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Biomedical subjects

A M Mastro

Publications and source records attributed to A M Mastro.

At least 73 records · Page 4Linked to original sources
PubMed1988

Comparative analysis of precipitating antibodies in White Rock and Fayoumi hens injected with bovine serum albumin or crude mite extract with resulting effects on northern fowl mite, Ornithonyssus sylviarum (Acari: Macronyssidae) population densities.

Precipitating antibody concentration responses to crude northern fowl mite extract (CME) and bovine serum albumin (BSA) injections were compared in White Rock and Fayoumi hens with two-dimensional immunoelectrophoresis and rocket electrophoresis. The effect of CME injections on northern fowl mite population development was also determined. White Rock and Fayoumi hens developed similar antibody concentrations in response to intramuscular injections of BSA according to serum samples analyzed with two-dimensional immunoelectrophoresis. Rocket electrophoresis analyses of pooled serum samples showed significant differences between slopes of White Rock and Fayoumi pools for CME and BSA injections, suggesting differences in antibody-antigen interactions. Fayoumi hens injected with CME, 78, 50, and 14 days prior to experimental infestation with 2,000 northern fowl mites/bird supported significantly fewer mites than BSA-injected hens, although mite populations were low on both treatment groups. Injections of CME had no effect on mite population development on White Rock hens, even though CME-specific antibodies were detected. Although White Rock hens supported significantly greater mite numbers than Fayoumi hens, the difference was not attributed to anti-CME antibody activity alone.

Animals↗
PubMed1987

Differentiation of peanut lectin positive suppressor T-cells from peanut lectin negative precursors in bovine cells by 12-O-tetradecanoylphorbol-13-acetate.

Although 12-O-tetradecanoylphorbol-13-acetate (TPA) can synergize with lectins to enhance lymphocyte proliferation, pretreatment of lymphocytes with TPA decreases their response. Pretreatment also inhibits the response to allogeneic cells in the mixed lymphocyte culture. In this study we determined that at least part of this inhibition was due to the generation of T-lymphocytes with the ability to suppress proliferation in vitro. By using populations enriched in lymphocytes or macrophages we determined that the interaction of TPA with lymphocytes, but not macrophages, was required to mediate the suppression. The number of macrophages present in culture (range, 0.5-10%) was irrelevant to the generation of inhibitory activity. Moreover, the TPA-induced suppressor activity copurified with T-cells. Furthermore, when peanut lectin (agglutinin) (PNA) was used to separate T-cells after treatment with TPA, essentially all of the activity copurified with the PNA positive cells. When PNA separations were carried out before treatment with TPA, the suppressor activity arose from the PNA negative fraction. Therefore, TPA appeared to cause phenotypically PNA negative T-cells to gain the PNA positive marker, as well as to function as suppressor cells in vitro. Suppressor activity was also found in the culture medium. Thus the suppression observed may be mediated through a soluble factor released by the TPA-treated cells. Although the suppressor cell activity induced by TPA can only partially account for its in vitro inhibition of lymphocyte proliferation, the development of suppressor cells merits further study with respect to lymphocyte phenotypic and functional differentiation. The results also suggest the possibility that similar processes could occur in vivo, possibly during the course of tumor promotion.

Animals↗
PubMed1987

Changes in protein kinase C and cAMP-dependent kinase in lymphocytes after treatment with 12-O-tetradecanoylphorbol-13-acetate or concanavalin A: quantitation of activities with an in situ gel assay.

