Abnormal functional connectivity in radiologically isolated syndrome: A resting-state fMRI study.

BACKGROUND: Radiologically isolated syndrome (RIS) patients might have psychiatric and cognitive deficits, which suggests an involvement of major resting-state functional networks. Notwithstanding, very little is known about the neural networks involved in RIS. OBJECTIVE: To examine functional connectivity differences between RIS and healthy controls using resting-state functional magnetic resonance imaging (fMRI). METHODS: Resting-state fMRI data in 25 RIS patients and 28 healthy controls were analyzed using an independent component analysis; in addition, seed-based correlation analysis was used to obtain more information about specific differences in the functional connectivity of resting-state networks. Participants also underwent neuropsychological testing. RESULTS: RIS patients did not differ from the healthy controls regarding age, sex, and years of education. However, in memory (verbal and visuospatial) and executive functions, RIS patients' cognitive performance was significantly worse than the healthy controls. In addition, fluid intelligence was also affected. Twelve out of 25 (48%) RIS patients failed at least one cognitive test, and six (24.0%) had cognitive impairment. Compared to healthy controls, RIS patients showed higher functional connectivity between the default mode network and the right middle and superior frontal gyri and between the central executive network and the right thalamus (pFDR < 0.05; corrected). In addition, the seed-based correlation analysis revealed that RIS patients presented higher functional connectivity between the posterior cingulate cortex, an important hub in neural networks, and the right precuneus. CONCLUSION: RIS patients had abnormal brain connectivity in major resting-state neural networks and worse performance in neurocognitive tests. This entity should be considered not an "incidental finding" but an exclusively non-motor (neurocognitive) variant of multiple sclerosis.

Real-Time H2 O2 Measurements in Bone Marrow Mesenchymal Stem Cells (MSCs) Show Increased Antioxidant Capacity in Cells From Osteoporotic Women.

Oxidative stress (OS) derived from an increase in intracellular reactive oxygen species (ROS) is a major determinant of aging and lifespan. It has also been associated with several age-related disorders, like postmenopausal osteoporosis of Mesenchymal stem cells (MSCs). MSCs are the common precursors for osteoblasts and adipocytes; appropriate commitment and differentiation of MSCs into a specific phenotype is modulated, among other factors, by ROS balance. MSCs have shown more resistance to ROS than differentiated cells, and their redox status depends on complex and abundant anti-oxidant mechanisms. The purpose of this work was to analyze in real time, H2 O2 signaling in individual h-MSCs, and to compare the kinetic parameters of H2 O2 management by cells derived from both control (c-) and osteoporotic (o-) women. For these purposes, cells were infected with a genetically encoded fluorescent biosensor named HyPer, which is specific for detecting H2 O2 inside living cells. Subsequently, cells were sequentially challenged with 50 and 500 µM H2 O2 pulses, and the cellular response was recorded in real time. The results demonstrated adequate expression of the biosensor allowing registering fluorescence from HyPer at a single cell level. Comparison of the response of c- and o-MSCs to the oxidant challenges demonstrated improved antioxidant activity in o-MSCs. This was further corroborated by measuring the relative expression of mRNAs for catalase, superoxide dismutase-1, thioredoxine, and peroxiredoxine, as well as by cell-surviving capacity under short-term H2 O2 treatment. We conclude that functional differences exist between healthy and osteoporotic human MSCs. The mechanism for these differences requires further study. J. Cell. Biochem. 118: 585-593, 2017. © 2016 Wiley Periodicals, Inc.

Characterization of Fatty Acid Composition in Bone Marrow Fluid From Postmenopausal Women: Modification After Hip Fracture.

