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Biomedical subjects

E L Cooper

Publications and source records attributed to E L Cooper.

At least 37 records · Page 2Linked to original sources
PubMed1997

Structure and differential target sensitivity of the stimulable cytotoxic complex from hemolymph of the Mediterranean mussel Mytilus galloprovincialis.

A cytotoxic protein complex of 320 kDA was isolated from dialyzed plasma of the edible mussel, Mytilus galloprovincialis. Constituted by the assembly of several different proteins, the complex exhibits selective killing against eukaryotic cells, including erythrocytes, mouse tumor cells and protozoan parasites. High variability, which was not correlated with protein concentration, suggested that the immune response of naive mussels was in various stages of activation. Stimulation assays by different treatments in vivo resulted in significant increases in the activity of the plasma suggesting that cytotoxic complexes are involved in immune defense. Lytic activity appears to involve binding of cytotoxic complexes onto target cell membranes and the formation of transmembrane pores. This research provides more evidence that the innate immune system of invertebrates involves large cytotoxic proteins with a broad range of recognitive specificities in addition to small antibacterial, antifungal peptides.

Animals↗
PubMed1997

Clavanins, alpha-helical antimicrobial peptides from tunicate hemocytes.

Hemocytes from the invertebrate Styela clava, a solitary tunicate, contained a family of four alpha-helical antimicrobial peptides that were purified, sequenced and named clavanins A, B, C and D. Each clavanin contained 23 amino acid residues and was C-terminally amidated. The tunicate peptides resembled magainins in size, primary sequence and antibacterial activity. Synthetic clavanin A was prepared and displayed comparable antimicrobial activity to magainins and cecropins. The presence of alpha-helical antimicrobial peptides in the hemocytes of a urochordate suggests that such peptides are primeval effectors of innate immunity in the vertebrate lineage.

Amino Acid Sequence↗
PubMed1996

A urochordate putative homolog of human EB1, the protein which binds APC1.

The human EBI protein has been cloned by virtue of its interaction with the C-terminus of the APC (adenomatous polyposis coli) protein, whose C-terminal truncated forms have been shown to accompany sporadic and familial forms of colorectal cancer. We have cloned a putative EBI homolog from Botryllus schlosseri (Urochordata. Ascidiacea). The deduced protein is 287 amino acids long, and is identical with 48% of the residues in human EBI and 24-25% in two yeast hypothetical proteins. We propose that such a high degree of conservation among EBI homologs is indicative of an essential regulatory mechanism in eukaryotic cells.

Amino Acid Sequence↗
PubMed1996

Earthworm leukocytes that are not phagocytic and cross-react with several human epitopes can kill human tumor cell lines.

Earthworm coelomocytes (leukocytes) effect cytotoxicity at significantly high levels against the NK-sensitive, human tumor cell line, K562, and the NK-resistant targets (U937, BSM, CEM). By cytofluorimetric analyses using mouse anti-human monoclonal antibodies and by morphological evaluations, two types of coelomocytes were identified: (1) small (8-11 micron) electron-dense cells (SC): CD11a+, CD45RA+, CD45RO+, CDw49b+, CD54+, beta 2-m+ and Thy-1+; (2) large (12-15 micron) electron-lucent cells (LC) that are negative for these markers. Both cell types were negative for other CD and MHC class I and class II markers. SC were active during recognition, rapidly binding to targets; LC were phagocytic. Release of 51Cr revealed rapid, significant, and equal levels of killing at 4 degrees, 20 degrees, and 37 degrees C. We propose that primitive NK-like activity appeared early in evolution.

Animals↗
PubMed1996

Earthworm coelomocytes in vitro: cellular features and "granuloma" formation during cytotoxic activity against the mammalian tumor cell target K562.

