observations that phorbol 12-myristate-13-acetate (PMA) mimics the biological effect of 12(S)-HETE in promoting the F-actin formation in B16a cells. As a final analysis, direct protein phosphorylation studies indicate that 12(S)-HETE treatment led to enhanced phosphorylation of myosin light chain, which may contribute to the increased stress fiber formation following 12(S)-HETE stimulation.

By whole-cell transmission electron microscopy (WCTEM), we recently demonstrated that carotenoid droplets are transported by elongating or retracting endoplasmic reticular cisternae in goldfish xanthophores. Here we report that permeabilized xanthophores demonstrate immunogold reactivity against several proteins involved in organelle translocation. The gold labeling against beta-tubulin and the intermediate filament protein p45a were found on microtubules and intermediate filaments. Labeling with anti-actin was found on nonidentifiable structures, on vesicles of unknown origin, occasional labeling on carotenoid droplets, and on occasional microfilaments. Immunoreactivity was demonstrated with anti-p57 on the carotenoid droplet surface, confirming previous results (Lynch et al., 1986a,b). Labeling with anti-PCD6 subunit (of the inositol trisphosphate/ryanodine receptor) was demonstrated on carotenoid droplets suggesting they possess calcium channels. Anti-MAP 1C (dynein) immunolabeling was generally seen on club-shaped structures in the cytomatrix and on carotenoid droplets. Finally, immunogold labeling with anti-MAP 2a + 2b was seen on a meshwork of microfilaments and intermediate filaments. Finally, this is the first report of a WCTEM technique for permeabilized cells that reveals immunoreactive elements, organelles, and cytomatrix components without the additional requirements of extraction or fracturing.

A modified procedure for whole mount transmission electron microscopy (WMTEM) was utilized, for electron microscopes with accelerating voltages of 80 to 100 kV, to view the outer 40% of goldfish (Carassius auratus [Linneaus]) xanthophores and swordtail fish (Xiphophorus hellerii [Heckel]) erythrophores. This viewing region covers most of the lamellae and all of the filopodia and cellular processes. In this region one can observe three of the four physiological states: pigment dispersing, pigment fully dispersed, and pigment aggregating. The pigment in the fourth state, fully aggregated, cannot be viewed because of thickness of the cell body and is not addressed in this report. In goldfish xanthophores and swordtail fish erythrophores, the primary pigmentary organelle, the carotenoid droplet, is not a discrete organelle but is either associated with or attached to an extended tubulovesicular structure. Carotenoid droplet translocations appear to be dependent upon external cues which result in the elongation or retraction of these tubulovesicular structures. Tubulovesicular structures are continous with membranous sacs, which we termed carotenoid bodies. Based upon DiOC6(3) staining for the endoplasmic reticulum, we propose that the tubulovesicular structure and the endoplasmic reticulum are the same organelles. By using inhibitors of microfilaments (cytochalasin B or D) and microtubules (nocodazole), we report results which suggest cytoskeletal involvement in the elongation or retraction of endoplasmic reticular cisternae. Results with the intermediate filament inhibitor, cycloheximide, demonstrated that intermediate filaments were eliminated from the cells. We believe that the results are inconclusive because cycloheximide primarily inhibits protein synthesis and any interpretation must be made with care. © 1993 wiley-Liss, Inc.

We previously reported that a lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], induced large vessel endothelial cell (EC) retraction and increased tumor cell adhesion to exposed extracellular matrix (Honn et al., FASEB J. 3, 2285-2293, 1989). Here, we present evidence that 12(S)-HETE induces the retraction of microvascular ECs in a time- and concentration-dependent manner. The EC retraction was observed 15 min after 12(S)-HETE treatment and reached a peak level between 1 and 2 h. The monolayer reformed by 24 h. Silver staining and "gap-FRAP" experiments suggest that 12(S)-HETE altered the normally apposed cell junctions and impaired gap junction-mediated cell-cell communication. It appears that the 12(S)-HETE effect was mediated by cytoskeletal alteration. The first observed alteration in EC cytoskeleton following 12(S)-HETE stimulation is vimentin bundling, followed by the rearrangement and disruption of vinculin-containing adhesion plaques and/or simultaneous redistribution of alpha-actinin and disruption of spectrin. These changes are accompanied by progressive microfilament dissolution. During the same time interval, alpha-actinin is mobilized to the cell periphery at cell "ruffles." However, 12(S)-HETE showed little or no effects on actin-binding proteins filamin and tropomyosin or on microtubules. 12(S)-HETE effects on these cytoskeletal elements were fully reversible by 24 h and appeared to be mediated through enhancing protein phosphorylation. Following 12(S)-HETE (0.1 microM) treatment increased phosphorylation of proteins that comigrated with myosin light chain (20 kDa), actin (42 kDa), and vimentin (57 kDa) were observed. The enhanced phosphorylation of these cytoskeletal proteins was confirmed by 2D gel analysis. The phosphorylation-promoting effect of 12(S)-HETE on cytoskeletal proteins could be totally abolished by calphostin C, partially inhibited by staurosporine, but was not influenced by N-[2-(methylamine)ethyl]-5-isoquinolinesilfonamide dihydrochloride (HS), suggesting that the 12(S)-HETE effect was mediated via protein kinase C. This was further substantiated by quantitative experiments demonstrating that calphostin C, but not H8, inhibited 12(S)-HETE-induced EC retraction.

