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Biomedical subjects

E R Tustanoff

Publications and source records attributed to E R Tustanoff.

At least 19 recordsLinked to original sources
PubMed1993

Evidence for bimodal distribution of breast carcinoma ER and PgR values quantitated by enzyme immunoassay.

Breast carcinoma oestrogen receptor (ER) and progesterone receptor (PgR) values obtained by radioligand binding assays have commonly been observed to have approximate log-normal distributions. We examined the distribution of log-transformed receptor values obtained by enzyme immunoassay for 5468 primary breast carcinomas in five Ontario laboratories. In each laboratory, it was found that the frequency histograms for the log transformed receptor values were not unimodal, and generally were suggestive of bimodality. This was not affected by stratification by age or inferred menopausal status (< or = 49, > or = 50 years), and could not be explained by kit characteristics. However, the low point in the distribution varied from 5 to 63 fmol/mg cytosol protein, depending on the receptor, patient age and laboratory. The tendency towards biomodality was more distinct for ER than for PgR. It remains to be determined whether the low points on the frequency histograms have clinical relevance for discriminating between hormone-sensitive and hormone-insensitive tumours.

Adult↗
PubMed1992

Amino acid transport in multidrug-resistant Chinese hamster ovary cells.

In the process of assessing the effect of anthracycline drugs on cellular membrane function in cultured multidrug resistant (MDR) and its parental cells, experiments were undertaken to investigate the kinetics of neutral amino acid membrane transport (the sodium dependent A and ASC systems). P-glycoprotein, a high molecular weight energy requiring integral membrane protein responsible for actively pumping drugs out of cells, has been shown to be overexpressed in MDR cells. It was our hypothesis that its presence might affect other membrane energy requiring systems such as amino acid transport. On establishing the concentrations of P-glycoprotein by western blotting in the two cell lines to be studied, the kinetics of membrane transport of the neutral amino acids alpha-aminoisobutyric acid (AIB) and serine (SER) were investigated using the CHRC5 multidrug resistant and AUX B1 parental Chinese hamster ovary (CHO) cells. In CHRC5 cells, the amount and rate (Vmax) of accumulated amino acids, was significantly depressed when compared to AUX B1 cells, however, there was no difference in the rates of amino acid efflux between these two cell lines. Using 1,6-diphenyl 1,3,5-hexatriene (DPH) polarization to evaluate the state of membrane fluidity in the two cell lines studied, it was seen that CHRC5 cells showed a slightly lower degree of polarization than that observed in AUX B1 cells. These results suggest, that the P-glycoprotein does not alter amino acid transport directly but may modify the activity or numbers of functional transport carriers.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Amino and iodotamoxifens: synthesis, estrogen receptor affinity and biodistribution.

Both geometrical isomers (E and Z) of an aminotamoxifen (2) have been prepared as precursors of the corresponding E and Z iodotamoxifens (1). The ability of E and Z-1 and 2 to compete with [3H]estradiol for estrogen receptors in rat uterine cytosol was measured relative to Z-tamoxifen and estradiol. The four tamoxifen derivatives showed affinities ranging from 50% to 1600% of that of tamoxifen. Under the same conditions, tamoxifen's relative binding affinity was 0.2% of that of estradiol. Preparative routes to the radioiodo-tamoxifens, [131I]-E and Z-1, were also developed and provided approximately 100 MBq of 'no carrier added' material in 40-60% radiochemical yield. Study of the biodistribution of these radioligands in tumor-bearing mice demonstrated significant radioactivity in the tumors and in the uterus. For [131I]-E-1, target to background ratios reached 28 for uterus/blood and 10 for tumor/blood; corresponding optimum ratios for [131I]-Z-1 were 10 and 5. A washout study using estradiol indicated selective uptake in the uterus of Swiss white mice. However, tumor uptake and image contrast in humans following intravenous administration of either [131I]-E or Z-1 were insufficient to allow diagnostic use of the radioiodotamoxifens.

Aged↗
PubMed1987

Changes in cyclic nucleotide profiles in proliferating multi-cell spheroids.