Earthworms possess specific, adaptive, cellular immunodefense as well as non-specific responses found in other complex metazoans. Here we characterized coelomocytes from the earthworm Eisenia foetida by electron microscopy and cytofluorimetric analyses, and investigated structural changes that occur when effector coelomocytes and target K562 erythromyeloid human tumor cells interact during cytotoxic activity. In in vitro cultures 1) the two earthworm cell types (i.e. small and large coelomocytes) retained their morphological features; 2) their DNA content was significantly less than that of human lymphocytes and the erythromyeloid human tumor cell line K562; 3) significant percentages of coelomocytes were found to be in S or G2/M phases of the cell cycle. When cultivated alone for up to 3 h, coelomocytes formed no aggregates. However, when mixed with K562, coelomocytes spontaneously killed tumor cells, and cytotoxic reactivity was accompanied by the formation of multiple aggregates similar to granulomas. These results are the first to describe this type of earthworm non-specific "inflammatory" response in vitro against tumor cells.

Animals↗
PubMed1995

Pilot study of labelling bovine muscle tissue with 14C.

A pilot study has been performed to assess the feasibility of labelling bovine muscle tissue with 14C, in order to produce material suitable for intercomparison exercises. Approximately 1 kg of tissue was prepared by grinding, blending, labelling with 14C-methylated bovine haemoglobin and thoroughly mixing by repeated blending. The product was sealed in cans and sterilized to preserve it. Portions of raw material were dried to test the homogeneity of moisture content. Homogeneity of 14C in the natural and labelled materials was tested by analyzing sub-samples of 2-3 g in weight of the freeze-dried materials. The 14C content was determined by combusting and collecting the CO2 for liquid scintillation counting. The material was found to be homogeneous to the extent of 2-4% (R.S.D.), which is comparable with the R.S.D. of the 14C measurements. The 14C concentration in the labelled material was 8190 +/- 220 Bq.kg-1 of carbon. It was concluded that bovine muscle prepared in this manner would be suitable for intercomparison exercises. The 14C level in unlabelled tissue was found to be 260 +/- 7 Bq.kg-1 of carbon, which is similar to the level of 14C previously determined in milk and both are consistent with levels measured in vegetation in Eastern Ontario.

Animals↗
PubMed1995

Mitochondrial mass and membrane potential in coelomocytes from the earthworm Eisenia foetida: studies with fluorescent probes in single intact cells.

Earthworm coelomocytes exist in two forms, i.e., small (SC) and large (LC) cells, as demonstrated by velocity sedimentation, electron microscopy, and FCM. However, we know little concerning the functional activities of various, important organelles, such as mitochondria. In comparison with SC, LC from Eisenia foetida have a higher number of mitochondria, and, accordingly, showed a greater fluorescence intensity when mitochondrial mass was measured by nonyl acridine orange and FCM. To measure MMP we used both the lipophilic cationic probe JC-1 and Rh123. The intracellular localization of JC-1 in SC and LC was observed by fluorescence microscopy. Using JC-1, MMP was analyzed separately on SC and LC by FCM, and significant percentages of coelomocytes (> 95% of SC and about 90% of LC) displayed a high MMP. Adding 0.1 microM VAL caused most SC to depolarize, while this occurred in only a few LC. Rh123 gave different results: no effects of VAL were observed either in SC or in LC. In coelomocytes there may be several energy-independent Rh123-binding sites whose role must still be elucidated. On the whole, these data indicate that it is possible to analyze mitochondrial parameters by FCM in intact invertebrate coelomocytes, and that the type of cell and the probe used have a critical importance.

Animals↗
PubMed1995

Autogeneic but not allogeneic earthworm effector coelomocytes kill the mammalian tumor cell target K562.