Previously, we have identified an alpha IIb beta 3-like integrin in tumor cells by using antibodies against platelet alpha IIb beta 3. However, alpha IIb beta 3 was considered to be expressed strictly in megakaryocyte lineage cells. In order to resolve this controversy, the alpha IIb beta 3-like integrin in murine B16 amelanotic melanoma (B16a) cells was characterized at DNA, RNA, and protein levels. The presence of alpha 5, alpha v, alpha IIb, beta 1, and beta 3 genes in B16a cells was confirmed by Southern analysis. mRNAs for all these integrins except alpha v were detectable by Northern blotting. The alpha IIb beta 3 protein was identified by Western blotting using subunit-specific antibodies and by immunoprecipitation using complex-specific antibody. The alpha IIb beta 3 integrin was localized intracellularly by immunocytochemistry. Finally, alpha IIb and beta 3 mRNAs were amplified by reverse transcription-polymerase chain reaction and the identity of alpha IIb was verified by sequencing. Partial DNA and deduced amino acid sequence analysis showed that B16a alpha IIb shares approximately 80% homology with the human alpha IIb and approximately 90% homology with the rat alpha IIb, whereas B16a alpha IIb shares only approximately 26% homology with the human alpha v. These experiments indicate that the alpha IIb beta 3-like protein in B16a cells is the authentic alpha IIb beta 3 and demonstrate, for the first time, that integrin alpha IIb beta 3 is not confined to megakaryocyte lineage cells.

Subpopulations of B16 amelanotic melanoma (B16a) cells, isolated by centrifugal elutriation from enzymatically dispersed solid tumors, demonstrated different abilities to form lung colonies when injected intravenously. In contrast, no differences in experimental metastasis were observed among subpopulations obtained from Lewis lung (3LL) tumors. Lung colonization by B16a and 3LL subpopulations correlated positively with observed differences (B16a) or lack of differences (3LL) in tumor cell ability to induce aggregation of homologous platelets, to adhere to subendothelial matrix or fibronectin, and with the percentage of cells in the G2/M phase of the cell cycle. Both B16a and 3LL cells express alpha IIb beta 3 integrin receptors; however, differences in the receptor expression level were found only among B16a subpopulations. Comparison of the amount of alpha IIb beta 3 receptor expressed on cell surface with tumor cell ability to induce platelet aggregation (TCIPA) and to adhere to fibronectin or subendothelial matrix revealed a positive correlation. Pretreatment of tumor cells with alpha IIb beta 3-specific antibodies inhibited tumor cell matrix adhesion, TCIPA, and lung colony formation. We propose that alpha IIb beta 3 integrin receptor expression, tumor cell matrix adhesion, and tumor cell-induced platelet aggregation can be important parameters to indicate the metastatic potential of some tumor cells and that the alpha IIb beta 3 is a multifunctional receptor involved in both tumor cell-matrix and tumor cell-platelet interactions. Further, the correlation among cell cycle phase, metastatic ability, and receptor expression suggests that metastatic propensity may be transiently expressed and/or increased in some tumor cell subpopulations.

The dispersion of carotenoid droplets in permeabilized goldfish xanthophores is dependent on ATP, F-actin, and cytosol. We report here that the motor (ATPase, translocator) resides with the permeabilized cell remnants and not in the cytosol. We also report that the carotenoid droplets have an ATPase that is not conventional myosin, dynein, or an ion pump. Its activity appears to correlate with the actin content of the carotenoid droplet preparation. A carotenoid droplet protein of Mr 72,000 (p72) is shown to be labeled by irradiation with 8-azido-ATP with concomitant loss of ATPase activity of the carotenoid droplets. We propose that this protein may be the ATPase responsible for carotenoid droplet dispersion.

Fish chromatophores serve as excellent study models for cytoskeleton-dependent organelle translocations because the distribution of pigmentary organelles can be observed against a time frame by microscopy. In this study the distribution of microfilaments along with microtubules in cultured melanophores of the killifish (Fundulus heteroclitus Linneaus) are examined using whole-cell transmission electron microscopy (WCTEM), fluorescence, and laser scanning confocal microscopy. Dispersing, dispersed, aggregating and aggregated states of pigment are induced by adding either caffeine (for dispersion) or epinephrine (for aggregation) to the cells in a standard culture medium. The cells that exhibited a random melanosome distribution in the standard culture media without these two reagents, served as the control. The results indicate that: (i) a structure considered to be the actin-filament organizing center (AFOC) is in close proximity to the microtubule-organizing center (MTOC); (ii) the radial layout of microfilaments remains similar over four physiological states of pigmentary response with the exception of epinephrine-aggregated pigment, in which the aggregate blocks the viewing of the AFOC and central microfilament rays, yet radial microfilaments, whether central and/or peripheral, are apparent in all physiological states of distribution; and (iii) microfilaments serve, together with microtubules, as scaffolding for melanosomes which migrate in bi-directional rows on cross-bridges, thus shedding light on the mechanisms for orderly melanosome translocations in a structural continuum.

Actin is a major cytoskeletal protein which is involved in many physiological cellular functions such as motility, cell shape, and adhesion. Recently, actin has also been reported to be cleaved by apoptotic proteases (i.e., caspases) and this cleavage is thought to contribute to the apoptotic process. However, conflicting data also exists as to whether actin represents a true caspase substrate during apoptosis induction in vivo (i.e., inside the cells). In this study, we critically examined the actin cleavage patterns during apoptosis of several tumor cell lines derived from three different species (i.e., mouse, rat, and human). Our findings demonstrate that: 1) actin cleavage in vivo is not a common phenomenon since apoptosis caused by multiple inducers in most cell types examined occurs without evidence of actin degradation; and 2) in certain cell types (e.g., U937), spontaneous, actin cleavage is observed which is not prevented by various specific chemical/peptide inhibitors of proteases such as caspases or serine proteases although apoptosis per se is retarded by some of these inhibitors. Our results conclude that actin is not a critical substrate for apoptotic proteases in vivo during apoptosis.

