Biphasic Activity Research Articles

Cyclophilin A as a Novel Biphasic Mediator of Endothelial Activation and Dysfunction

Inflammation-mediated endothelial cell (EC) dysfunction likely contributes to the pathogenesis of several vascular diseases including atherosclerosis. We found that stimulation of human umbilical vein ECs with lipopolysaccharide induced secretion of cyclophilin (CyPA) an intracellular protein belonging to the immunophilin family. We then found that when added exogenously CyPA has direct effects on ECs in vitro. At low concentrations (10 to 100 ng/ml) CyPA increased EC proliferation, migration, invasive capacity, and tubulogenesis. Gelatin zymography indicated increased secretion of active matrix metalloproteinase-2, a mediator of cell migration and angiogenesis. At high concentrations (eg, 2 microg/ml) CyPA had opposite effects, decreasing EC migration and viability, possibly in relation to induction of Toll-like receptor-4 expression, detected by immunocytochemistry and flow cytometry. In vivo CyPA expression was not detectable in the luminal ECs of normal mouse carotid arteries but was rapidly induced after systemic lipopolysaccharide injection. In an experimental mouse model of atherosclerosis, CyPA expression was detected in the ECs of neocapillaries of carotid artery lesions, supporting its association with pathological angiogenesis suggested by our in vitro results. In conclusion, we found that CyPA has a biphasic activity on ECs in vitro and is up-regulated in vivo in ECs under pathological states. Our results suggest that CyPA is a novel paracrine and autocrine modulator of EC functions in immune-mediated vascular disease.

Investigations of the methanolic leaf extract of Costus afer. Ker for pharmacological activities in vitro and in vivo.

The methanolic leaf extract of Costus afer. Ker (family: Zingiberaceae) was investigated for some pharmacological effects in vivo and in vitro. Brine shrimp lethality test showed that the extract was significantly (p < 0.05) cytotoxic with LC50 of 21.3 ppm. The extract showed moderate local anesthetic property, about twice less than lignocaine of the same concentration, on guinea pig wheal test. The extract contracted the guinea pig ileum in a concentration-dependent manner, but had no effect on pleuripara and nullipara non-gravid uteri at progestogenic and estrogenic phases respectively. The contractile effect on the guinea pig ileum was partially inhibited by atropine but completely reversed by adrenaline. The extract induced expulsion of whole fetuses still enveloped within the placental membrane at the 3rd trimester of pregnancy. The extract exhibited a biphasic antihyperglycemic activity. At 200 mg/kg body wt., p.o., it decreased the blood glucose level by 50% in Streptozotocin-induced hyperglycemia in male rats in 60 minutes post dosing. However, doses above 200 mg/kg body wt., p.o., caused increase in blood glucose level, potentiating the action of STZ. At 10 microg/ml the extract induced about 98% glucose uptake in differentiated 3T3-L1 adipocytes when compared with insulin (340 nm).

Lack of dose-responsive effect of dietary phyto-oestrogens on transepithelial calcium transport in human intestinal-like Caco-2 cells.

Ca absorption has been shown to be unaffected by high luminal concentrations of two commonly consumed soyabean phyto-oestrogens (PO) (genistein and daidzein) in Caco-2 cells grown under oestrogen-depleted conditions. However, these compounds exhibit dose-dependent biphasic effects in some tissues, such as reproductive tissue and bone. Thus, in light of this biphasic activity, the effect of lower concentrations of genistein and daidzein on Ca absorption requires further investigation. Therefore, the aim of the present study was to investigate the effect of a range of concentrations of genistein and daidzein on Ca absorption in the human Caco-2 intestinal-like cell model. Caco-2 cells were seeded onto permeable filter supports and allowed to differentiate into monolayers. On day 21, the Caco-2 monolayers (n 12 per treatment), grown in oestrogen-deplete media, were then exposed to 10 nm-1,25-dihydroxycholecalciferol (1,25 (OH)2D3), or 1, 10 and 50 microm-genistein or -daidzein for 24 h. After exposure, transepithelial and transcellular transport of (45)Ca and fluorescein transport were measured. As expected, 1,25 (OH)2D3 stimulated Ca absorption in Caco-2 cells, by up regulating transcellular transport. Ca absorption was unaffected by either PO at luminal concentrations of 1, 10 or 50 microm, typical of intakes by Western and Asian populations as well as supplemental levels, respectively. The results of this model suggest that the proposed beneficial effects of supplemental levels of these PO compounds on bone mass in postmenopausal women more probably arise from direct effects on bone cells, and not by an indirect effect of these compounds on Ca absorption.

