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S Tamir

Publications and source records attributed to S Tamir.

At least 19 recordsLinked to original sources
PubMed2001

Estrogen-like activity of glabrene and other constituents isolated from licorice root.

Licorice root extract and its major isoflavan, glabridin, exhibited varying degrees of estrogen receptor (ER) agonism in different tissues in vitro and in vivo. Animals fed with licorice extract, compared with estradiol and glabridin, showed an increase in creatine kinase (CK) activity, a known marker for estrogen responsive genes, which was higher than expected from the levels of glabridin in the extract. This led us to test for other components that may contribute to this strong estrogen agonist activity. Results indicated that glabrene and isoliquiritigenin, (2',4',4-three hydroxy chalcone) (ILC) in the licorice extract can bind to the human ER with higher affinity (IC50, 1 and 0.5 microM) than glabridin (IC50, 5 microM). The stimulatory effects of glabrene in vivo were tissue specific and similar to those of estradiol. The effect of increasing concentrations of glabrene and ILC on the growth of breast tumor cell were biphasic. Both showed an ER-dependent growth-promoting effect at low concentrations (10 nM-10 microM), and ER-independent antiproliferative activity at concentrations >15 microM. This is the first study to indicate that glabrene, an isoflavene exerted varying degrees of ER agonism in different tissues.

Animals↗
PubMed2000

Estrogenic and antiproliferative properties of glabridin from licorice in human breast cancer cells.

There is an increasing demand for natural compounds that improve women's health by mimicking the critical benefits of estrogen to the bones and the cardiovascular system but avoiding its deleterious effects on the breast and uterus. The estrogenic properties of glabridin, the major isoflavan in licorice root, were tested in view of the resemblance of its structure and lipophilicity to those of estradiol. The results indicate that glabridin is a phytoestrogen, binding to the human estrogen receptor and stimulating creatine kinase activity in rat uterus, epiphyseal cartilage, diaphyseal bone, aorta, and left ventricle of the heart. The stimulatory effects of 2.5-25 microg/animal glabridin were similar to those of 5 microg/animal estradiol. Chemical modification of glabridin showed that the position of the hydroxyl groups has a significant role in binding to the human estrogen receptor and in proliferation-inducing activity. Glabridin was found to be three to four times more active than 2'-O-methylglabridin and 4'-O-methylglabridin, and both derivatives were more active than 2',4'-O-methylglabridin. The effect of increasing concentrations of glabridin on the growth of breast tumor cells was biphasic. Glabridin showed an estrogen receptor-dependent, growth-promoting effect at low concentrations (10 nM-10 microM) and estrogen receptor-independent antiproliferative activity at concentrations of > 15 microM. This is the first study to indicate that isoflavans have estrogen-like activities. Glabridin and its derivatives exhibited varying degrees of estrogen receptor agonism in different tests and demonstrated growth-inhibitory actions on breast cancer cells.

Animals↗
PubMed1998

Etheno adducts in spleen DNA of SJL mice stimulated to overproduce nitric oxide.

In order to investigate specific DNA damage caused by nitric oxide (NO) induced lipid peroxidation, levels of promutagenic etheno adducts 1,N6-ethenodeoxyadenosine (epsilondA) and 3,N4-ethenodeoxycytidine (epsilondC) were measured in spleen DNA of SJL mice induced to produce high levels of NO by injection of RcsX (pre-B-cell lymphoma) cells. epsilondA and epsilondC levels were quantified by an ultrasensitive immunoaffinity-32P-post-labeling method. Spleen DNA of control mice (n = 5) had background levels of 9.2+/-5.4 epsilondA adducts per 10(9) dA and 13.1+/-5.7 epsilondC adducts per 10(9) dC. In RcsX cell-injected mice (n = 7), levels of these adducts were elevated approximately 6-fold, i.e. 53.9+/-39.4 epsilondA per 10(9) dA and 83.5+/-57.8 epsilondC per 10(9) dC (P < 0.05). Mice injected with RcsX cells and also treated with NG-methyl-L-arginine (NMA), an inhibitor of inducible nitric oxide synthase (n = 6), had significantly reduced levels (P < 0.05) of both epsilondA and epsilondC (13.5+/-5.7 epsilondA per 10(9) dA and 28.2+/-15.7 epsilondC per 10(9) dC). These findings constitute the first available evidence of formation of etheno adducts associated with NO overproduction in vivo. The adducts were presumably formed from lipid peroxidation products such as trans-4-hydroxy-2-nonenal (HNE), generated via oxidation of lipids by peroxynitrite. The results suggest that etheno-DNA adducts, among other types of damage, may contribute to the etiology of cancers associated with chronic infection/inflammation in which NO is overproduced.

