Biomedical subjects

J D Taylor

Publications and source records attributed to J D Taylor.

At least 91 records · Page 5Linked to original sources

PubMed1990

Actin-dependent carotenoid droplet dispersion in permeabilized cultured goldfish xanthophores.

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.

Actins↗

PubMed1990

Partial characterization of a carotenoid droplet ATPase and its possible significance in carotenoid droplet dispersion in goldfish xanthophores.

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.

Adenosine Triphosphatases↗

PubMed1990

Effect of urine pH on the stability of doxorubicin and its recovery from bladder instillations.

Doxorubicin (1 mg/ml) was shown to be stable when added to urine samples with a mean natural pH of 5.4 and in urine buffered to a mean pH of 4.6. However, at alkaline pH (mean = 8.1) there was a biphasic degradation of doxorubicin (mean t1/2 = 3.24 and 89 h respectively). The data indicate that buffering intravesical doxorubicin to pH 4.6 (acetate buffer) or pre-dosing of patients with ammonium chloride may minimise loss of active drug during the time for which the drug is retained in the bladder. Recovery of doxorubicin following 1 hour's retention in the bladder was similar (77%) for doses of 38/48 or 78 mg. It is suggested that a dose of 50 mg (1 mg/ml) is sufficient to ensure an adequate delivery of active drug to the bladder wall.

Administration, Intravesical↗

PubMed1990

Chronic urinary retention postpartum.

Three cases of chronic urinary retention postpartum are reported. All patients were primigravidas. Unrecognized urinary retention following epidural anaesthesia in labour was the likely cause. Abdominal ultrasound may have a place in early recognition of this complication. The early use of a suprapubic catheter hastens bladder recovery.

Adolescent↗

PubMed1990

Case four. Synergistic strategy development.

Mercy Medical Center is a 467 bed, not-for-profit teaching hospital affiliated with a multispecialty group practice and health maintenance organization through contractual arrangements. Historically, strategy development was not helping the hospital remain viable while accomplishing its mission. Strategic planning was fragmented, not focused toward concern for balance of the overall product line, total growth of the organization or the best use of resources. The hospital did not involve the affiliated group practice or HMO in its planning. This last case in Chapter One describes the pursuit of synergy through strategy formulation. The case provides a good example of what Kanter describes as a post-entrepreneurial organization, striving for advantage through collaboration and linkage with "PALs."

Decision Making, Organizational↗

PubMed1990

Case seven. Managing mature operations in a competitive environment.

In this case, a medical center learns how to evaluate each of its services along a Product Life Cycle curve. Each stage of the cycle has characteristics that suggest appropriate strategic growth and action orientations. When services were plotted along the Product Life Cycle curve, many of the services were found to be mature or making the transition from growth to maturity. At the time of the case, Memorial Medical Center's competitors were entering many of its traditional markets creating a strong competition for market share. Greater competitive emphasis on performance and price reduced profits in the medical center's mature services. The case reports the medical center's approach to modifying its action orientation in light of a better understanding of organizational competence.

Decision Making, Organizational↗

PubMed1989

Discrimination between DNA sequences by the EcoRV restriction endonuclease.

The EcoRV restriction endonuclease cleaves not only its recognition sequence on DNA, GATATC, but also, at vastly reduced rates, a number of alternative DNA sequences. The plasmid pAT153 contains 12 alternative sites, each of which differs from the recognition sequence by one base pair. The EcoRV nuclease showed a marked preference for one particular site from among these alternatives. This noncognate site was located at the sequence GTTATC, and the mechanism of action of EcoRV at this site was analyzed. The mechanism differed from that at the cognate site in three respects. First, the affinity of the enzyme for the noncognate site was lower than that for the cognate site, but, by itself, this cannot account for the specificity of EcoRV as measured from the values of kcat/Km. Second, the enzyme had a lower affinity for Mg2+ when it was bound to the noncognate site than when it was bound to its cognate site: this appears to be a key factor in limiting the rates of DNA cleavage at alternative sites. Third, the reaction pathway at the noncognate site differed from that at the cognate site. At the former, the EcoRV enzyme cleaved first one strand of the DNA and then the other while at the latter, both strands were cut in one concerted reaction. The difference in reaction pathway allows DNA ligase to proofread the activity of EcoRV by selective repair of single-strand breaks at noncognate sites, as opposed to double-strand breaks at the cognate site. The addition of DNA ligase to reactions with EcoRV made no difference to product formation at the cognate site, but products from reactions at noncognate sites were no longer detected.