Possible mechanisms of action of the neutral extract from Bidens pilosa L. leaves on the cardiovascular system of anaesthetized rats.

The aim of this study was to investigate the hypotensive and cardiac effects of the neutral extract from Bidens pilosa leaves. Intravenous administration of the extract resulted in a biphasic dose-related hypotensive activity. In normotensive rats (NTR), B. pilosa decreased systolic blood pressure by 18.26%, 42.5% and 30% at doses of 10, 20 and 30 mg/kg, respectively. In spontaneously hypertensive rats (SHR), the decrease in systolic blood pressure was 25.77%, 38.96% and 28.64% at the above doses, respectively. These doses induced hypotension by 27%, 34.13% and 18.73% respectively in salt-loaded hypertensive rats. In NTR, B. pilosa reduced heart rate by 23.68% and 61.18% at doses of 20 and 30 mg/kg, respectively. The force of contraction of the heart was only affected at 30 mg/kg. The initial phase of hypotensive response was partially inhibited by atropine while propranolol increased this effect. These results suggest that B. pilosa exhibited its fi rst hypotensive effects by acting on the cardiac pump efficiency and secondly through vasodilation.

Bioactivity of falcarinol and the influenceof processing and storage on its content in carrots (Daucus carota L)

AbstractThe concentration‐dependent activity of the polyacetylene falcarinol ((9Z)‐heptadeca‐1,9‐dien‐4,6‐diyn‐3‐ol), isolated from carrots, was investigated in a bioassay with primary mammary epithelial cells in collagen gels and compared with that of β‐carotene, the orange pigment in carrots. Falcarinol showed biphasic activity, having stimulatory effects between 0.01 and 0.05 µg ml−1 and inhibitory effects between 1 and 10 µg ml−1, whereas β‐carotene showed no effect in the concentration range 0.001–100 µg ml−1. The results are discussed in relation to the health‐promoting effects of carrots and related vegetables. Falcarinol was quantified in the carrot cultivars Bolero, Rodelika and Fancy by analytical reverse phase HPLC, subjected to various processing and storage conditions in order to study how long‐term storage, blanching, freezing and boiling influence the content of falcarinol. Long‐term storage of raw carrot cubes (1 cm3) reduced the falcarinol content by almost 35%. A similar reduction was found in steam‐blanched carrot cubes (1 cm3). Long‐term storage at −24 °C of steam blanched carrot cubes did not reduce the falcarinol content further. A reduction of almost 70% in the falcarinol content was found in carrot pieces boiled in water for 12 min compared with raw carrots. Copyright © 2003 Society of Chemical Industry

Dose-dependent Biphasic Activity of tRNA Synthetase-associating Factor, p43, in Angiogenesis

Mammalian aminoacyl tRNA synthetases form a macromolecular protein complex with three non-enzymatic cofactors. Among these factors, p43 is also secreted to work as a cytokine on endothelial as well as immune cells. Here we investigated the activity of p43 in angiogenesis and determined the related mediators. It promoted the migration of endothelial cells at low dose but induced their apoptosis at high dose. p43 at low concentration activated extracellular signal-regulating kinase, which resulted in the induction and activation of matrix metalloproteinase 9. In contrast, p43 at high concentration activated Jun N-terminal kinase, which mediated apoptosis of endothelial cells. These results suggest that p43 is a novel cytokine playing a dose-dependent biphasic role in angiogenesis.

Epaxial muscle function in trotting dogs.