Aoptosis, or programmed cell death, is a genetically encoded cell suicide program defined by characteristic morphologic, biochemical, and molecular changes resulting in nonpathologic cell loss. A large number of distinct cellular phenotypes set apoptosis apart from another cell death process, i.e., necrosis.1,2 Apoptosis plays a key role in physiological processes such as embryonic development, maturation of the host immune system, and in maintaining tissue and organ homeostasis. Apoptosis has also been implicated in a variety of pathological conditions exemplified by cardiac infarction, atherosclerosis, Alzeimer’s disease and other neurodegenic diseases, HIV, tumorigenesis and tumor progression. A multitude of factors have been implicated in regulating/modulating apoptosis; these include: (i) oncogenes/tumor suppressor genes exemplified by p53, bcl-2 family (bcl-2, bcl-XL, bcl-Xb, bcl-XS, bax, BAG-1, bad, bak, Al, Mcl-1), myc, ras, abl, raf, Rb-1, and Waf-1; (ii) growth factor/growth factor receptors represented by NGF/NGF receptor, TNF-α/Fas, TGF-β/TGF receptor, IGF-1/IGF receptor, and PDGF/PDGF receptor; (iii) intracellular signal transducers such as protein kinase C, tyrosine kinases and protein phosphatases, lipid signaling molecules such as ceramide, and Ca2+; (iv) cell cycle regulators exemplified by cdc-2 and E2F; (v) reactive oxygen species; (vi) extracellular matrix regulators/signal transducers (extracellular matrix proteins such as fibronectin and transmembrane integrin receptors); (vii) specific endonucleases such as Ca2+- and Mg2+-dependent DNase; and (viii) cytoplasmic proteases typified by ICE (interleukin 1-converting enzyme) family.1-10 The major impact of apoptosis on cancer research is manifested primarily in three areas: oncogenesis, tumor homeostasis, and the mechanism of action of cytotoxic antitumor drugs.11 Most anti-tumor agents such as radiation (by generating oxygen radicals) and chemotherapeutic drugs kill tumor cells by inducing apoptosis. Likewise, development by tumor cells of resistance to these treatments is mostly a result of loss of response to apoptosis induction.1-11

In metastatic B16a murine melanoma cells, alpha IIb beta 3 integrin was shown to be one of the key adhesion molecules responsible for matrix adhesion and spreading. Upon stimulation, alpha IIb beta 3 can be upregulated at the cell surface due to translocation of the receptor to the plasma membrane from an intracellular pool. Here we have characterized this integrin pool as a tubulovesicular structure (TVS) corresponding to endosomes. TVS was found to be associated temporarily with microtubules and intermediate filaments especially after protein kinase C (PKC) stimulation with a lipoxygenase metabolite of arachidonic acid, 12-(S)-hydroxyeicosatetraenoic acid [12-(S)-HETE]. After PKC stimulation, the predominantly vesicular TVS became elongated and alpha IIb beta 3 appeared at the apical plasma membrane and microvilli. Disruption of either the microtubules or intermediate filaments prevented the 12-(S)-HETE effect both on vesicular to tubular transition of TVS as well as on surface expression of this integrin. The connection with the Golgi system of the integrin-containing TVS was proved by a Golgi-inhibitor (brefeldin A) pretreatment, which prevented the PKC-stimulation-induced TVS elongation and subsequent receptor-upregulation at the cell surface. After a soluble ligand binding (mAb to the alpha IIb beta 3 complex) the surface receptor endocytosed back to the TVS indicating the presence of a dynamic, cytoskeleton associated integrin pool in melanoma cells.

Lipoxygenase metabolites of arachidonic acid (AA) are responsible for alterations in membrane receptor expression, regulated secretion of cellular products as well as in cell motility (1). These alterations involve extensive cytoskeletal rearrangements as translocations of cytoplasmic proteins stored in transport-organelles are mediated by microtubules or microfilaments (2,3). Intermediate filaments are considered as structural components of the cytoskeleton without active involvement in cytoskeletal rearrangement (4). The levels of phosphorylation of individual cytoskeletal proteins affect their conformation and interaction with other cytoskeletal proteins (s). Therefore kinases (PTK, PKC, PKA or MLCK) play an important role. 12-(S)-HETE was shown to induce cytoskeleton-dependent integrin upregulation in tumor cells (6,7) resulting in an increased matrix adhesion as well as in endothelial cell retraction (8). More recently it was reported that 12-(S)-HETE induces PKC activation and translocation to the plasma membrane (9). Here we provide morphological and biochemical evidences for the effect of 12-(S)-HETE on the tumor cell cytoskeleton.

The fatty acid 12(S)-HETE may be a new second messenger capable of activating PKC. In tumor cells 12(S)-HETE stimulates cytoskeleton-dependent cellular responses such as adhesion and spreading. Analysis of 12(S)-HETE effects on B16a melanoma cell cytoskeleton revealed reversible rearrangement of microtubules, microfilaments, the actin-binding proteins, vinculin, myosin heavy (MHC) and light chains (MLC), as well as bundling of vimentin intermediate filaments. The alterations in microfilaments and intermediate filaments occurred very rapidly, i.e., 5 min after exposure of tumor cells to 12(S)-HETE. The 12(S)-HETE-induced cytoskeletal alterations were accompanied by centrifugal organelle-translocation. Interestingly, MLC exhibited clear association with the cytoplasmic organelles. Biochemical analysis of the 12(S)-HETE effect indicated a PKC-mediated reversible hyperphosphorylation of MLC, vimentin, and a 130 kD cytoskeletal-associated protein. Optimal effects were obtained after 5 min treatment with 12(S)-HETE at 0.1 μM concentration. 12(S)-HETE pretreatment induced tumor cell spreading on a fibronectin matrix which required the intactness of all three major cytoskeletal components. The spreading process was dependent upon the activity of PKC. Our data suggest that 12(S)-HETE is a physiological stimulant of PKC. Further, it induces rearrangement of the cytoskeleton of tumor cells in interphase resulting in the stimulation of cytoskeleton-dependent cell activity such as spreading. © 1993 Wiley-Liss, Inc.