Aldehydes↗
PubMed1997

Nitrotyrosine formation, apoptosis, and oxidative damage: relationships to nitric oxide production in SJL mice bearing the RcsX tumor.

In SJL mice, growth of RcsX lymphoma cells results in activation of macrophages in the spleen and lymph nodes to produce high levels of NO radical (NO.). We used this experimental model system to study the toxicology of NO. in vivo. To characterize spatial relationships between sites of NO. production and tissue damage, immunohistochemical techniques were developed for simultaneous detection of inducible NO. synthase (iNOS), 3-nitrotyrosine, and apoptosis in spleen and lymph nodes of tumor-bearing animals. Elevated expression of iNOS, presumed to reflect increased NO. production, was associated with a significant increase in frequency of apoptotic nuclei. Both apoptotic nuclei and 3-nitrotyrosine staining were found in cells juxtaposed to iNOS-expressing (ie., NO.-producing) macrophages and also within the macrophages themselves. To assess the extent of DNA damage associated with the response, 8-oxoguanine levels were quantified in DNA extracted from spleens of tumor-bearing mice. No increase in levels of this marker of oxidative DNA damage was found in tissues in which apoptosis and 3-nitrotyrosine levels were highly elevated within specific subsets of cells. Collectively, our results indicate that under the pathophysiological conditions existing in the RcsX tumor-bearing SJL mouse, cellular damage caused by NO. and/or other reactive species produced by activated macrophages is highly localized within cells in close proximity to the activated macrophages.

Animals↗
PubMed1997

A mechanistic analysis of nitric oxide-induced cellular toxicity.

Nitric oxide (NO.)-induced toxicity was investigated in two different cell lines, Chinese hamster ovary (CHO-AA8) and human lymphoblastoid (TK6), over a range of NO. doses (0-9 mM) delivered for an exposure of 2 h. To determine both short-term and delayed effects leading to death, a range of assays was employed to decipher the major mechanisms of cytotoxicity. Examples of damage parameters measured in this study include inhibition of DNA synthesis, damage to mitochondria, loss of cell membrane integrity, apoptosis, changes in cell cycle distribution, and the occurrence of DNA strand breaks. Our results indicate that NO.-induced toxicity is an extremely complex process involving multiple pathways generally leading to apoptotic cell death. Results consistently demonstrate that TK6 cells are much more susceptible to NO.-induced toxicity than CHO-AA8 cells. This difference in sensitivity could be seen for all types of cellular damage examined. The earliest observable effect of NO. exposure is inhibition of DNA synthesis which is not the result of inhibition of ribonucleotide reductase but may be the result of DNA damage leading ultimately to cell cycle arrest.

Animals↗
PubMed1996

Nitric oxide production in SJL mice bearing the RcsX lymphoma: a model for in vivo toxicological evaluation of NO.

SJL mice spontaneously develop pre-B-cell lymphoma that we hypothesized might stimulate macrophages to produce nitric oxide (NO.). Transplantation of an aggressive lymphoma (RcsX) was used to induce tumor formation. Urinary nitrate excretion was measured as an index of NO. production and was found to increase 50-fold by 13 days after tumor injection. NO. production was prevented by the addition of a nitric oxide synthase (NOS) inhibitor. The expression of inducible NOS (iNOS) in various tissues was estimated by Western blot analysis and localized by immunohistochemistry. The synthase was detected in the spleen, lymph nodes, and liver of treated but not control mice. To assess whether the iNOS-staining cells were macrophages, spleen sections from ResX-bearing animals were costained with anti-iNOS antibody and the anti-macrophage antibody moma-2. Expression of iNOS was found to be limited to a subset of the macrophage population. The concentration of gamma-interferon, a cytokine known to induce NO. production by macrophages, in the serum of tumor-bearing mice, was measured and found to be elevated 25-fold above untreated mice. The ability of ResX-activated macrophages to inhibit splenocyte growth in primary culture was estimated and macrophage-derived NO. was found to inhibit cell division 10-fold. Our findings demonstrate that ResX cells stimulate NO. production by macrophages in the spleen and lymph nodes of SJL mice, and we believe this experimental model will prove useful for study of the toxicological effects of NO. under physiological conditions.