Bone marrow adipose tissue (BMAT) is associated with low bone mass, although the functional consequences for skeletal maintenance of increased BMAT are currently unclear. BMAT might have a role in systemic energy metabolism, and could be an energy source as well as an endocrine organ for neighboring bone cells, releasing cytokines, adipokines and free fatty acids into the bone marrow microenvironment. The aim of the present report was to compare the fatty acid composition in the bone marrow supernatant fluid (BMSF) and blood plasma of postmenopausal women women (65-80 years old). BMSF was obtained after spinning the aspirated bone marrow samples; donors were classified as control, osteopenic or osteoporotic after dual-energy X-ray absorptiometry. Total lipids from human bone marrow fluid and plasma were extracted, converted to the corresponding methyl esters, and finally analyzed by a gas chromatographer coupled with a mass spectrometer. Results showed that fatty acid composition in BMSF was dynamic and distinct from blood plasma, implying significance in the locally produced lipids. The fatty acid composition in the BMSF was enriched in saturated fatty acid and decreased in unsaturated fatty acids as compared to blood plasma, but this relationship switched in women who suffered a hip fracture. On the other hand, there was no relationship between BMSF and bone mineral density. In conclusion, lipid composition of BMSF is distinct from the circulatory compartment, most likely reflecting the energy needs of the marrow compartment. J. Cell. Biochem. 117: 2370-2376, 2016. © 2016 Wiley Periodicals, Inc.

Osteoporosis-associated alteration in the signalling status of BMP-2 in human MSCs under adipogenic conditions.

Postmenopausal osteoporosis is characterized by decreased bone quality and mineral density. Mesenchymal stem cells (MSCs) found in the bone marrow, are pluripotent cells able to differentiate into several phenotypes, including osteoblasts and adipocytes. In osteoporosis, MSCs' commitment and differentiation into osteoblast/adipocyte is unbalanced, favoring adipocyte formation. The osteo and adipogenic processes are modulated by the bone morphogenetic protein-2 (BMP-2). This cytokine regulates the expression of transcription factors PPARγ and Runx 2, but its action on cells under adipogenic conditions is poorly understood. In this work we studied BMP-2 signaling in MSCs obtained from bone marrow of control or osteoporotic volunteer postmenopausal women. MSCs were cultured under basal, adipogenic (AD) or AD plus BMP-2 conditions. The protein content of PPARγ, p-PPARγ, Runx2, bone morphogenetic receptor IA (BMPR IA), phosphorylated Smad-1/5/8 (p-Smad) and Smad 4 were determined by specific western blots. mRNA level for BMPRs was determined by PCR and cell localization of p-Smad-1/5/8 were detected by immunocytochemistry. Control MSCs showed a differential response to both AD and AD plus BMP-2 treatments: BMP-2 exerted an anti-adipogenic effect increasing both transcription factors analyzed. Moreover, p-Smads-1/5/8 were detected in nuclei after short term BMP-2 treatment. Osteoporotic MSCs showed no response to exogenous added BMP-2, as shown by p-PPARγ/PPARγ ratio and Runx2 levels, although BMPR-IA level was significantly higher in osteoporotic than in control MSCs. In addition, staining for p-Smad-1/5/8 in o-MSCs was observed around nuclei at all experimental conditions. Taken together results demonstrate failure of BMP-2 signaling in osteoporotic MSCs.

Preparation of synthetic micro- and nano plastics for method validation studies.

Microplastic (MP) pollution is a persisting global problem. Accurate analysis is essential in quantifying the effects of microplastic pollution and develop novel technologies that reliably and reproducibly measure microplastic content in various samples. The most common methods for this are FTIR and Raman spectroscopy. Coloured, standardized beads are often used for method validation tests, which limits the conclusions to a very specific case rarely observed in the natural environment. This study focuses on the preparation of reference micro- and nanoplastics via cryogenic milling and shows their use for FTIR and Raman method validation studies. MPs can now be reproducibly milled from various plastics, offering the advantages of a better representation of MPs in real environment. Moreover, this study highlights issues with the current detection methods, up to now considered as the most reliable ones for MP detection and identification. Such issues, e.g. misidentification, will need to be addressed in the future. Additionally, milled MPs were used in experiments with commercial high-resolution imaging device, enabling a possible in-situ optical detection of microplastics. These experiments represent a step forward in understanding MPs in a water sample and provide a basis for a more accurate detection and identification directly from water, which would considerably reduce the time of analysis.

An umbrella review of systematic reviews on interventions of physical activity before pregnancy, during pregnancy, and postpartum to control and/or reduce weight gain.