Tumor cell — extracellular matrix interactions consist of attachment, spreading, migration and digestion of matrix ligands. These interactions are critical determinants during metastasis (1,2) and are mediated by receptors consisting of integrins, cell adhesion molecules (CAM) and proteoglycans (3,4). The first step in matrix interaction is a passive phase characterized by initial ligand/receptor interactions, while the second step, the spreading process, is more complex and includes redistribution of matrix receptors, activation of the signal transduction pathways and rearrangement of the cytoskeleton (5). These events are prerequisites for subsequent tumor cell migration and matrix lysis. The regulatory mechanism(s) of cell spreading is not known. However, those mechanisms which control matrix receptor expression, e.g., TGFβ and other cytokines (i.g., IL-I, TNF) may be involved (6). Previous studies indicated that a 12-LOX metabolite of arachidonic acid (AA), 12-(S)-HETE, serves as a positive regulator of integrin αllbβ3 expression and function in murine tumor cells (7,8). However, it was not clear in these studies which phase (i.e., attachment or spreading) of tumor cell matrix interaction was affected by 12-(S)- HETE. Therefore we have analyzed the effects of 12-(S)-HETE on the spreading of tumor cells on a defined matrix protein, fibronectin (FN).

Using the substracted probe approach, we had previously isolated eight cDNAs whose corresponding RNAs are more abundant in pigmented (melanized) than in closely related unpigmented goldfish cell lines. We report here that two of these are of mitochondrial origin, suggesting that pigmentation is accompanied by higher content (activity) of mitochondria. We also present the complete nucleotide sequence of the full length cDNA for the large mitochondrial rRNA, showing the presence of a polyA tail, two polyadenylation signals and a long open reading frame potentially encoding for a polypeptide of 166 amino acids and with no known protein homologue. It is however unknown whether such a polypeptide is actually produced.

In 1925, Wilson listed, in his classic third edition of Cell in Development and Heredity, four theories for the morphological and physiological characteristics of cytoplasm; each theory provided some sort of explanation as to the mechanism(s) of organelle translocations. During the past twenty years, cell biologists have focused their attentions on the cell's cytoskeleton, microtrabecular lattice, and associated mechanochemical motors which drive organelles along cytoskeletal tracks. A number of cell types have been used to study organelle translocations, but chromatophores, pigment cells, from cold-blooded vertebrates have been one of the more popular models. This article reviews some of the research findings during the past twenty years, particularly those involving cytoplasmic elements: i.e, microfilaments, intermediate filaments, microtubules, and mechanochemical motors. In addition, it contrasts the proposed involvement of these elements in organelle translocations with the endoplasmic reticulum, a tubulovesicular organelle, which we recently demonstrated is responsible, through its elongation or retraction, for the translocations of carotenoid droplets in goldfish xanthophores and swordtail fish erythrophores. Here, the carotenoid droplets are not free in the cytoplasm and do not translocate via cytoskeletal tracks, but instead are attached to or are a part of the endoplasmic reticulum. On the other hand, carotenoid droplets of squirrel fish erythrophores are free in the cytoplasm and appear to translocate via microtubules. Finally, the rates of pigmentary organelle translocations are reviewed in light of the participation of the cytoskeletal elements with the endoplasmic reticulum.

Integrin receptors are mediators of cell-extracellular matrix and cell-cell interactions. Biochemical and immunocytochemical evidence shows that the platelet integrin receptor alpha IIb beta 3 is present on the cell surface, at focal adhesion plaques and in the perinuclear region of metastatic B16a murine melanoma cells. Antibody to the fibronectin receptor alpha 5 beta i, inhibits basal adhesion by approx. 30%, whereas antibodies to alpha IIb beta 3 are ineffective. The surface immunoreactivity of tumor cells for alpha IIb beta 3 can be enhanced by pre-treatment (5 min) with a lipoxygenase metabolite of arachidonic acid [i.e. 12-(S)-HETE] in a dose-dependent manner (max. effect approx. 0.1 microM). Other lipoxygenase metabolites are ineffective. B16a cells possess a large intracellular pool of alpha IIb beta 3, from which the receptor complex translocates to the cell surface following 12-(S)-HETE pretreatment. This pre-treatment of tumor cells enhances their adhesion to fibronectin, which is mediated exclusively by alpha IIb beta 3 receptors. 12-(S)-HETE also facilitates the redistribution of alpha IIb beta 3 in the plasma membrane with localization at the focal adhesion plaques. The cytoskeleton of the B16a cell is characterized by an absence of distinct microtubules in interphase cells and the presence of prominent microfilaments and vimentin intermediate filaments. In B16a cells, the disruption of intermediate filaments and/or microfilaments prevents the 12-(S)-HETE-induced increase in plasma membrane alpha IIb beta 3 and enhanced tumor-cell adhesion to fibronectin. The microtubule-disrupting agent, colchicine, is ineffective in both respects. We conclude that the lipoxygenase metabolite of arachidonic acid, 12-(S)-HETE, regulates the surface expression and function of the alpha IIb beta 3 integrin in B16a cells. Further, these data support the hypothesis that microfilaments and intermediate filaments have a profound role in regulating the expression of a multifunctional integrin in B16a tumor cells.

Organelle translocations are essential cellular processes. Although much progress has been made with regards to microtubule-dependent organelle translocations, little is known about actin-dependent organelle translocation(s) except cytoplasmic streaming in Nitella. On the other hand, there is indirect evidence that actin-dependent organelle translocation may be involved in secretion. We now present evidence that the dispersion of the pigment organelles carotenoid droplets in goldfish xanthophores is apparently actin dependent and that this process may be related to secretory processes. We show that, in digitonin-permeabilized goldfish xanthophores, the pigment organelles can be induced to disperse by a combination of cAMP, ATP, and xanthophore cytosol. This induced dispersion is inhibited by DNase I, phalloidin, or anti-actin, but not by anti-tubulin or anti-intermediate filament proteins, suggesting a dependence on F-actin. Since the dispersion of carotenoid droplets and secretion both involve outward translocation of organelles, we tested the possibility that cytosols of secretory tissues have similar activity. Such activity was indeed found in different tissues, apparently in parallel with the secretory activity of the tissues, suggesting that pigment dispersion in xanthophores and some secretory processes may share a common component.