Animals↗
PubMed1996

The role of nitric oxide (NO.) in the carcinogenic process.

The inflammatory process has long been known to be a risk factor for human cancers, particularly of the lung, bladder, colon, stomach, and female breast. Earlier hypothesis cited production of oxygen radicals, release of cytokines, and synthesis of prostaglandins and leukotrienes as biochemical modulators of the carcinogenic process. The discovery of NO. as a product of cells in the immune system has implicated this chemical in the mechanism of carcinogenesis, particularly when NO. is overproduced over a long period of time. After briefly reviewing the important chemical reactions of NO. under physiological conditions, we examine how the chemistry of its key reactants toward biologically important molecules relate to DNA damage and cytotoxicity. In these two processes, NO may play an important role in currently accepted models of multistage carcinogenesis.

Apoptosis↗
PubMed1996

Isolation, characterization, and properties of a trypsin-chymotrypsin inhibitor from amaranth seeds.

A trypsin-chymotrypsin inhibitor was isolated from the seeds of amaranth--a highly nutritious protein source. The purification of the inhibitor (AmI) was carried out by affinity chromatography on trypsin-Sepharose and by HPLC. AmI is a single-chain protein of 8 kD, as determined by electrophoresis on SDS-polyacrylamide gels and by gel exclusion on Sephadex G-50 column. It is stable at neutral and alkaline pH and is relatively thermostable. AmI inhibits trypsin and chymotrypsin from the digestive system of insects such as Tribolium castaneum and Locusta migratoria, supporting the hypothesis that inhibitors may have evolved as defense mechanisms of seeds against insects. AmI lost its inhibitory activities when submitted to limited proteolysis with trypsin, while limited proteolysis with chymotrypsin had almost no effect. The partial amino acid sequence of 45 amino acids from the amino terminus of AmI differs significantly from the known sequences of legume-seed and cereal-grain protease inhibitor families. Differences in the chemistry at the inhibitory site(s) and in the amino acid sequence of AmI in comparison to that of other cereal and legume inhibitors suggest that AmI is a member of a new family of serine protease inhibitors. AmI was found to inhibit the anchorage-independent growth of MCF-7 breast cancer cells, suggesting that AmI may have anticarcinogenic activity.

Amino Acid Sequence↗
PubMed1996

NO-induced oxidative stress and glutathione metabolism in rodent and human cells.

Nitric oxide (NO.), a radical species produced by many types of cells, is known to play a critical role in both regulatory processes and cell defense, yet it may also participate in collateral reactions, leading to DNA damage and cell death in both NO-generating and neighboring cells. Glutathione has been shown to protect cells from the toxic effects of free radicals and reactive oxygen species. The goal of this study was to investigate whether differences in glutathione metabolism could account for the resistance or sensitivity to cell killing by NO.. The cytotoxic effect of NO. was examined in CHO-AA8 (Chinese Hamster Ovary) cells and TK6 (human lymphoblastoid) cells pretreated with L-buthionine SR-sulfoximine (BSO), a potent inhibitor of gamma-glutamylcysteine synthetase, and with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), an irreversible inhibitor of glutathione reductase. The consequences resulting from the depletion of glutathione levels and from the arrest of oxidoreduction allowed us to show the involvement of glutathione in protecting cells from NO. and to investigate the importance of changes in glutathione metabolism on NO-induced toxicity. In CHO-AA8 cells, we found that treatment with NO. resulted in the oxidation of reduced glutathione (GSH) to oxidized glutathione (GSSG) and to mixed glutathione disulfides (GSSR). The resulting depletion of GSH stimulated its de novo synthesis, enabling the cells to resist killing by NO.. A slight difference in GSH metabolism was observed in TK6 cells. NO. led to an increase in GSSG levels similar to that observed in CHO-AA8 cells, however, a decrease in GSH levels, no change in GSSR levels, and higher levels of toxicity were also found, suggesting that NO-treated TK6 cells are not as competent in GSH homeostasis as CHO cells. We conclude that GSH is involved in protecting cells from killing by NO. and that both de novo synthesis of GSH and GSSG reduction are important in maintaining an adequate level of protection for the cells.