Base Sequence↗

PubMed1989

Protein phosphorylation and the two stages of pigment organelle dispersion in permeabilized xanthophores: organelle protein phosphorylation alone supports only the first stage.

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

Adenosine Triphosphate↗

PubMed1989

In vivo characterization of combination antitumor chemotherapy with calcium channel blockers and cis-diamminedichloroplatinum(II).

We have examined nifedipine, a dihydropyridine class calcium channel blocker, for ability to overcome cis-diamminedichloroplatinum(II) (cisplatin) resistance in a murine tumor line variant, B16a-Pt, which we developed for resistance to cisplatin. Nifedipine significantly enhanced the antitumor actions of cisplatin against primary subcutaneous B16a-Pt tumors and their spontaneous pulmonary metastases. We have characterized, in vivo, the pharmacokinetics and dose-response interactions between nifedipine and cisplatin. We now report our studies designed to compare, in vivo, the efficacy of nifedipine and other calcium active compounds including: (a) structurally similar calcium channel blockers (nimodipine, nicardipine) from the dihydropyridine class, (b) structurally different calcium channel blockers from the benzothiazepine (diltiazem) and the phenylalkylamine (verapamil) classes, and (c) calmodulin antagonists (trifluoperazine and calmidazolium) for ability to enhance the antitumor action of cisplatin. Nifedipine was included as the standard or reference compound. In these studies verapamil and diltiazem failed to enhance the antitumor actions of cisplatin as did both calmodulin antagonists. Our findings suggest that nifedipine has a greater degree of specificity for B16a-Pt cells than structurally different calcium channel blockers from other chemical classes (i.e., diltiazem and verapamil), or the two calmodulin antagonists (i.e., trifluoperazine and calmidazolium). We concluded that nifedipine interacts with specific target site(s) which are not accessible by verapamil, by diltiazem, or by the calmodulin antagonists. Surprisingly, the two dihydropyridine class calcium channel blockers, nimodipine and nicardipine, also failed to enhance cisplatin's antitumor actions despite the fact that their specificity and kinetics for binding to the dihydropyridine receptor component of the calcium channel favors them (nimodipine and nicardipine) over nifedipine. Therefore, we postulate that the synergism between cisplatin and nifedipine is independent of the latter's effect on the voltage sensitive, slow inward calcium channel. We suggest that cisplatin cytotoxicity is enhanced by nifedipine's interaction with an as yet unidentified specific "target site," as opposed to nonspecific interactions with the tumor cell plasma membrane or specific interactions with calmodulin or the P-glycoprotein (which is responsible for pleiotropic resistance).

Animals↗

PubMed1989

Acute hydrogen sulfide poisoning. Demonstration of selective uptake of sulfide by the brainstem by measurement of brain sulfide levels.

The possibility of measuring sulfide levels in the central nervous system (CNS) opens up many avenues for exploration. In acute hydrogen sulfide (H2S) poisoning, death results from loss of central respiratory drive. To date, however, measurement of brain sulfide has not been possible. By employing gas dialysis and ion chromatography coupled to electrochemical detection, rat brain sulfide levels could be measured either following inhalation of H2S or after injection of sodium hydrosulfide (median lethal dose, [LD50] = 14.6 +/- 1.00 mg/kg). Accumulation of brain sulfide was linearly proportional to the dose over the range 0.50 LD50 to 3.33 LD50 units, and was strongly correlated with mortality data (R = 0.947). Furthermore, analysis of untreated (control) brain showed an endogenous sulfide level of 1.57 +/- 0.04 micrograms/g (mean +/- SE; N = 16). Studies on various rat brain regions (brainstem, cerebellum, hippocampus, striatum and cortex) showed that the endogenous sulfide level of brainstem, 1.23 +/- 0.06 micrograms/g, was significantly lower than that of the other brain regions. Net uptake of sulfide was greatest in the brainstem (3.02 micrograms/g) compared to the other regions as was the selective accumulation of sulfide as calculated from normalized blood flow rates. The results of subcellular fractionation demonstrated that sulfide was detectable in fractions enriched in myelin, synaptosomes and mitochondria. Approximately one-quarter of the endogenous sulfide content of whole rat brain was found in the mitochondrial fraction. The sulfide content of these fractions increased 2- to 3-fold after 50 mg/kg NaHS, the greatest increases occurring in myelin- and mitochondrial-enriched fractions.

Analysis of Variance↗

PubMed1989

Bidirectional control of membrane expression and/or activation of the tumor cell IRGpIIb/IIIa receptor and tumor cell adhesion by lipoxygenase products of arachidonic acid and linoleic acid.