BACKGROUND: The increasing prevalence of overweight and obesity worldwide represents a (chronic) complex public health problem. This is also seen among women of childbearing age despite increased efforts to promote physical activity (PA) interventions. Excessive gestational weight gain (GWG) is associated with negative health outcomes for both mothers and offspring. OBJECTIVES: To summarize current systematic reviews (SRs) on PA interventions during pregnancy and postpartum to prevent excessive GWG and identify the most effective approaches. SEARCH STRATEGY: A literature search was conducted on major electronic databases (MEDLINE/Pubmed, Cochrane, Web of Science, Epistemonikos) from inception to March 2023. SELECTION CRITERIA: This study included SRs and meta-analyses of studies involving women aged 18 years or older from diverse ethnic backgrounds, who were either in the preconception period, pregnant, or within 1 year postpartum and who had no contraindications for exercise. Women with chronic diseases, such as pre-existing diabetes (type 1 or type 2) were excluded. DATA COLLECTION AND ANALYSIS: Two reviewers extracted data from selected studies assessing the impact of PA in preconception, pregnancy, and postpartum. Methodologic quality was assessed with the AMSTAR-2 tool. A narrative summary of results addresses relationships between PA and weight before, during, and after pregnancy, informing future research priorities for preventing excessive weight gain. This study is registered on PROSPERO (CRD420233946666). MAIN RESULTS: Out of 892 identified articles, 25 studies were included after removing duplicates, unrelated titles, and screening titles and abstracts for eligibility. The results demonstrate that PA can help prevent excessive GWG and postpartum weight retention. Structured and supervised moderate-intensity exercise, at least twice a week, and each session lasting a minimum of 35 min seems to provide the greatest benefits. CONCLUSIONS: Women who comply with the PA program and recommendations are more likely to achieve adequate GWG and return to their pre-pregnancy body mass index after delivery. Further research is warranted to explore how preconception PA influences pregnancy and postpartum outcomes given the absence of identified preconception-focused interventions.

Isolation, characterization, and immunomodulatory activity evaluation of probiotic strains from colostrum and canine milk.

Background: This study aimed to characterize potential probiotic strains for use in dogs to prevent infectious enteropathies. Lactic acid bacteria (LAB) isolated from canine milk and colostrum were characterized according to their functional properties, including their resistance to gastrointestinal conditions, inhibitory effect against pathogens, and intestinal adhesion. Methods: The immunomodulatory effects of the strains were also analyzed in in vitro and in vivo studies. Among the strains evaluated, two LAB strains (TUCO-16 and TUCO-17) showed remarkable resistance to pH 3.0, bile salts, and pancreatin, as well as inhibitory effects against pathogenic Escherichia coli, Salmonella sp., and Clostridium perfringens. Results: The TUCO-16 and TUCO-17 strains induced a significant increase in the expression of TNF-α, IL-8, and TLR2 in canine macrophages. The oral administration of TUCO-16 and TUCO-17 strains to mice significantly augmented their resistance to pathogenic E. coli or Salmonella intestinal infections. Both canine strains reduced intestinal damage and pathogen counts in the liver and spleen and avoided their dissemination into the bloodstream. These protective effects were related to the ability of TUCO-16 and TUCO-17 strains to differentially modulate the production of IFN-γ, IFN-ß, TNF-α, IL-6, KC, MCP-1, and IL-10 in the intestinal mucosa. Conclusion: Both strains, TUCO-16 and TUCO-17, are potential probiotic candidates for improving intestinal health in dogs, particularly for their ability to inhibit the growth of Gram-negative pathogens common in gastrointestinal infections and modulate the animal's immune response. Further studies are required to effectively demonstrate the beneficial effects of TUCO-16 and TUCO-17 strains in dogs.

In osteoporosis, differentiation of mesenchymal stem cells (MSCs) improves bone marrow adipogenesis.

The formation, maintenance, and repair of bone tissue involve close interlinks between two stem cell types housed in the bone marrow: the hematologic stem cell originating osteoclasts and mesenchymal stromal cells (MSCs) generating osteoblasts. In this review, we consider malfunctioning of MSCs as essential for osteoporosis. In osteoporosis, increased bone fragility and susceptibility to fractures result from increased osteoclastogenesis and insufficient osteoblastogenesis. MSCs are the common precursors for both osteoblasts and adipocytes, among other cell types. MSCs' commitment towards either the osteoblast or adipocyte lineages depends on suitable regulatory factors activating lineage-specific transcriptional regulators. In osteoporosis, the reciprocal balance between the two differentiation pathways is altered, facilitating adipose accretion in bone marrow at the expense of osteoblast formation; suggesting that under this condition MSCs activity and their microenvironment may be disturbed. We summarize research on the properties of MSCs isolated from the bone marrow of control and osteoporotic post-menopausal women. Our observations indicate that intrinsic properties of MSCs are disturbed in osteoporosis. Moreover, we found that the regulatory conditions in the bone marrow fluid of control and osteoporotic patients are significantly different. These conclusions should be relevant for the use of MSCs in therapeutic applications.