One of the features that distinguish mammals from other groups of terrestrial vertebrates is the structure and relative size of their epaxial muscles. Yet we have only a superficial understanding of the role these muscles play in locomotion. To address their locomotor function, we recorded the electrical activity of the iliocostalis, longissimus dorsi and multifidus muscles of trotting dogs. Activity was monitored at both lumbar and thoracic sites. To develop and evaluate hypotheses of epaxial muscle function, we quantified footfall patterns and sagittal trunk kinematics from high-speed videos, and the magnitude and orientation of ground reaction forces from force-plate recordings. All three epaxial muscles tended to exhibit a double-bursting (biphasic) activity pattern, with the exception of the iliocostalis muscle at the thoracic site (which was uniphasic). In general, a large burst of activity in each muscle occurred during the second half of the support phase of the ipsilateral hindlimb, and was active for an average of 30% of the locomotor cycle. A smaller burst of activity occurred during the second half of the support phase of the contralateral hindlimb, and was active for an average of 15% of the locomotor cycle. Analysis of ground reaction forces and sagittal trunk kinematics led us to the hypothesis that the epaxial muscles do not directly stabilize the trunk against the vertical and horizontal components of the ground reaction force. Instead, the epaxial muscles appear to counteract the tendency of the trunk to rebound (flex) in the sagittal plane during the latter half of the support phase. This hypothesis of epaxial muscle function was supported by loading experiments performed on the longissimus dorsi muscle in the lumbar region.

The C1 domain of protein kinase C as a lipid bilayer surface sensing module.

The activity of membrane-associated protein kinase C (PKC) is tightly controlled by the physical properties of the membrane lipid bilayer, in particular, curvature stress, which is induced by bilayer-destabilizing lipid components. An important example of this is the weakened lipid headgroup interactions induced by phosphatidylethanolamine (PE) and cholesterol. In this work our previous observation with a mixed isoform PKC showing a biphasic dependence of activity as a function of membrane curvature stress [Slater et al. (1994) J. Biol. Chem. 269, 4866-4871] was here extended to individual isoforms. The Ca(2+)-dependent PKCalpha, PKCbeta, and PKCgamma, along with Ca(2+)-independent PKCdelta, but not PKCepsilon or PKCzeta, displayed a biphasic activity as a function of membrane PE content. The fluorescence anisotropy of N-(5-dimethylaminonaphthalene-1-sulfonyl)dioleoylphosphatidylserine (dansyl-PS), which probes the lipid environment of PKC, also followed a biphasic profile as a function of PE content for full-length PKCalpha, PKCbetaIotaIota, and PKCgamma as did the isolated C1 domain of PKCalpha. In addition, the rotational correlation time of both PKCalpha and PKCdelta C1-domain-associated sapintoxin D, a fluorescent phorbol ester, was also a biphasic function of membrane lipid PE content. These results indicate that the C1 domain acts as a sensor of the bilayer surface properties and that its conformational response to these effects may directly underlie the resultant effects on enzyme activity.

Impact of certain flavonoids on lipid profiles--potential action of Garcinia cambogia flavonoids.

Flavonoids from Cocos nucifera, Myristica fragrance, Saraka asoka and Garcinia cambogia exerted hypolipidaemic activity in rats. Lipid lowering activity was maximum in rats administered flavonoids (10 mg/kg BW/day) from Garcinia cambogia. A dose response study revealed biphasic activity. Higher doses were less effective in reducing lipid levels in serum and tissues, although devoid of toxic effects.

Cell Cycle Regulation of NF-κB-Binding Activity in Cells from Human Glioblastomas

Glioblastoma multiforme is a highly malignant and anaplastic tumor of the central nervous system representing more than 50% of all malignant gliomas. The cell origin of this highly undifferentiated tumor remains obscure, although it is postulated that glioblastomas are developed from astrocytes. The rapid growth of the glioma and the state of its undifferentiation are attributed to the deregulation of several signal transduction pathways and cell cycle events. Recent studies showed diverse functions for the NF-κB/Rel family of inducible transcription factors including differentiation, apoptosis, oncogenesis, and cell cycle regulation. We sought to examine the level of NF-κB activity throughout the glioma's cell cycle. Results from band-shift studies indicated a biphasic NF-κB DNA-binding activity in the nuclei of cycling glioblastoma cells. We showed that NF-κB-binding activity maximizes in nuclear extracts at specific cell cycle stages including G0/G1, mid-late G1, and S phase. Results from Northern blotting studies revealed that the differential expression of the NF-κB subunits, p50 and p65, may not be responsible for cell cycle stage-specific association of NF-κB subunits with DNA. However, results from Western blotting analysis utilizing nuclear extracts from glioma cells throughout the cell cycle demonstrated that the nuclear accumulation of p50 and p65 perfectly correlates with their DNA-binding activity. These observations suggest that the nuclear translocation of the p50/p65 subunit of NF-κB in glioma cells is cell cycle stage-dependent and that is distinct from the differential mRNA expression of these genes during glioma cell cycling. The possible role of NF-κB in glioma cell formation and regulation of cellular genes by NF-κB in these tumor cells is discussed.