Treatment of goldfish xanthophores with adrenocorticotropin (ACTH) or cyclic AMP (cAMP) induces the centrifugal movement of their pigment organelles from the center of the cells. Using purified xanthophores pulse labeled with 32Pi, we have shown that the dispersion of the organelles is accompanied by the phosphorylation of a pair of polypeptides, termed p57. After fractionation on sucrose gradients, nearly all of the p57 is found associated with the pigment organelles. The phosphorylation induced by ACTH or cAMP apparently occurs at multiple sites on p57. The minimal effective doses of ACTH or cAMP required to induce full pigment dispersion also fully stimulate the phosphorylation of p57. Increased phosphorylation of p57 is detectable within a minute after stimulating the cells and appears to be near completion during the early phases of pigment dispersion. Upon withdrawal of ACTH, these events are reversed; the pigment organelles reaggregate toward the center of the cells and p57 is dephosphorylated. Again, dephosphorylation commences soon after ACTH is withdrawn and is complete before the organelles have completely reaggregated. These results suggest a novel mechanism for governing the movement of these organelles which acts on the organelles themselves through the phosphorylation and dephosphorylation of p57.

The objectives of this paper are to describe 1) the development of new research systems for biochemical comparison of cellular traits between normal and tumorous pigment cells of fish origin, 2) the similarity and dissimilarity between these two categories of pigment cells with regard to growth, differentiation and pigment translocating activities and 3) the potentials of these tumorous pigment cells to manifest multiple differentiation. The development of research systems has been achieved by the establishment of 1) methods to obtain homogenous populations of viable, cultivatable xanthophores (erythrophores) and melanophores from goldfish skins, 2) permanent cell lines from goldfish erythrophoromas (tumors derived from erythrophores) and from Nibe croaker irido-melanophoromas (tumors composed of mixed populations of iridophore- and melanophore-like cells) and 3) procedures to induce differentiation in normal and tumorous stem cells (including the formation of pigments and ability to undergo pigment translocation in response to cAMP, to the neurotransmitter epinephrine and to the hormones ACTH and melatonin). Two kinds of tumorous pigment cell lines examined herein have the ability to form, in addition to various pigments, structures similar to dermal skeletons and lentoid bodies. These findings strongly suggest the possibility that these fish pigment cell tumors are neural crest stem cell tumors in nature.

The fundamentally diverse vertebrate pigment cells, melanophores, xanthophores, and iridophores, contain pigmentary organelles known, respectively, as melanosomes, pterinosomes, and reflecting platelets. Their pigments are mealanins pteridines, and purines. Mosaic pigment cells containing more than one type of organelle have been observed and mosaic organelles containing more than one type of pigment have been discovered. It is proposed that the various pigment cells are derived from a stem cell that contains a primordial organelle of endoplasmic reticular origin. This primordial organelle can differentiate into any of the known pigmentary organelles.

Using a goldfish-derived melanized cell line, we attempted to determine the identity of the signal transduction system/second messenger for epinephrine-induced aggregation of melanosomes in a goldfish cell line. The results show that the second messenger is unknown. It is not 1) influx of extracellular calcium, 2) release of intracellular stored calcium via the phosphoinositide pathway, 3) cGMP, or 4) decrease of cAMP. These results suggest that there is an unknown second messenger for this activity of epinephrine.

A novel organelle movement is described in goldfish xanthophores. Smooth endoplasmic reticulum, containing carotenoids pigments, undergo MSH or c-AMP-induced dispersion. This dispersion is independent of RNA and protein synthesis, not modulated by c-GMP and inhibited by cytochalasin B but only at the relatively high concentration of 10 mug/ml. Epinephrine induces aggregation, a process that is inhibited by colchicine. These results suggest, but do not prove, the involvement of microfilaments in dispersion and microtubules in the aggregation of carotenoid-containing endoplasmic reticulum.

Triton-insoluble cytoskeleton of nonpigment cells has bound protein kinase that phosphorylates, with or without added cAMP, tubulins and the intermediate filament proteins p60, p56, p53, and p45a to give multiple charge variants. In the absence of 8-Br-cAMP, Triton-insoluble cytoskeletons from xanthophores also phosphorylate p60, p56, and p45a, but not p53; tubulin phosphorylation may also be reduced. In the presence of 8-Br-cAMP, p53, as well as several other peptides, are phosphorylated. One of these latter peptides was identified as the carotenoid droplet (pigment organelle) protein p57, whose phosphorylation and dephosphorylation precede pigment dispersion and aggregation respectively (Lynch et al.: J. Biol. Chem. 261:4204-4211, 1986). The amount of pp57 produced depends on the state of pigment distribution in the xanthophores used to prepare the cytoskeletons for labeling. With cytoskeletons from xanthophores with aggregated pigment, pp57 is a major labeled phosphoprotein seen in two-dimensional gels. With cytoskeletons prepared from xanthophores with dispersed pigment, the yield of labeled pp57 is greatly reduced (by at least 90%). Together with earlier results, we propose that, in the aggregated state, p57 serves to bind carotenoid droplets to the cytoskeletons, most likely the microtubules. The significance of other cAMP-dependent phosphorylation reactions is unknown but may be related to cAMP-induced cytoskeleton rearrangement in intact xanthophores.