Primary lymphocytes can be stimulated to proliferate by mitogenic lectins such as concanavalin A (Con A). While the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) alone is not mitogenic for these cells, it can enhance the response to Con A. Previously, protein kinases and phosphorylation have been reported to be important in lymphocyte proliferation. More recently TPA has been found to bind and activate protein kinase C. Therefore, we examined kinase activity in lymphocytes stimulated with the complete mitogen Con A and the comitogen TPA. In order to monitor more than one kinase we used an in situ gel assay and developed the system to compare both protein kinase C and cAMP-dependent kinases. When total cell extracts were assayed in the presence of histone five major bands of activity were detected by autoradiography of the gel. The bands corresponding to protein kinase C and to cAMP-dependent kinases were identified by partial purification of the enzymes, by binding of [20-3H(N)]7-phorbol-12, 13-dibutyrate (3H-PDBU), and by photoaffinity labelling with 8-azidoadenosine-3':5'-cyclic monophosphate (8-N3-[32P]cAMP). Differential extraction of cell lysate allowed comparison of soluble and particulate kinases. We found that when the preparations from either TPA- or Con A-treated lymphocytes were assayed, protein kinase C activity increased three- to four-fold in the particulate fraction within 5 min after treatment. A concurrent decrease of 30-50% occurred in the cytosol. In contrast, cytosolic cAMP-dependent protein kinase II increased 1.4-fold in the same period with Con A. PKI and PKII showed the most significant changes after 24 h of stimulation by Con A when the activity of the holoenzyme decreased to half that of the unstimulated cells. Therefore, although TPA and Con A separately can affect protein kinase C this alone is not sufficient for proliferation to occur.

Affinity Labels↗
PubMed1987

A microtiter plate assay for protein kinase C.

We report the development of a microtiter plate assay for protein kinase C. Reaction components and enzyme samples (protein kinase C purified by phosphatidylserine/cholesterol affinity or DEAE-Sephacel ion-exchange chromatography) were added to wells of a 96-well microtiter plate. The assay was started by the addition of [gamma-32P]ATP with a repeating pipet. After a 3-min incubation at 30 degrees C the wells were sampled six at a time with a 12-channel pipet and spotted onto phosphocellulose filter paper rectangles which were washed with tap water and acetone and counted for radioactivity. The microtiter plate method was more rapid than but gave results similar to those of a standard assay performed in plastic test tubes individually incubated in a 30 degrees C water bath. The microtiter plate procedure gave an intraassay (within one plate) variation of less than 9% and an interassay (between plates) variation of less than 5%. It was linear with time of incubation for 20 min and with amount of enzyme. This method can be used to expedite the assaying of column chromatography fractions for protein kinase C (and other kinase) activity.

Cholesterol↗
PubMed1987

Induction of suppressor activity by tumor-promoting phorbol esters in primary cultures of lymph node cells.

We have observed the induction of suppressor activity in primary cultures of lymphocytes by the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Suppressor activity was detected as the ability of TPA-treated lymphocytes to inhibit proliferation in mixed lymphocyte cultures (MLC). Induction of this activity was dependent on the dose of TPA and was maximal after approximately 12 h of incubation. Neither the induction of the activity, nor the activity itself was inhibited by indomethacin or interleukin 2 (IL2). A comparison of addition of TPA directly to the MLC, addition of TPA pretreated cells as participants in the MLC and addition of cells treated with TPA to induce suppressor activity suggested that the suppressor activity was only one of the ways that TPA could inhibit proliferation. This suppressor activity may account for some of the reported effects of TPA in immunological systems in vitro and suggests that suppressor cells could play a role in TPA-mediated promotion in vivo.

Animals↗
PubMed1986

DNA synthesis and production of interleukin 1 by lymph node macrophages in culture.

When bovine lymph node cells are cultured for several days the adherent macrophage population increases by as much as tenfold. This increase in cell number is primarily due to cell division, which reaches a maximum on day 4 or 5 of culture. Although the presence of the nonadherent cells seems required for cell division, we have been unable to detect a macrophage growth factor in either the nonadherent cell populations. The adherent cells were identified as macrophages based on positive esterase staining, the presence of Fc receptors, beta-glucuronidase activity, and phagocytosis. Moreover, these adherent cells produced interleukin 1 (IL1) after exposure to lipopolysaccharide in serum-free medium. Approximately 10(7) macrophages were stimulated to produce about 900 units of IL1 in a 24-hr period. Thus, the bovine lymph node preparation is a potential source of a large number of macrophages capable of dividing in culture and of producing IL1.