The intracellular concentration of cyclic adenosine 3':5'-mono-phosphate (cAMP) has been shown to be related to each developmental phase of the cell cycle. Highest levels of this nucleotide are evident during the S-phase (the DNA synthetic phase) which has also been shown to be radiation-sensitive. The relationship between the levels of cyclic nucleotides, cAMP and guanosine 3':5'-monophosphate (cGMP), and the proliferation of cells in a tumor model system was investigated using V79-171b Chinese hamster lung cells grown both as monolayer and as three dimensional cell clusters (spheroids). The spheroid which is more radiation-resistant than its monolayer counterpart, has been used by many radiobiologists as an in vitro tumor model. Our results indicate that the yin-yang hypothesis of a opposing regulatory relation between the two different classes of cyclic nucleotides only held true for monolayer cultures (both exponential and plateau phase) but could not be demonstrated in the tumor model where the levels of both nucleotides increased directly with the diameter of the growing spheroid mass.

Animals↗
PubMed1986

CA 125 surveillance and second-look laparotomy in ovarian carcinoma.

CA 125 was evaluated as a tumor marker in 31 patients undergoing treatment for ovarian carcinoma, 17 of whom had second-look laparotomies. At the time of second-look laparotomy, 14 patients had CA 125 values in the normal range. Six of these patients had a positive second-look laparotomy. Although normal CA 125 values do not obviate the need for second-look laparotomy in treatment planning, rising or falling trends reflected clinical disease progression or regression in 80% of the cases.

Adult↗
PubMed1986

Role of phospholipid fatty acids on the kinetics of high and low affinity sites of cytochrome c oxidase.

The nature of the interactions between cytochrome c oxidase and the phospholipids in mitochondrial membranes has been investigated by varying the nature of the fatty acyl components of Saccharomyces cerevisiae. A double fatty acid yeast mutant, FAI-4C, grown in combinations of unsaturated (oleic, linoleic, linolenic, and eicosenoic) and saturated (lauric and palmitic) fatty acids, was employed to modify mitochondrial membranes. The supplemented fatty acids constituted a unique combination of different acyl chain lengths with varying degrees of unsaturation which were subsequently incorporated into mitochondrial phospholipids. Phosphatidylethanolamine and cardiolipin, the predominant phospholipids of the inner mitochondrial membrane, were characterized by their high levels of supplemented unsaturated fatty acids. Increasing the chain length or the degree of unsaturation of mitochondrial membrane phospholipids had no effect on altering the nature of the phospholipid polar head group but did result in a profound change on the specific activity of cytochrome c oxidase. When studied under conditions of different ionic strengths and pHs the enzyme's activity, as documented by Eadie-Hofstee plots, showed biphasic kinetics. The kinetic parameters for the low affinity reaction were greatly influenced by the changes in the membrane fatty acids and only marginal effects were noted at the high affinity reaction site. The discontinuities in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, monitored at increasing temperatures, suggested that changes in membrane fluidity were conditioned by alterations in mitochondrial membrane fatty acid constituents. These results indicate that the lipid changes affecting the low affinity binding site of cytochrome c oxidase may be the result of lipid-protein interactions which lead to enzyme conformational changes or may be due to gross changes in membrane fluidity. It may, therefore, follow that this enzyme site may be embedded in or be juxtaposed to the outer surface of the inner mitochondrial membrane bilayer in contrast to the high affinity site which has been shown to be significantly above the membrane plane.

Electron Transport Complex IV↗
PubMed1985

Membrane glycoprotein and surface free energy changes in hypoxic fibroblast cells.

Hypoxia affects the biochemistry of mammalian cells and thus alters their sensitivity to subsequent chemo- and radiotherapy. When V79 Chinese hamster lung fibroblasts were grown under conditions of extreme hypoxia (less than 10 ppm O2) there was a significant shift in the membrane glycoprotein composition. Scanning electron microscopy revealed altered cell surface morphology including loss of pseudopodial projections. Experiments to determine changes in interfacial free energy of these cells using equilibrium two phase systems of poly(ethylene glycol) (PEG) and dextran were carried out. Test fluid droplets of the denser dextran-rich phase were formed on layers of cells in the PEG-rich phase as the bathing medium, and the contact angles the droplets made with the cell layers were measured from photomicrographs. The contact angles on cells in the plateau phase increased significantly with time of exposure to hypoxia, from 25 degrees (zero time) to 35 degrees (6 h) to 60 degrees (9 h). Contact angles on cells in the exponential phase increased from 80 degrees (zero time) to 150 degrees after 20 h of hypoxia. It appears that the altered contact angles reflect changes in cell surface hydrophobicity that may, in part, reflect alterations in the membrane glycoprotein composition.