Animals↗
PubMed1995

Kinetic analysis of the fate of nitric oxide synthesized by macrophages in vitro.

To investigate the fate of nitric oxide (NO) synthesized by activated macrophages, the concentrations of NO and its principal reaction products, nitrite (NO2-) and nitrate (NO3-), were measured as a function of time in suspension cultures of RAW264.7 macrophages attached to microcarrier beads. Synthesis of NO became evident 2-5 h after stimulation of the cells, and steady concentrations of NO were achieved after about 9 h. The appearance of NO in the extracellular fluid coincided with the appearance of NO2- and NO3-, which were formed thereafter at approximately equal and constant rates. Using a kinetic model based on rate constants measured previously in cell-free systems, only half of the NO2- formed could be accounted for by the reaction of NO with O2. It is known that NO reacts with superoxide (O2.) to give peroxynitrite and that NO also reacts with peroxynitrite to yield NO2-, so that the latter reaction may explain the "excess" NO2- formation. Adding superoxide dismutase to the medium markedly reduced the ratio of NO3- to NO2-, consistent with the hypothesis that NO3- in the medium results primarily from the extracellular reaction of NO with O2-.. The addition of morpholine, a model amine, resulted in formation of N-nitrosomorpholine, concurrent with the other products. Measured rates of nitrosomorpholine formation were 6-fold lower than predictions based on kinetics in simple solutions, suggesting that in the cell culture system there were additional reactions that lowered the concentration of nitrous anhydride, the principal nitrosating agent formed from NO and O2.

Cell Line↗
PubMed1995

An improved method to create nitrocellulose particles suitable for the immobilization of antigen and antibody.

A method is described for producing 1-3 microns sized particles of nitrocellulose (NC) which are able to absorb protein. Protein is absorbed onto preformed particles made by first dissolving a sheet of nitrocellulose paper in DMSO, and then precipitating it with sodium carbonate/bicarbonate buffer. The efficiency of binding is the same as that of an equivalent sheet of non-processed NC filter paper. Antibodies absorbed onto preformed particles are not exposed to DMSO and carbonate buffer and therefore retain a high antigen binding capacity. Antigen and antibody-absorbed NC preformed particles were used to capture antibody and antigen, respectively. Using lysis buffer, the captured antibodies and antigens were readily released from the NC particles. This makes the latter an appropriate matrix for immunoprecipitation assays either for an antigen or for specific antibody. Antigen-coated NC particles were specifically aggregated ('agglutinated') by specific antibodies and thus can be used in semi-quantitative tests.

Animals↗
PubMed1995

Nitric oxide production in relation to spontaneous B-cell lymphoma and myositis in SJL mice.