Animals↗
PubMed1986

Phorbol ester circumvents the need for macrophages as well as for mitogenic lectins in the stimulation of lymphocytes with wheat germ agglutinin or the calcium ionophores A23187 or ionomycin.

Numerous biochemical events precede the proliferation of primary lymphocytes stimulated by mitogenic lectins in the presence of macrophages. Various compounds can activate parts of this response. Specifically the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, (TPA), can replace the requirement for macrophages, apparently by mimicking the macrophage product interleukin 1 (IL1). Wheat germ agglutinin (WGA), itself a non-mitogenic lectin, is reported to cause a calcium flux, phosphatidylinositol turnover, and enhance interleukin 2 (IL2) synthesis. In spite of these positive responses, WGA inhibits DNA synthesis caused by mitogenic lectins. Nevertheless, in this study, we tested the possibility that together TPA and WGA could complement and bring about DNA synthesis. This prediction turned out to be true. The combination of two non-mitogenic compounds resulted in lymphocyte proliferation. The TPA overcame the inhibitory effects of WGA. Moreover, macrophages were not required. The TPA also synergized with the calcium ionophores A23187 or ionomycin to cause lymphocyte proliferation in the absence of macrophages. WGA and the ionophores together did not cause proliferation, a finding which suggested that they fulfill the same roles. These observations led us to conclude that at least two signals were required for lymphocyte stimulation. One signal caused the mobilization of calcium and the other signal circumvented the need for macrophages or macrophage products possibly by mimicking diacylglycerol, the activator of protein kinase C.

Animals↗
PubMed1986

Mitogenic activity of snake venom lectins.

Five lactose-inhibitable lectins have been isolated from snake venoms. These five share certain biochemical properties but are not identical (Gartner, Stocker & Williams, 1980; Gartner & Ogilvie, 1984). In this study the lectins were tested for their ability to stimulate lymphocytes to undergo DNA synthesis. We found that three of the lectins were comparable in mitogenic activity to the T cell lectin, concanavalin A (Con A). The mitogenic activity was blocked by lactose, a sugar which also blocks the haemagglutination activity of these lectins. Although mitogenic response appeared to be due to T cells, it depended on the presence of accessory cells in the culture. This requirement for macrophages could be replaced by the phorbol ester tumour promoter, 12-o-tetradecanoylphorbol-13-acetate (TPA).

Animals↗
PubMed1985

Mitogen and co-mitogen stimulation of lymphocytes inhibited by three Ca++ antagonists.

The Ca++ requirement for in vitro lymphocyte stimulation by lectins is well known and can be demonstrated by the use of Ca++ chelators. In this study, three Ca++ antagonists were examined for their effects on lymphocyte proliferation. [3H]-thymidine incorporation was employed to measure DNA synthesis in several systems. Stimulation and proliferation were achieved by the addition of one of the following: the mitogenic lectin concanavalin A (ConA); the combination of two co-mitogens, the calcium ionophore A23187 and the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA), neither of which is mitogenic alone; or the non-mitogenic lectin, wheat germ agglutinin (WGA) with TPA. These mitogenic systems were tested for their sensitivity to the Ca++ channel blockers verapamil and nicardipine and the intracellular Ca++ antagonist TMB-8. We found that the ConA and WGA plus TPA treated cells were inhibited approximately 50% by 10 microM verapamil, nicardipine or TMB-8. The stimulation caused by A23187 and TPA was only inhibited by TMB-8 and nicardipine. The inhibitory effects caused by the Ca++ antagonists could not be reversed by the addition of exogenous Ca++ (0.1-1.5 mM), but were reversed by repeated washings in antagonist free media. Using TMB-8 we saw an apparent intracellular Ca++ dependence throughout the G1 phase. Previous studies using Ca++ chelators or Ca++ antagonists suggested an endpoint at about halfway through this period.