C-Terminal glycine is crucial for hyperalgesic activity of nociceptin/orphanin FQ-(1–6)

A C-terminal analog of the hexapeptide orphanin FQ/nociceptin-(1–6), [Ala 6]-orphanin FQ/nociceptin-(1–6), and a pentapeptide orphanin FQ/nociceptin-(1–5) were tested in vivo for their analgesic/hyperalgesic activity in the hot-plate test with rats. Replacement of the C-terminal glycine by l-alanine (Phe-Gly-Gly-Phe-Thr-Ala) in orphanin FQ/nociceptin-(1–6) abolished the hyperalgesic potency of native orphanin FQ/nociceptin-(1–6) (Phe-Gly-Gly-Phe-Thr-Gly), but analgesic activity was retained and was diminished by naloxone. Removal of the C-terminal amino acid (glycine or alanine) from orphanin FQ/nociceptin-(1–6) caused a significant loss of analgesic activity. It is anticipated that glycine plays a crucial role in the biphasic activity of orphanin FQ/nociceptin-(1–6). This may suggest the existence of a mechanism for terminating the biological action of orphanin FQ/nociceptin.

Home Range and Habitat Use of Male Rafinesque's Big-eared Bats (Corynorhinus rafinesquii)

Abstract We examined home range size and habitat use of four reproductively active male Rafinesque's big-eared bats (Corynorhinus rafinesquii) in an area of the Upper Coastal Plain of South Carolina during August and September 1999. Corynorhinus rafinesquii had biphasic activity patterns, with most foraging activity occurring during the first 4 h after sunset and 2 h before sunrise. Mean home range size calculated using the adaptive kernel method with a 95% use distribution was 93.1 ha. Although large contiguous tracts of mature bottomland hardwoods were common in the study area, most foraging activity occurred in young pine stands. Only 9% of foraging areas were in bottomland hardwoods.

Biphasic regulation of NF-kappa B activity underlies the pro- and anti-inflammatory actions of nitric oxide.

Expression of inducible NO synthase (iNOS) by macrophages is a prerequisite for the production of high output NO, which mediates many bactericidal and tumoricidal actions of these immune cells. The expression of iNOS in mammalian cells is governed predominantly by the transcription factor, NF-kappa B, which regulates the expression of many host defense proteins. In the present study, we characterize a novel, biphasic effect of NO on NF-kappa B activity in murine macrophages. This mechanism depends on the local concentration of NO and enables it both to up- and down-regulate the expression of host defense proteins including iNOS, cyclooxygenase-2, and IL-6. This biphasic activity of NO appears to play a pivotal role in the time course of activation of these immune cells and, by inference, in facilitating the initiation of a defense response against pathogenic stimuli and in its termination to limit tissue damage. This mechanism may explain at least in part the reported ability of NO to act in both a pro- and anti-inflammatory manner.

Activation of mitochondrial ATP-dependent protease by peptides and proteins.

We examined the effect of peptides or protein on the proteolytic and ATPase activities of mitochondrial ATP-dependent LON protease purified from bovine adrenal cortex. Peptides/proteins including angiotensin I which stimulated ATPase activity without hydrolysis of any peptide bonds stimulated proteolysis of 125I-labeled substrates at low concentrations; whereas at high concentrations they competitively inhibited proteolysis, thus displaying a biphasic activity profile. All peptides and proteins thus examined stimulated degradation of 125I-labeled substrates. When an ATP analog was substituted for ATP, only inhibition; i.e., no stimulation, of proteolysis by unlabeled peptides was observed. Without activator peptides, degradation of [125I] peptides was higher in the presence of an ATP analog than that in the presence of ATP. ADP, a product of the ATPase reaction, inhibited the proteolytic activity in the absence of an activator peptide but not in its presence. From analogy to E. coli ATP-dependent protease La (LON), we suggest that the activator peptides stimulated the proteolysis by releasing enzyme-bound ADP.