SJL mice spontaneously develop B cell lymphomas (historically described as reticulum cell sarcomas) by 12 months of age and inflammatory muscle disease (myositis) by 6 months of age. Tumors originate in mesenteric lymph nodes and in Peyer's patches and resemble human germinal center lymphomas. The growth of reticulum cell sarcomas is completely dependent on cytokine production by normal T cells. The spontaneous myositis, which resembles human idiopathic myositis, is characterized by various abnormalities in skeletal muscle, including infiltration with inflammatory cells consisting primarily of macrophages. The participation of different cytokines in the pathogenesis of the lymphoma and the massive invasion of macrophages into muscle tissues led us to investigate the possible involvement of nitric oxide (NO.), which is known to be synthesized by activated macrophages under inflammatory conditions. Elevated NO. production, measured by urinary nitrate excretion, by SJL mice in comparison with BALB/c control mice was observed as early as 7 weeks of age. Both aging and degree of spontaneous myositis correlated with increased nitric oxide production. Oral administration of N-monomethyl-L-arginine, an inhibitor of nitric oxide synthase (NOS), reduced urinary nitrate excretion and also the severity of myositis. Immunohistochemical analysis revealed the presence of inducible NOS (iNOS) in cells in the spleen, lymph nodes, and skeletal muscle. The iNOS is primarily responsible for the enhanced nitric oxide production. Morphology of cells that stained positive for iNOS was similar to that of macrophages infiltrating into the affected tissues. Chronic production of elevated amounts of nitric oxide by the SJL mice, therefore, provides a useful in vivo model for future studies of cellular damage resulting from endogenously produced NO.in combination with oxygen radicals.

Amino Acid Oxidoreductases↗
PubMed1994

Nitric oxide production is intensely and persistently increased in tissue by thermal injury.

NO is a major messenger molecule which regulates immunity and vascular tone, serves as a neurotransmitter and participates in wound healing. It has also been known to be increased in vivo by thermal injury. Urinary nitrate excretion and tissue levels of NO synthase activity were measured after a non-lethal thermal injury. Urinary nitrate excretion decreased by 90% 24-48 h after injury, but dramatically increased by 10-fold thereafter for 30-40 days after injury and remained elevated until the wound healed. This response was inhibited by a competitive inhibitor of NO synthase. These rates of urinary nitrate excretion suggest that NO production is dramatically affected by injury in a pattern that is distinct from that observed after lipopolysaccharide administration. On the basis of these findings, we suggest that the hyperdynamic cardiovascular and hypermetabolic responses seen to continue weeks after thermal injury could be a result of the autocrine and paracrine effects of NO generated locally within the tissues in addition to that generated by inflammatory cells.

Amino Acid Oxidoreductases↗
PubMed1993

Separation of rare cell subpopulations with the aid of biotin-labelled ligands.

A universal method for selection of surface marker-positive cells is described. The cells, admixed with an excess of surface marker-negative cells, are first labelled with a specific biotinylated ligand and then isolated with the aid of monoclonal, anti-biotin coated beads. The method enables selection and isolation of cells with a frequency as low as 10(-4). The ligand can be an antigen (for selection of infrequent antibody-producing cells), an antibody (for selection of surface antigen-positive cells) or other molecules (for selection of specific receptor-positive cells).

Animals↗
PubMed1993

alpha 1-Antitrypsin- and anchorage-independent growth of MCF-7 breast cancer cells.

alpha 1-Antitrypsin (alpha 1-AT) and alpha 1-antichymotrypsin (alpha 1-ACHY) are closely related protease inhibitors, synthesized primarily by the liver, which play major roles in modulation of the inflammatory response. Previously, we had shown that MCF-7 human breast cancer cells were able to synthesize active alpha 1-AT and alpha 1-ACHY and that the synthesis of both inhibitors varied among different MCF-7 sublines. We now show that when MCF-7(ML) cells (a subline synthesizing low levels of alpha 1-AT) are grown in soft agar in medium depleted of its trypsin inhibitory capacity (i.e. alpha 1-AT-free), addition of alpha 1-AT (50 micrograms/ml) significantly reduces colony formation in both the presence and absence of estradiol (34% and 44%, respectively). Under these conditions, incubation with 10(-7) M estradiol alone increased colony formation 2- to 3-fold. Colony formation was also significantly reduced by serum leukocyte protease inhibitor, which, like alpha 1-AT, is a potent inhibitor of elastase-like enzymes. We also found that a variety of inflammatory mediators, cytokines, and steroid hormones are able to stimulate synthesis of alpha 1-AT and alpha 1-ACHY by MCF-7 cells. Stimulation by interleukin-6 (IL-6; 200 U/ml), epidermal growth factor (4 nM), and estradiol (10(-7) M) was 2- to 3-fold, whereas stimulations by tetradecanoylphorbol-13-acetate (TPA; 80 nM) and IL-1 (10 U/ml) were 2- to 5-fold and 5- to 10-fold, respectively. In each instance, protein synthesis, monitored by immunoprecipitation of 35S-labeled proteins followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and steady state mRNA levels, monitored by Northern blot analysis with specific cDNA probes, increased to the same extent. Consistent with their ability to stimulate alpha 1-AT synthesis, TPA and IL-1 reduced colony formation in the absence of estradiol by 65% and 63%, respectively. In addition, the effects of both TPA and IL-1 could be reversed by antibody to alpha 1-AT. These results suggest that local synthesis of alpha 1-AT and possibly other protease inhibitors may be important in regulating the tumorigenic potential of MCF-7 breast cancer cells.