Animals↗
PubMed1985

Changes in a T-cell subpopulation marker induced by tumor-promoting phorbol esters.

We observed a significant reduction in one specific T-cell rosetting marker after treatment of bovine lymph node lymphocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA). There was a dose-dependent reduction in the formation of neuraminidase-treated sheep erythrocyte rosettes (En), but not aminoethylisothiouronium bromide-treated sheep rosettes (Ea) after as little as 10 min of incubation with TPA. A maximum of approximately 50% reduction was reached after 1 h. When several phorbol esters and mezerien were tested, we found that the reduction in En rosetting induced by the compounds correlated with their in vivo tumor-promoting activity. The reduction in En rosetting appears to be approximately 50% reversible for up to 6 h of treatment with TPA when the cells were incubated in TPA-free medium for an additional 24 h, but it was irreversible after at least 12 h of treatment. Addition of the tumor promoters directly to the rosetting assay had no detectable effect on the sheep erythrocytes or the percentage of Ea rosettes. This specific change in En rosetting marker may represent the maturation of a T-cell subpopulation to T-suppressor cells in the presence of tumor-promoting phorbol esters.

Animals↗
PubMed1984

Visualization of protein kinases in lymphocytes stimulated to proliferate with concanavalin A or inhibited with a phorbol ester.

Stimulation of lymphocytes with a mitogenic lectin such as concanavalin A (ConA) results in differentiation and cell division. Among the changes which occur after stimulation are increases in phosphorylation of proteins and in protein kinase activity. We used a high-resolution, nondenaturing gel system to separate and visualize protein kinases in situ. We have clearly identified both autophosphorylating and substrate-dependent kinases. One band of cyclic AMP-dependent kinase activity was significantly enhanced in lectin-stimulated cells. In contrast, treatment of the cells with phorbol ester under conditions which depress stimulation caused a decrease in the activity of one kinase.

Animals↗
PubMed1984

Characterization of protein kinases in mitotic and meiotic cell extracts.

A number of protein kinases have been separated and identified in extracts from mitotic and interphase culture cells and from mature and immature amphibian oocytes using nondenaturing polyacrylamide gel electrophoresis followed by in situ phosphorylation assays. Certain of these protein kinase activities appear to correlate with the biological activity of extracts, assayed by their ability to induce meiotic maturation following injection into Xenopus oocytes. These results are consistent with the notion that protein phosphorylation/dephosphorylation may be integral to the mechanisms of both nuclear membrane breakdown and chromosome condensation, events common and distinctive to mitosis and meiosis.

Animals↗
PubMed1984

Diffusion of a small molecule in the cytoplasm of mammalian cells.

Electron spin resonance was used to measure the diffusion of a small (Mr 170) spin label in the aqueous cytoplasm of mammalian cells. Translational and rotational motion were determined from the same spectra. Based on measurements made in model systems, it was hypothesized that calculations of the apparent viscosity from either rotational or translational motion would distinguish between the effects of cytoplasmic viscosity or cytoplasmic structure on diffusion. The diffusion coefficient calculated from spin label collision frequency, averaged 3.3 X 10(-6) cm2/sec in several cell lines. It was greater in growing cells and in cells treated with cytochalasin B than in quiescent cells. The viscosity of the cytoplasm calculated from the translational diffusion coefficient or the rotational correlation time was 2.0-3.0 centipoise (1 P = 0.1 Pa X sec), about 2-3 times that of the spin label in water. Therefore, over the dimensions measured by the technique, 50-100 A, solvent viscosity appears to be the major determinant of particle movement in cells under physiological conditions. However, when cells were subjected to hypertonic conditions, the translational motion decreased by 67%, while the rotational motion changed less than 20%. These data suggested that the decrease in cell volume under hypertonic conditions was accompanied by an increase in cytoplasmic barriers and a decrease in the spacing between existing components. In addition, a comparison of reported values for diffusion of a variety of molecules in water and in cells indicates that cytoplasmic structure plays an important role in the diffusion of proteins such as bovine serum albumin.