Effect of Age and Gender in the Control of Elbow Flexion Movements

In previous studies of rapid elbow movements in young healthy men, characteristic task-dependent changes in the patterns of muscle activation when movement speed or distance was varied have been reported. In the present study, the authors investigated whether age or gender is associated with changes in the patterns of muscle activity previously reported in young men. Arm movements of 10 healthy older and 10 healthy younger participants (5 men and 5 women in each group) were studied. Surface electromyograms (EMGs) from agonist (biceps) and antagonist (triceps) muscles, kinematic and kinetic parameters, as well as anthropometric and strength measures were recorded. All 4 groups of participants showed similar task- (distance or speed) dependent changes in biphasic EMG activity. Similar modulation of the initial rate of rise of the EMG, integrated agonist and antagonist EMG activity, as well as their relative timing were observed in all 4 groups. Those results suggest that older individuals of both genders retain the control strategies for elbow movements used by young individuals. Despite the qualitative similarities in the patterns of muscle activation, the men moved more quickly than the women, and younger participants moved more quickly than older participants. Those performance differences could not be explained in terms of differences in body size and strength alone.

Depression of neuronal firing rates in somatosensory and posterior parietal cortex during object acquisition in a prehension task.

Prehension is an object-oriented behavior consisting of four components: reach, grasp, manipulation, and release. To determine how such actions are represented in primary somatosensory (S-I) and posterior parietal cortex (PPC), we used digital video to synchronize spike trains of neurons recorded in Brodmann's areas 3b, 1, 2, 5, and 7 with the hand kinematics as monkeys performed a prehension task. Statistical analyses indicated that one-third of task-modulated neurons showed significantly depressed firing rates during object acquisition and/or manipulation. This population was dominated by neurons innervated by deep receptors that sensed extension movements of the fingers, or by tactile receptors in hairy skin sensing stretch. Grasp-inhibited responses were the most common type. Tonic firing rates of these cells dropped significantly during approach as the hand was preshaped for grasping, or at contact when grasp was initiated, and persisted until hand motion ceased or as the grip relaxed. Maximum suppression of firing occurred at grasp completion. Their lack of specificity for particular hand behaviors formed the inhibitory counterpart of broadly tuned cells that fired prolonged bursts during grasp and manipulatory stages of prehension. The remainder of the task-inhibited population showed biphasic responses. Firing rates were significantly depressed during grasping and manipulation when the hand interacted directly with the object, but were enhanced prior to contact, when the hand was preshaped (approach-tuned), or upon relaxation of grasp and release of the object from the hand (loweror relax-tuned). Grasp-inhibited responses occurred primarily in S-I, whereas biphasic inhibitory activity was recorded mainly in PPC. Suppression of activity within these populations may thereby increase the saliency of excitatory responses to acquisition and manipulation of objects. Reduction of firing during prehension might also signal the flexed postures used to retain objects in the hand, rather than a generalized gating of sensory information. The similarity of responses to active and passive extension movements suggests that the inhibitory responses may provide important postural and motor information about the hand kinematics when performing skilled tasks.

Sex differences in locomotor activity following β-endorphin in the ventrolateral periaqueductal gray

Both morphine and selective opioid receptor agonists produce typical biphasic effects upon activity in male rats with initial reductions in activity followed by subsequent increases in activity. Sex differences have been observed in a number of opioid-mediated responses, including the observation that microinjection of the opioid peptide, β-endorphin into the ventrolateral periaqueductal gray (vlPAG) produces more pronounced analgesia in male rats relative to female rats. The present study evaluated whether β-endorphin (5.2–26 μg) in the vlPAG produced biphasic activity effects in male and female rats. Both male and female rats displayed initial reductions in total, ambulatory, and stereotypic activity following β-endorphin in the vlPAG. Whereas male rats displayed subsequent (90–120 min) increases in total and ambulatory activity following β-endorphin in the vlpAG, female rats tested during the estrous stage of their estrous cycle failed to display any changes. These sex differences in the opioid modulation in the pattern of activity measures in the vlPAG are discussed in terms of the roles of sex hormones in opioid peptide processing within the vlPAG.