Breast Neoplasms↗
PubMed1990

Regulation of antitrypsin and antichymotrypsin synthesis by MCF-7 breast cancer cell sublines.

We have examined the synthesis of the protease inhibitors alpha 1-antitrypsin (alpha 1-AT) and alpha 1-antichymotrypsin (alpha 1-ACHY) by variants of the MCF-7 human breast cancer cell line. Spent medium from MCF-7 203P cells, grown in the absence of serum, was found to contain immunoreactive alpha 1-AT and alpha 1-ACHY by Western blotting. In the presence of 10(-8) M estradiol, levels of both inhibitors were increased 3- to 6-fold. Incubation of spent medium with [125I]trypsin or [125I]chymotrypsin resulted in the formation of stable 75- and 90-kDa complexes identical to the complexes formed between these proteases and the protease inhibitors in plasma, showing the release of active protease inhibitors by MCF-7 cells in culture. Immunoprecipitation of 35S-labeled proteins from the medium of cells grown in the presence of [35S]methionine yielded comparable results, confirming hormonally sensitive synthesis of both protease inhibitors. Northern blot analysis suggests that stimulation of estradiol occurs at the level of transcription. Tetradecanoyl phorbol acetate (50 ng/ml) also stimulated alpha 1-AT and alpha 1-ACHY synthesis 2- to 4-fold, suggesting the involvement of protein kinase-C. Comparison studies with MCF-7 cell sublines ML, BK, 203P, and 300P (a variant spontaneously appearing after 100 passages of 203P) show a wide variation in synthesis of alpha 1-AT and alpha 1-ACHY proteins; sublines 203P and 300P synthesize both inhibitors, the ML subline synthesizes detectable amounts only of alpha 1-ACHY, while no detectable synthesis of either inhibitor was seen in the BK subline. Similar results were obtained for protease inhibitor mRNA transcription by Northern blotting, although low levels of alpha 1-AT mRNA transcription by the ML subline and of alpha 1-AT and alpha 1-ACHY mRNA transcription by the BK subline could be detected.

Blotting, Western↗
PubMed1988

Human monoclonal anti-idiotypic antibodies. I. Establishment of immortalized cell lines from a tumor patient treated with mouse monoclonal antibodies.

Patients who undergo immunotherapy with a murine anti-colon carcinoma mAb (mAb17-1A) generate high titers of anti-idiotype and anti-isotype antibodies. Specifically selected anti-idiotypic antibodies that elicit in vivo a humoral and a cellular immune response against the nominal Ag can be used as surrogate Ag for immunization. We established from the B lymphocytes of a treated patient a series of EBV-transformed cell lines. Three weeks after immortalization, the cells were selected for production of antibodies (Ab2) against the Fab fragment of the murine mAb17-1A. The selected cells were cloned and screened by ELISA for specific anti-mAb17-1A idiotypic antibodies. Thirty-six out of 89 clones were anti-idiotypes. Cell culture supernatants and the purified Ig derived from 10 clones completely inhibited the specific binding of radiolabeled mAb17-1A to HT-29 colon carcinoma cells thus resembling Ab2-gamma anti-idiotypes. These cell lines which grow now in culture for 18 mo, continuously secrete IgG,K anti-Ab1-idiotype mAb. Human anti-idiotypic mAb might be candidates for vaccines when the nominal Ag itself is not available or cannot be used as such.

Adenocarcinoma↗