Animals↗
PubMed1985

Changes in ultrastructure and function of hypoxic V79 fibroblast cells after treatment with misonidazole.

Morphologic and enzymatic changes due to exposure to the radiosensitizing chemical, misonidazole, have been identified in V79 cells grown in a system in which oxygen tensions and culture density have been controlled. Misonidazole prevented the increase in mitochondrial size normally seen during exposure of these cells to conditions of moderate hypoxia (2 X 10(3) ppm O2). Mitochondrial size was also significantly decreased in cells from exponential cultures exposed to 1 mmol/l misonidazole. Morphologic changes to the mitochondria that varied from data reported elsewhere were also noted. Misonidazole caused a significant initial decrease in cytochrome oxidase activity after 4 hours of exposure of aerobic and moderately hypoxic cultures that did not return to normal in chronically hypoxic cells during continued exposure to the drug.

Animals↗
PubMed1985

Ultrastructural changes in V79 hamster lung fibroblasts during hypoxic exposure.

Cells in tumors that are deprived of their blood supply become hypoxic. These stressed cells adapt to their new environments by altering their metabolic regimen which in time induces cellular structure changes. The morphologic make-up of these O2-deprived cells is the focal point of this electron microscopy study. V-79 hamster lung fibroblast cells grown as monolayer cultures were examined under controlled culture density and oxygen tensions - normal aerobia (2.1 X 10(5) ppm O2), and extreme hypoxia (less than 10 ppm O2). Electron micrographs of these cells demonstrated a loss of structural mitochondrial integrity accompanied with large increases in both mitochondrial and lipid vacuole size following exposure to extreme hypoxia. When these cells were reoxygenated, those mitochondria which had not become degenerate returned to their normal state however, lipids still continued to accumulate in vacuoles for a further 6 h. Addition of 1 mM palmitic acid to aerobic cultures evoked similar lipid and mitochondrial irregularities as were observed in hypoxic cells although, the latter were not as marked. When this saturated fatty acid was added to hypoxic cells no further structural alterations were seen. The cellular changes manifested during this study were subjected to quantitative measurements and these results have given an insight into the scope and variety of ultrastructural changes which have resulted from exposure of cultured cells to hypoxic conditions.

Animals↗
PubMed1985

Inter-laboratory quality control of estrogen and progesterone receptor assays in breast cancer tissue using lyophilised cytosols.

In 1981 a quality control (QC) program for estrogen and progesterone receptor assays was organized among six laboratories in Ontario, Canada. Twenty-three vials of lyophilised cytosol prepared from human breast tumor tissues were analysed by each laboratory over a two-year period. Samples of each batch of QC material were analysed at least twice: either in the same batch or on separate occasions. The present study demonstrates the stability of the QC material, defines the relative accuracy of the receptor assays, and provides estimates of within-batch and between-batch precision of the receptor assays.

Breast Neoplasms↗
PubMed1984

Iodination of [Tyr8]-bradykinin-comparison of chloramine-T and lactoperoxidase techniques.

Antigen-antibody kinetics were studied using a hapten which was iodinated by two unique procedures. Using bradykinin, a vasopressor hormone as a model peptide, radioactive iodination (125I) of its 8-tyrosyl analogue was carried out both enzymatically and chemically using modified procedures. Two distinct chemical species were obtained which were characterized on a chromatographic, chemical as well as charge basis as a mono-iodinated form of [Tyr8]-bradykinin using the lactoperoxidase procedure and a di-iodinated entity using chloramine-T technique. The addition of a second iodine atom to the antigen lowers its immunoreactivity for its antibody and thus alters the kinetics of this reaction. Further experiments on the stability (temperature, time of storage, and chemical environment) of these iodinated peptides are described.