Animals↗
PubMed1984

Diffusion in the aqueous compartment.

Measurements of diffusion of molecules in cells can provide information about cytoplasmic viscosity and structure. In a series of studies electron-spin resonance was used to measure the diffusion of a small spin label in the aqueous cytoplasm of mammalian cells. Translational and rotational motion were determined from the same spectra. Based on measurements made in model systems, it was hypothesized that calculations of the apparent viscosity of the cytoplasm from both rotational and translational motion would distinguish between the effects of viscosity and structure on diffusion. The diffusion constant measured in several cell lines averaged 3.3 X 10(-6) cm2/s. It was greater in growing cells and in cells treated with cytochalasin B than in quiescent cells. The viscosity of the cytoplasm calculated from the translational diffusion constant or the rotational correlation time was 2.0-3.0 centipoise, about two to three times that of the spin label in water. Therefore, over the dimensions measured by the technique, 50-100 A, solvent viscosity appears to be the major determinant of particle movement in cells under physiologic conditions. However, when cells were subjected to hypertonic conditions, the translational motion of the spin label decreased threefold, whereas the rotational motion changed by less than 20%. These data suggest that the decrease in cell volume under hypertonic conditions is accompanied by an increase in cytoplasmic barriers and a decrease in the space between existing cytoplasmic components without a significant increase in viscosity in the aqueous phase. In addition, a comparison of reported diffusion values of a variety of molecules in water and in cells indicates that cytoplasmic structure plays an important role in the diffusion of proteins such as bovine serum albumin.

Animals↗
PubMed1984

Measurement of glutathione requiring enzymes involved in arachidonic acid cascade of rat basophilic leukemia cells.

Rat basophilic leukemia cells have frequently been employed for investigating the pathways of leukotriene biosynthesis, a class of biologically active arachidonic acid metabolites. However, information is lacking on the levels of selenium-dependent glutathione peroxidase (Se-GSH-Px), non-Se-GSH-Px and glutathione S-transferases (GSH-S-Trs), key enzymes involved in fatty acid hydroperoxide metabolism and leukotriene biosynthesis in these cells. Both GSH-S-Trs and non-Se-GSH-Px reactions are catalyzed by the same enzyme. In the present studies, we have measured the enzyme activities of GSH-Px(s) and GSH-S-Trs in the 105,000 X g supernatant fraction of sonified RBL-1 cells. The specific activities for GSH-Px(s) toward H2O2, cumene hydroperoxide, and 15S-hydroperoxy-eicosatetraenoic acid (15S-HPETE) are 12.6, 17.9 and 26.9 nmoles X min-1 X mg-1 protein, respectively. A specific activity of 18.9 nmoles X min-1 X mg-1 protein with 1-chloro-2,4-dinitrobenzene was estimated for the GSH-S-Trs. Therefore, the cell fraction that exhibits 5-lipoxygenase activity also contains selenium and non-selenium glutathione peroxidases.

Animals↗
PubMed1983

Calcium-dependent activation of lymphocytes by ionophore, A23187, and a phorbol ester tumor promoter.

The phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore, A23187, have similar effects on many different cells. For example, both show mitogenic and comitogenic activities for lymphocytes. It had been suggested that some of TPA's effects are due to its ability to act as a calcium ionophore. In order to test this idea, we compared the ability of TPA and ionophore to synergize with concanavalin A (Con A) in a two-phase system of lymphocyte mitogenesis. We found that ionophore was most comitogenic with Con A when present in the early phase of stimulation. TPA was only comitogenic when present in the late phase. Ionophore and TPA could not replace one another in the system. However, both ionophore and TPA together could replace Con A and stimulate DNA synthesis when they were presented to the cells in the sequential order of ionophore followed by TPA. Both compounds required the presence of external calcium to be effective.

Animals↗