Impact of splenectomy and immunochemotherapy on survival following gastrectomy for carcinoma: Covariate interaction with immunosuppressive acidic protein, a serum marker for the host immune system

The role of the spleen in tumor immunology is still controversial in that it can either enhance or suppress the antitumor immune response depending on the tumor-bearing host. To clarify this biphasic effect of the spleen, a clinical evaluation of splenectomy in conjunction with immunotherapy and the host immune status was performed in gastric cancer patients. The effect of splenectomy and immunotherapy in 253 gastric cancer patients enrolled in a prospective randomized trial (SIP) was analyzed using the Cox's proportional hazards model in terms of the covariate interaction of the preoperative immunosuppressive acidic protein (IAP) level. In patients with high IAP levels (>580 microg/ml) with predicted negative antitumor immune reactions, splenectomy improved the prognosis. In patients with lower IAP values, conversely, the preservation of the spleen and immunotherapy demonstrated a significant benefit to survival. The spleen was shown to have a biphasic activity in terms of its antitumor immune response depending on the IAP level of the patient. The effect of immunotherapy is significantly influenced by the activity of spleen cells. The preoperative IAP level is therefore considered to be a possible indicator for the effectiveness of splenectomy and immunotherapy in curatively resected gastric cancer patients.

Biphasic extracellular proteolytic enzyme activity in benthic water and sediment in the northwestern mediterranean Sea

In this study, we used the fact that bacteria are able to cleave a fluorogenic substrate analog (L-leucine-7-amido-4-methylcoumarin) to determine the maximal ectoproteolytic activities (Vm) and affinities (Km) of natural benthic microbial communities by the multiconcentration kinetic method. This investigation was performed during the winter and summer of 1997 with a set of 36 samples of near-bottom water and sediment collected from a coastal area and an offshore area in the western part of the Gulf of Lions. The existence of biphasic microbial ectoproteolysis was statistically confirmed for both the near-bottom water and the sediment, regardless of the spatial and seasonal conditions. Globally, 72.2% of the entire set of bacterial consortia collected at the water-sediment boundary layer showed biphasic microbial kinetics. A specific estimator of the biphasicity indicated that deep benthic bacterial consortia responded better with episodic nutrient supplies than shallower benthic bacterial consortia responded.

Meso-1,2-Bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine]dichloroplatinum(II), a Compound with a Specific Activity on Hormone-Sensitive Breast Cancers - Evidence for a Diethylstilbestrol-Like Mode of Action

[meso-1,2-Bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine]-dichloroplatinum(II) (meso-1-PtCl2), an estrogenic and cytotoxic platinum complex, shows activity against ER+ but not against ER-breast cancers in vivo (ER: estrogen receptor; ER+ and ER- indicate the presence or absence of the ER). To clarify whether its estrogenic or its cytotoxic potency or both properties are the cause of this specific inhibitory effect, we tested meso-1-PtCl2 comparatively in vivo on an ER+ and an ER- murine breast cancer (MXT-M-3.2 MC and MXT-M-3.2(ovex) MC, respectively), and in vitro on two cell lines derived from the former in vivo models (MXT+ and MXT- , respectively). The estrogens diethylstilbestrol (DES) and the ligand of meso-1-PtCl2 (meso-1), responsible for the hormonal effect of meso-1-PtCl2, and the cytotoxic drug cisplatin (cDDP) were used as comparative substances. Meso-1-PtCl2, DES and cDDP showed a strong and comparable activity on the ER+ MXT-M-3.2 MC in vivo, meso-1 being somewhat less inhibitory. In experiments on the murine, ER- MXT-M-3.2(ovex) MC only cDDP caused a marked inhibitory effect. The other compounds were inactive or only marginally active. In accordance with the in vivo results cDDP was also very active on the MXT+ and MXT- breast cancer cell line. In contrast to this meso-1-PtCl2, meso-1, and DES proved to be only weakly active or inactive on both cell lines. From these results it can be concluded that there is only little if any contribution of the cytotoxic PtCl2 moiety of meso-1-PtCl2 to the anti-breast cancer activity in vivo. On the ER+ MXT-M-3.2 MC transplanted into ovariectomized mice meso-1-PtCl2 yielded a biphasic dose activity curve, i.e. an increase of the tumor growth at low doses followed by a decrease at high doses, identical with those of the estrogens DES and meso-1. These results indicate that meso-1-PtCl2 inhibits ER+ breast cancers by its estrogenicity in the same manner as meso-1 and DES. The complex mechanism of anti-breast cancer active estrogens involves presumably the endocrine and/or the immune system. Its investigation is the subject of further studies.