Earthworm coelomocytes have been used as effector cells against the human tumor target, K562. To first assess the viability of effectors, incorporation of [3H]-thymidine was tested and was higher in autogeneic (A<==A, self) than in allogeneic (A<==>B, nonself) coelomocytes. A<==>A showed significantly greater numbers in S, G2, or M phases than A<==>B coelomocytes. When A<==>A or A<==>B were cultured, no significant cell killing occurred in either, as measured in a 4-hr 51Cr release assay. A<==>A but not A<==>B killed K562 target cells. Cytotoxicity was dependent upon membrane binding between small, electron-dense coelomocytes and targets; it was enhanced by adding PHA. The heat labile supernatant from A<==>A but not from A<==>B killed K562 targets after cultivation for 10 min at 22 degrees C, but not immediately after washing. Recognition of, binding to, and killing of foreign cells in a natural killer cell-like reaction may reflect natural immunity in earthworms.

Animals↗
PubMed1995

PCBs increase molecular-related activities (lysozyme, antibacterial, hemolysis, proteases) but inhibit macrophage-related functions (phagocytosis, wound healing) in earthworms.

Both humoral and cellular immunodefense responses of the earthworms, Eisenia fetida andrei, Eisenia hortensis, and Lumbricus terrestris, have been compared after exposure to the PCB Aroclor 1254. Responses mediated by free factors, detected by in vitro assays for lysozyme, hemolysis, and proteases, were increased in both Eisenia. Antibacterial activity directed against pathogenic bacteria was increased in E.f. andrei. The resistance of L. terrestris against non-pathogenic bacteria was decreased, confirming that the bacteria were treated by different systems according to their pathogenicity. Nonspecific cellular functions, including phagocytosis and those related to wound healing, decreased dramatically in all earthworms.

Aeromonas hydrophila↗
PubMed1995

Killing of intrafamilial leukocytes by earthworm effector cells.

When Lumbricus and Eisenia coelomocytes are cultured together in intrafamilial xenogeneic combinations, significant cytotoxicity occurs at 24 h but not at 5 nor 72 h, as shown by trypan blue assay. In a 4.5-h assay, measuring 51Cr release, using an effector/target ratio of 25:1, unpooled cells from a single Lumbricus killed Eisenia cells at levels of 6% and 14%. However, Eisenia coelomocyte survival was high and identical in either cell-free xenogeneic (Lumbricus) coelomic fluid or in artificial medium. In this 1-way assay, earthworm (Lumbricus) coelomocytes act as effector cells that kill non-self target cells, even those of other earthworms. Comparisons with previous results reveal greater reliability and consistently repeatable results when the 51Cr release assay is used to measure cytotoxicity regardless of the targets.

Animals↗
PubMed1995

Tributyltin affects phagocytic activity of Ciona intestinalis hemocytes.

Organotin compounds have been used in marine anti-fouling paints as biocides. Because tunicates are vulnerable to these compounds in their natural habitats, we used Ciona intestinalis to establish an assay for phagocytosis in vitro of yeast by hemocytes after exposure to different concentrations (0.0015, 0.015, 0.15 and 1.5 microM) of four organotin compounds: tributyltin (TBT), triphenyltin (TPT), dibutyltin (DBT) and diphenyltin (DPT). To evaluate the phagocytic activity, we used a method based on fluorescence excitation of yeast pre-treated with eosin-Y. The percentage of phagocytosis decreased from 45.1 +/- 3.49 to 22.4 +/- 5.14 at 1.5 microM of TBT (P < 0.001); it was significantly reduced in presence of the ionophore A23187. TPT, DPT and DBT did not show significant effects on phagocytosis. Because the effect of TBT was irreversible, differences between the inhibitory mechanisms of ionophore and TBT are suggested. These results indicate that for future analyses, tunicates should become excellent models for dissecting events such as phagocytosis that are associated with immunosuppression after exposure to xenobiotics.

Animals↗
PubMed1995

Spontaneous cytotoxic earthworm leukocytes kill K562 tumor cells.