Immunofluorescence and phase-contrast microscopic studies of goldfish xanthophores with aggregated or dispersed pigment show two unusual features. First, immunofluorescence studies with anti-actin show punctate structures instead of filaments. These punctate structures are unique for the xanthophores and are absent from both goldfish dermal non-pigment cells and a dedifferentiated cell line (GEM-81) derived from a goldfish xanthophore tumor. Comparison of immunofluorescence and phase-contrast microscopic images with electron microscopic images of thin sections and of Triton-insoluble cytoskeletons show that these punctate structures represent pterinosomes with radiating F-actin. The high local concentration of actin around the pterinosomes results in strong localized fluorescence such that, when the images have proper brightness for these structures, individual actin filaments elsewhere in the cell are too weak in their fluorescence to be visible in the micrographs. Second, whereas immunofluorescence images with anti-tubulin show typical patterns in xanthophores with either aggregated or dispersed pigment, namely, filaments radiating out from the microtubule organizing center, immunofluorescence images with anti-actin or with anti-intermediate filament proteins show different patterns in xanthophores with aggregated versus dispersed pigment. In cells with dispersed pigment, the punctate structures seen with anti-actin are relatively evenly distributed in the cytoplasm, and intermediate filaments appear usually as a dense perinuclear band and long filaments elsewhere in the cytoplasm. In cells with aggregated pigment, both intermediate filaments and pterinosomes with associated actin are largely excluded from the space occupied by the pigment aggregate, and the band of intermediate filaments surrounds not only the nucleus but also the pigment aggregate. The patterns of distribution of the different cytoskeleton components, together with previous results from this laboratory, indicate that formation of the pigment aggregate depends at least in part on the interaction between pigment organelles and microtubules. The possibility that intermediate filaments may play a role in the formation/stabilization of the pigment aggregate is discussed.

In intact goldfish xanthophores, the phosphorylation of a pigment organelle (carotenoid droplet) protein, p57, appears to play an important role in adrenocorticotropin (ACTH)- or cAMP-induced pigment organelle dispersion while the dephosphorylation of this protein upon withdrawal of ACTH or cAMP is implicated in pigment aggregation. In this paper, we report the cAMP-dependent phosphorylation of this protein in cell-free extracts of xanthophores as determined by the incorporation of 32P from [gamma-32P]ATP. As is the case in intact cells, p57 is the predominant protein phosphorylated in the presence of cAMP. The cAMP-dependent protein kinase which phosphorylates p57 is not bound to the isolated organelles but is found in the soluble portion of the cell extracts. Hence, the phosphorylation of p57 requires the carotenoid droplets bearing the substrate, soluble extract containing the kinase, cAMP (half-maximal activation at 0.5 microM), and Mg2+ (optimal at 5 mM or higher). The presence of protein phosphatase(s) in these extracts was shown indirectly by the stimulation of phosphorylation by fluoride. The phosphorylation of p57 does not appear to require a cell-specific kinase as soluble extracts of goldfish dermal nonpigment cells also phosphorylate p57 associated with isolated carotenoid droplets. Furthermore, using a constant amount of carotenoid droplets, a linear relationship was demonstrated between the rate of p57 phosphorylation and the amount of extract present in the assays. These results suggest that p57 is phosphorylated directly by a cAMP-dependent protein kinase and that the activity of this enzyme is important in regulating the intracellular movement of the pigment organelles of the xanthophore.

Cell lines with stable melanized phenotypes were isolated from a goldfish erythrophoroma cell line. These cell lines include several interesting phenotypes: a) reversible dedifferentiation and redifferentiation in response to withdrawal and addition of fish serum; b) irreversible dedifferentiation in response to withdrawal of fish serum; c) independence on fish serum for melanization; d) dependence on fish serum for growth; and e) pigment aggregation in response to epinephrine. We also report that cryopreservation of all the melanized cell lines, but not any of the unmelanized cell lines, requires the presence of fish serum. This raises the possibility that there may be advantages to use autologous serum for the cryopreservation of sensitive cell lines.

Two key modifications of the previously reported method for isolation of goldfish xanthophores allowed the isolation and establishment of primary cultures of terminally differentiated melanophores from the Black Moor goldfish (Carassius auratus). First, pretreatment with 10(-4) M epinephrine causing aggregation of the melanosomes and collapse of the dendrites, prevents damage to the melanophores during tissue dissociation and melanophore isolation. Second, maintenance of these cells in culture was successful only when the culture medium was supplemented with fish serum. The purified melanophores attached, flattened, and were maintained in culture for up to 3 mo. Although the morphology of the cultured melanophores is less dendritic than their in vivo counterparts, the melanophores translocate melanosomes in a normal manner except that they exhibit enhanced sensitivity to epinephrine. This epinephrine-induced pigment aggregation, as well as the redispersion of pigment after the removal of epinephrine, can occur in the presence of ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and absence of Ca2+.

Walker 256 carcinosarcoma cells induce the aggregation of rat platelets and concomitant production of eicosanoid metabolites (e.g., 12-hydroxyeicosatetraenoic acid, thromboxane A2). Cyclooxygenase inhibitors, but not lipoxygenase inhibitors, were able to inhibit platelet aggregation induced in vitro by low concentrations of agonists. At high agonist concentrations, neither cyclooxygenase nor lipoxygenase inhibitors affected platelet aggregation; however the combination of both inhibitors resulted in inhibition of aggregation. Also, a low concentration of agonist induced minimal eicosanoid metabolism, whereas a high concentration resulted in increased eicosanoid metabolism. These inhibitors, at the doses tested, did not inhibit protein kinase C activity.