Lewis lung carcinoma cells express a plasma membrane receptor (i.e., IRGpIIb/IIIa) which is immunologically and functionally related to the platelet aggregation receptor complex (i.e., GpIIb/IIIa). Both fluorescence microscopy and flow cytometric analysis reveal that surface expression and/or activation of this tumor cell receptor is enhanced by a phorbol ester [i.e., 12-O-tetradecanoylphorbol-13-acetate (TPA)] and a lipoxygenase metabolite of arachidonic acid; 12-hydroxyeicosatetraenoic acid (i.e., 12-HETE). TPA-enhanced expression appears to be mediated by a lipoxygenase metabolite, as this effect can be reversed by lipoxygenase inhibitors but not by cyclooxygenase inhibitors. In parallel with these results both TPA and 12(S)-HETE [but not 12(R)-HETE] enhance tumor cell adhesion to endothelial cells, subendothelial matrix and fibronectin, but not to type IV collagen. TPA-enhanced adhesion can be reduced by lipoxygenase inhibitors but not by cyclooxygenase inhibitors and in addition, stimulated adhesion can be blocked by pretreatment of tumor cells with specific polyclonal or monoclonal antibodies which react against IRGpIIb/IIIa. 12(S)-HETE-enhanced adhesion can also be inhibited by these same antibodies. In contrast, a lipoxygenase product of linoleic acid, 13(S)-hydroxyoctadecadienoic acid, inhibited TPA and 12(S)-HETE-enhanced tumor cell adhesion to endothelial cells, subendothelial matrix, and fibronectin. These results suggest that (a) IRGpIIb/IIIa is a multifunctional receptor which mediates tumor cell adhesion to a variety of biological substrata, (b) TPA enhances surface expression and/or activation of this receptor possibly via a lipoxygenase metabolite of arachidonic acid, and (c) these effects are opposed by a lipoxygenase metabolite of linoleic acid.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1989

Rearrangements of pterinosomes and cytoskeleton accompanying pigment dispersion in goldfish xanthophores.

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

Adrenocorticotropic Hormone↗

PubMed1989

cAMP-independent and cAMP-dependent protein phosphorylations by isolated goldfish xanthophore cytoskeletons: evidence for the association of cytoskeleton with a carotenoid droplet protein.

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.

Animals↗

PubMed1989

Immunofluorescence evidence for cytoskeletal rearrangement accompanying pigment redistribution in goldfish 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.

Actin Cytoskeleton↗

PubMed1989

Purification of anterogin, a protein factor necessary for the dispersion of carotenoid droplets in permeabilized xanthophores of goldfish.

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.

Adenosine Triphosphatases↗

PubMed1989

Isolation of melanized cell lines with stable phenotypes from a goldfish erythrophoroma cell line and cryopreservation of these cells by the use of autologous serum.

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.

Animals↗

PubMed1989

Reversible dedifferentiation and redifferentiation of a melanized cell line from a goldfish tumor.

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

Animals↗

PubMed1989

Enhanced tumor cell adhesion to the subendothelial matrix resulting from 12(S)-HETE-induced endothelial cell retraction.

A 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE), which is produced by platelets and tumor cells, was tested for its ability to induce retraction of endothelial cell monolayers. The induction of endothelial cell retraction is a critical step in tumor cell metastasis. Endothelial cells demonstrated reversible retraction in response to 12(S)-HETE, but did not respond to the stereoisomer 12(R)-HETE or to unrelated 5-lipoxygenase (i.e., 5[S]-HETE) or 15-lipoxygenase (i.e., 15[S]-HETE) metabolites. Endothelial cells did not demonstrate loss of viability in response to 12(S)-HETE. The induction of retraction was both dose and time dependent. Scanning electron microscopy confirmed that 12(S)-HETE induced endothelial cell retraction and revealed collapsed filopodia on their surface, the appearance of spaces between endothelial cells and the underlying subendothelial matrix, in addition to large gaps between adjacent endothelial cells. Tumor cell adhesion to endothelial cell monolayers was enhanced 1 h after pretreatment of monolayers with 12(S)-HETE but not after pretreatment with other lipoxygenase metabolites. Tumor cell adhesion to endothelial cell monolayers 36 h after pretreatment with 12(S)-HETE was not different from adhesion to untreated monolayers. Therefore we suggest that 12(S)-HETE generated during tumor cell-platelet-endothelial cell interactions may induce reversible endothelial cell retraction, allowing tumor cell access to the subendothelial matrix, which is a critical step in their eventual extravasation from the microvasculature during hematogenous metastasis.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