Earthworm coelomocytes may act as effector cells which destroy targets in vitro. In a 51Cr release assay, Lumbricus coelomocyte effectors showed lytic activities of 3-14% against K562 human tumor cells when incubated 1-4 hr at 23 degrees C or 37 degrees C. Cytotoxicity was correlated with effector: target ratio. However, targets were not killed by incubating them in cell-free, 0.2 micron filtered coelomic fluid. The supernatant from coelomocytes cultured alone failed to kill K562 targets but coelomocyte lysates were toxic to target cells in a concentration-dependent manner. Coelomocytes were examined using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). When effectors and targets were examined under TEM, we found close apposition of effector granulocytic coelomocytes and target cell membranes but not with coelomocytes nor eleocytes at up to 15 min incubation. By SEM, effector cells appeared not only to be in close contact with targets, but instances of target lysis were observed. These results suggest that effector cell/target cell contact is essential for cytotoxicity to occur.

Animals↗
PubMed1994

Invertebrates can tell us something about senescence.

Senescence is a ubiquitous phenomenon, i.e., all vertebrates and invertebrates will ultimately manifest it. Any attempt to answer the question of adaptive significance of the aging process must take into account the universality of and change in the DNA molecule that governs, integrates, regulates and ensures the vitality of all organisms. With invertebrates and from the comparative viewpoint, there are examples of: 1) rapid senescence and sudden death; 2) gradual senescence with definite life span; 3) negligible senescence; 4) genetic influence on life span, mortality rates, and age-related diseases. Although these characteristics are ascribed to invertebrates and vertebrates, this need not force upon invertebrates the organization, structure and eventual features of vertebrate senescence. "Invertebrate gerontologists" can thus, freely delve into certain unique aspects of what may be the more primitive mechanisms of aging in invertebrates. In contrast, using the opposite strategy that is still problematic, i.e., linking invertebrate and vertebrate aging, seems to give us an approach to universality that might eventually reveal more readily obvious and homologous kinship.

Aging↗
PubMed1993

Implementation of a radiochemical procedure for the analysis of isotopes of Pu, Am and Cm in food and environmental samples.

A radiochemical procedure for the analysis of isotopes of Pu, Am and Cm was implemented during an expert mission to Thailand sponsored by the International Atomic Energy Agency. This method was to be used for the analysis of food and environmental samples, particularly those of marine origin. The project was initiated by evaluating a procedure which had recently been published by the IAEA. This procedure could not handle the wide range of matrix materials used in the testing and a program was undertaken to develop a modified procedure. Plutonium was radiochemically separated using an anion exchange procedure which gives clean separations from the matrix. Americium and curium came off the column unseparated from most of the other elements in the matrix and they were separated by using a number of steps, which were selected to handle the specific chemical and radiochemical interferences in a particular matrix material. Sources were prepared for alpha spectrometry by coprecipitation with CeF3. The procedure was tested with a wide variety of sample types and good chemical yields and separations were obtained in most cases.

Americium↗
PubMed1993

Invertebrate cytokines. III: Invertebrate interleukin-1-like molecules stimulate phagocytosis by tunicate and echinoderm cells.

Phagocytosis is the predominant defense mechanism of invertebrates. Here we show that phagocytosis by echinoderm bladder amoebocytes and tunicate granular amoebocytes can be enhanced by invertebrate interleukin-1-like molecules. As little as 5 ng/ml of invertebrate interleukin-1 produced a significant stimulation of echinoderm and tunicate amoebocyte phagocytosis. Stimulation of phagocytosis by echinoderm interleukin-1-like molecules was inhibited by antisera to vertebrate interleukin-1. Invertebrate interleukin-1 also acted as an opsonin when preincubated with erythrocytes or yeast. In addition, the cellular mechanisms of invertebrate phagocytosis were studied using pharmacologic agents to inhibit echinoderm amoebocyte phagocytosis. The energy requirements and involvement of cellular cytoskeletal elements in phagocytosis by bladder amoebocytes were similar to those of mammalian macrophages. These results demonstrate a role for interleukin-1 in invertebrate host defense mechanisms.

Animals↗