Hormone-induced pigment translocation studies were conducted at both the light and electron microscopic levels on cultured dermal iridophores from the Mexican leaf frog, Pachymedusa dacnicolor. Two distinct types of dermal iridophores were characterized which differed in (1) their in vivo locations, (2) their overall morphologies in vitro, (3) their responses to alpha-MSH, ACTH, c-AMP or theophylline, (4) their physical alterations of light, and (5) certain ultrastructural features. One iridophore (Type I) was found to be physiologically responsive to the above hormones or agents by a reversible retraction of cellular processes and a thickening of the cell body, an event which is inhibited by cytochalasin B. The other iridophore (Type II) appeared to be unresponsive. Type I iridophores contain cube-like pigmentary organelles, refractosomes, while Type II iridophores contain larger, bar-shaped refractosomes. In addition, both iridophore types contain 60 and 100 A microfilaments as well as microtubules. By in large, micorfilaments were found within microvilli, beneath and parallel to the plasma membrane and in the perinuclear region. Occasionally, bundles of 100 A microfilaments were found between layers of refractosomes in Type I iridophores. These results are discussed in relation to hormone-induced changes in cell shape.

Previous experiments have shown that treatment of the melanophores of Pachymedusa (Agalychnis) dacnicolor with Melanophore-Stimulating Hormone (MSH) + puromycin causes the nuclear envelope to breakdown leading to the formation of discontinuous vesicles and the hyperdispersion of chromatin. We show here that these cells recover, reform their nuclear envelopes, and recondense their chromatin, both in the presence and in the absence of actinomycin D (actD). After recovery, these cells respond to MSH by melanosome dispersion. From these results, the following conclusions or observations are drawn: 1. 1, Reformation of nuclear envelope does not require that the chromatin be condensed into chromosomes as in mitosis. 2. 2, The new nuclear envelope is derived primarily from reutilization of the membrane vesicles produced during nuclear envelope breakdown, somewhat similar to mitosis. There may also be contributions from other membranous organelles. 3. 3, The hyperdispersed chromatin appears not to be subject to extensive attack by endogenous nucleases as the recovered cells are of good ultrastructure and can respond tropically to MSH. 4. 4, The presence of actD appears not to prevent the conversion of the hyperdispersed chromatin into the normal pattern.

A method is described for the subcellular fractionation of goldfish xanthophores. The procedure produces relatively pure fractions of caroteniod droplets, pterinosomes, cytosol and what appears to be plasma membrane. The presence of a distinct pattern of proteins is shown to be associated with the carotenoid droplets. Treatment of the xanthophores with ACTH affects the buoyant density of some carotenoid droplets and stimulates the phosphorylation of a polypeptide associated with the carotenoid droplets.

Evidence is presented for the following hypothesis dealing with dibutyryl-c-AMP induction of neural differentiation in Pleurodeles waltlii embryos: (1) Dibutyryl-c-AMP induces the determination for neural differentiation. (2) Additional dibutyryl-c-AMP is not needed for cytodifferentiation. (3) Cytodifferentiation of induced cells can only take place after morphogenetic movements (in vivo) or extensive cell migrations (in vitro). It is suggested that cell migrations, both in vivo and in vitro, lead to the separation of the induced cells from one another or from unidentified inhibitory cells.

The ACTH-induced pigment dispersion in primary cultures and in suspensions of xanthophores of the goldfish, Carassius auratus L., has been shown to include the formation of rosette-like membranous structures and plasma membrane invaginations (pits). The rosettes and pits are probably similar structures sectioned at different angles. Horseradish peroxidase studies demonstrate that these structures are eventually converted into small spherical vesicles and long smooth elements, similar in appearance to spherical and “tubular” endoplasmic reticulum, respectively. In addition, these studies show that once vesicles are formed they are no longer continuous to the outside of the cell.

Rapid color changes of amphibians are mediated by three types of dermal chromatophores, xanthophores, iridophores, and melanophores, which comprise a morphologically and physiologically distinct structure, the dermal chromatophore unit. Xanthophores, the outermost element, are located immediately below the basal lamella. Iridophores, containing light-reflecting organelles, are found just beneath the xanthophores. Under each iridophore is found a melanophore from which processes extend upward around the iridophore. Finger-like structures project from these processes and occupy fixed spaces between the xanthophores and iridophores. When a frog darkens, melanosomes move upward from the body of the melanophore to fill the fingers which then obscure the overlying iridophore. Rapid blanching is accomplished by the evacuation of melanosomes from these fingers. Pale coloration ranging from tan to green is provided by the overlying xanthophores and iridophores. Details of chromatophore structure are presented, and the nature of the intimate contact between the chromatophore types is discussed.

Bright integumental pigmentation of amphibians is imparted by the action of reflecting pigment cells (iridophores), yellow pigment cells (xanthophores), or red pigment cells (erythrophores). Iridophores are sensitive to intermedin and respond by concentrating their pigment. Common features of the intermedin molcule are involved in the stimulation of both iridophores and melanophores. The purines, guanine, hypoxanthine, and adenine are the primary iridophore pigments and are found in discrete organelles, the reflecting platelets. Intermedin markedly affects the form of reflecting platelets as well as the amounts of purine they contain. Xanthophores and erythrophores contain pteridine and carotenoid pigments. Pteridines are found within an organelle, the pterinosome, a spheroid approximately 500 mμ in diameter, formed by a series of concentric lamellae. Carotenoids appear to be contained in droplets interspersed between the pterinosomes. Migration of pigment within xanthophores is not generally controlled by intermedin although such may be the case in adult Hyla arenicolor . The carotenoid content of skin is higher in normal larvae than in hypophysioprivic larvae. Pteridine content of xanthophores is much higher in normal larvae than in hypophysioprivic larvae. Pteridine content of xanthophores is much higher in normal larvae than in hypophysioprivic animals. In the latter, administration of intermedin elevates pteridine levels. Profound changes in pteridine content occur at metamorphosis under the direct control of thyroxine. Adult amphibians which can change their colors rapidly, do so by means of a group of dermal chromatophores constituting the “dermal chromatophore unit”. Cytological and endocrinological relationships of the unit are discussed in terms of color change mechanisms.