Antibody Affinity↗
PubMed1979

Immunological and biological activities of fragments and analogs of bradykinin.

Using a number of analogs and fragments of a short-chain peptide bradykinin, a series of experiments have been carried out to assess the effect of modifications to the basic structure of the parent molecule on its myotropic and immunoreactive properties. Binding kinetics of both an antibody raised against the authentic nonapeptide and its specific biological receptor found in the guinea pig ileum were used to study these alteration effects. Peptide derivatives of bradykinin with an extension at the N-terminal (Lys- and Met-Lys-bradykinin) cross-react with the antibody raised to bradykinin 59 and 70% respectively. On the other hand, internal fragments with intact C-termini (2-9 and 3-9 bradykinin) react with this same antibody to an extent of 250 and 875% respectively, indicating that they are more potent antigens than the vasopressor molecule itself. Other internal fragments, as well as 9-substituted analogs effectively and not interact. These results indicated that the C terminal arginine of bradykinin is indeed essential in the binding mechanism with its antibody. This in turn illustrates the role of the carrier ovalbumin in the development of antiserum to the ovalbumin-toluene-diisocyanate-bradykinin complex. The physiological experiments with the guinea pig bioassay preparations lead to similar conclusions. Most internal fragments of bradykinin are devoid of activity, whereas N-terminal fragments (2-9, 3-9, and 5-9 bradykinin) have retained some activity again indicating a need for an intact arginine residue at the C-terminus of the molecule. Any modification in position 9 results in severe impairment of biological activity. Thus, the C-terminal residue of bradykinin must be conserved in order that the molecule may retain its immunological and physiological activities. Any extensions, deletions, or modifications of this site will severely retard these functions.

Amino Acid Sequence↗
PubMed1978

Effect of radiosensitizing agents on electron transport systems.

Experiments have been carried out to study the interaxtion between chemical radiosensitizing agents and model electron transport systems. Using an NAD(P)H:O2 oxidoreductase enzyme as such a model, it was demonstrated that radiosensitizers can act as intermediates in the transfer of electrons from NADH to O2, even in the presence of classical inhibitors of electron transport, with anefficiency related to both their redox potentials and their radiosensitizing abilities. This work which was further confirmed in mammalian mitochondria and microsomes as well as in a cultured cell system indicated that these sensitizers can accept electrons from a variety of organelle systems. This action was shown to be related to the concentration of reduced pyridine nucleotides present both in vivo and in vitro. Of the electron-affinic agents tested, those whose redox potential was more negative than -0.39 V may possibly serve as better radiotherqpeutic mediators.

Animals↗
PubMed1975

The biogenesis of mitochondrial membranes in the yeast Saccharomyces cerevisiae.

Membrane lipids of yeast mitochondria have been enriched by growing yeast cells in minimal medium supplemented with specific unsaturated fatty acids as the sole lipid supplement. Using the activity of marker enzymes for the outer (kynurenine hydroxylase) and inner (cytochrome c oxidase and oligomycin-sensitive ATPase) mitochondrial membranes, Arrhenius plots have been constructed using both promitochondria and mitochondria obtained from O2-adapting cells in the presence of a second unsaturated fatty acid (i.e. linoleate (N2) to elaidic (O2)). Transition temperatures which reflect the unsaturated fatty acid enrichment of the new membranes reveal interesting features involved in the mechanism of the assembly of these two mitochondrial membranes. This approach was further enforced with both lipid depletion and mitochondrial protein inhibition studies. Kynurenine hydroxylase which does not require fatty acid for its continued synthesis during aerobiosis seems to be incorporated into the preformed linoleate-anaerobic outer membrane. The newly synthesized activities of inner mitochondrial membrane enzymes on the other hand, appear to integrate their activity into newly formed aerobic-elaidic-rich inner membrane. These latter enzymes show a distinct dependence on fatty acid supplement for their continued synthesis during their aerobic phase. This suggests that O2-dependent proteo-lipid precursors are formed before these enzymes are integrated into their membrane mosaic. Two separate models are proposed to explain these results, one for the lipid-rich outer mitochondrial membrane and another for the protein-rich inner mitochondrial membrane.

Adenosine Triphosphatases↗