Using the conditions for vimentin filament recycling, intermediate filaments (approximately 10 nm) were prepared from the cytoskeleton of a goldfish tumor cell line (erythrophoroma or xanthophoroma). 2-D analysis showed unusual protein composition, with four proteins of molecular weights of 60, 45, 56 and 51 kilodaltons in ratios of approximately 4:4:1:1. These correspond to four of the major cytoskeletal proteins of both the tumor cells and normal xanthophores.

Light and electron microscopic examination of the nurse shark adrenocortical gland revealed the presence of regional cellular ultrastructural differences; three zones are present. The zona germinativa externa is characterized by cells with specific mitochondrial and vesicular types and the lack of lipid droplets; all cells display intense basophilia. The zona progressa intermedia is characterized by cells with membrane specializations, lipid droplets, and specific mitochondrial and vesicular types, and some cellular degeneration; all cells display intense basophilia. The zona terminata centralis is characterized by cells with specific mitochondrial and vesicular types, lipid droplets, and extensive cellular degeneration; all cells display weak basophilia. Squaliform adrenocortical cells possess several ultrastructural details common to other vertebrate adrenocortical cells. However, differences including intercellular microvillar and ciliar connections and intercellular junctional complexes are present. In addition, the presence of cell debris in sinusoids and zonal limitations in regard to cellular degeneration suggests an active holocrine secretory mechanism.

The hormone-induced pigment dispersion in primary cultures of xanthophores of goldfish (Carassius auratus L.) has been shown to involve the dispersion of not only carotenoid droplets but also of smooth endoplasmic reticulum. The dispersion of these organelles is inhibited by cytochalasin B and is accompanied by thinning of the cell body, thickening of the processes, and also overall changes in cellular morphology (process extension) under certain conditions. Electron microscopic examination of heavy meromyosin treated glycerinated xanthophores in scales revealed the presence of actin filaments in these cells.

We have previously shown that the dispersion and aggregation of carotenoid droplets in goldfish xanthophores are regulated, respectively, by phosphorylation and dephosphorylation of a carotenoid droplet protein p57. There is a basal level of p57 phosphorylation of p57 in unstimulated cells, which is greatly stimulated by adrenocorticotropic hormone (ACTH) or cyclic adenosine monophosphate (cAMP) acting via cAMP-dependent protein kinase. We have also observed that, in permeabilized xanthophores, pigment dispersion can be induced when cAMP is replaced by fluoride. Since p57 has multiple phosphorylation sites, there is the question of whether all p57 phosphorylation is by cAMP-dependent protein kinase or whether phosphorylation by cAMP-independent protein kinase coupled with inhibition of phosphatase activity by fluoride can replace cAMP-dependent protein kinase and that the ability of fluoride to replace cAMP for pigment dispersion in permeabilized cells is probably due to activation of adenylcyclase. We also show that ACTH causes an approximately threefold increase in the level of cAMP in these cells.

We reported previously that the dispersion of carotenoid droplets in permeabilized xanthophores requires cAMP, ATP, and a cytosolic factor present in several secretory tissues as well as in xanthophores. We have now purified this factor from beef liver to apparent/near homogeneity. It appears to be a heterodimer with Mr approximately 125,000. The purified factor has little or no ATPase activity, with or without the presence of actin. Nor does it stimulate the ATPase activity of carotenoid droplets. Its exact function in carotenoid droplet dispersion is thus unclear. Since dispersion of carotenoid droplets is an anterograde translocation, we propose the name anterogin for this protein. We also report that yeast cytosol has anterogin activity.

The movement of calcium ions across cellular membranes (e. g., plasma membrane, endoplasmic or sarcoplasmic reticulum) can serve as a molecular messenger that modulates biochemical processes appropriate to the specific cell type. Calcium antagonists by preventing release of Ca2+ from intracellular stores, by peventing influx of external Ca2+ or by inhibiting the effects of Ca2+ through interactions with Ca2+-binding proteins such as calmodulin can interfere with these biochemical functions. The primary action of one subgroup of calcium antagonists is to interact with the channels in the cell membrane through which calcium enters the cells. This class of compounds originally described by Fleckenstein has been called by a variety of names including Ca2+-channel blockers, Ca2+-entry blockers, Ca2+-channel inhibitors and slow-channel blockers (for review see 1–3). We will use the terminology calcium channel blockers (CCB) in this chapter.

Aggregation of rat platelets was induced in vitro by homologous rat Walker 256 carcinosarcoma cells and the extent of tumor cell-platelet interactions examined ultrastructurally. By 30s there was surface contact between unstimulated platelets and tumor cell microvilli. By midphase aggregation (2-3 min) tumor cells became enmeshed within expanding platelet aggregates. Tumor cell microvilli and platelet pseudopodia interdigitated as aggregation progressed. During the later stages of aggregation (6-10 min) tumor cells formed large processes which penetrated deep into the aggregate. Platelet activation (i.e. degranulation) occurred in gradient fashion and was concentrated near tumor cell membrane sites involved in process formation. At these later stages tumor cells near the aggregate periphery were found to have engulfed platelets or platelet fragments. Tumor cell-platelet interactions in the pulmonary microvasculature were also studied in vivo following injection of murine Lewis lung carcinoma, 16C mammary adenocarcinoma, and B16 amelanotic melanoma tumor cells into the tail vein. Platelets demonstrated a biphasic association with arrested tumor cells with peak interactions occurring at 10-30 min and 4-24 h. Ultrastructurally, tumor cells exhibited newly formed processes which interdigitated with the platelet aggregate. Such processes formed only in areas of contact with platelets and not in areas of contact with endothelial cells or other blood elements (i.e. erythrocytes, polymorphonuclear leukocytes). Numerous tumor cell mitochondria were concentrated in the areas of greatest platelet-tumor cell process activity. At early time intervals (2-10 min), intravascular platelet degranulation was observed primarily in platelets associated with tumor cell processes. Tumor cells also were found to have engulfed platelet fragments in vivo.