The increased expression of receptor activator of nuclear-kappaB ligand (RANKL) of multiple myeloma bone marrow stromal cells is inhibited by the bisphosphonate ibandronate.

The receptor activator of nuclear factor-kappaB ligand (RANKL) and interleukin-1beta are osteoclast activating factors which are abnormally expressed in bone marrow stromal cells and plasma cells of multiple myeloma patients. In this work we analyzed RANKL expression in human bone marrow mesenchymal stromal cells and the effect of the bisphosphonate ibandronate on RANKL expression after IL-1beta activation of ERK pathway. Mesenchymal stromal cells were obtained from bone marrow iliac aspirates from multiple myeloma patients at stages II/III and non-osteoporotics control donors; these cells were maintained under long-term culture conditions. Cells were cultured in the presence or the absence of 5 ng/ml IL-1beta and/or 5 microM ibandronate, during selected periods. mRNA for RANKL and protein levels were assayed by RT-PCR and Western blot, respectively. Human bone marrow stromal cell line HS-5 was used for assessing IL 1beta- and ibandronate-ERK phosphorylation responses. Multiple myeloma mesenchymal stromal cells differentiate from control cells by increased basal RANKL expression. IL-1beta up regulated RANKL expression showed dependent on activated MEK/ERK pathway. Finally, the bisphosphonate ibandronate, that hindered activation of the MEK/ERK pathway significantly inhibited both basal and IL-1beta dependent RANKL expression by cells. Results indicate that RANKL expression involves the MEK/ERK pathway in multiple myeloma mesenchymal stromal cells, and that early obstruction of this path, such as that achieved with ibandronate, significantly deters RANKL protein expression.

Update and New Epidemiological Aspects about Grapevine Yellows in Chile.

To date, phytoplasmas belonging to six ribosomal subgroups have been detected to infect grapevines in Chile in 36 percent of the sampled plants. A new survey on the presence of grapevine yellows was carried out from 2016 to 2020, and 330 grapevine plants from the most important wine regions of the country were sampled and analyzed by nested PCR/RFLP analyses. Phytoplasmas enclosed in subgroups 16SrIII-J and 16SrVII-A were identified with infection rates of 17% and 2%, respectively. The vineyards in which the phytoplasma-infected plants were detected were further inspected to identify alternative host plants and insects of potential epidemiological relevance. Five previously unreported plant species resulted positive for 16SrIII-J phytoplasma (Rosa spp., Brassica rapa, Erodium spp., Malva spp. and Rubus ulmifolius) and five insect species were fully or partially identified (Amplicephalus ornatus, A. pallidus, A. curtulus, Bergallia sp., Exitianus obscurinervis) as potential vectors of 16SrIII-J phytoplasmas. The 16SrVII-A phytoplasmas were not detected in non-grape plant species nor in insects. This work establishes updated guidelines for the study, management, and prevention of grapevine yellows in Chile, and in other grapevine growing regions of South America.

Feasibility and learning curve of unsupervised colorectal endoscopic submucosal hydrodissection at a Western Center.

OBJECTIVES: Colorectal endoscopic submucosal dissection (CR-ESD) is an evolving technique in Western countries. We aimed to determine the results of the untutored implementation of endoscopic submucosal hydrodissection for the treatment of complex colorectal polyps and establish the learning curve for this technique. METHODS: This study included data from 80 consecutive CR-ESDs performed by a single unsupervised western therapeutic endoscopist. To assess the learning curve, procedures were divided into four groups of 20 each. RESULTS: En bloc resection was achieved in 55, 75, 75 and 95% cases in the consecutive time periods (period 1 vs. 4, P = 0.003). Curative resection was achieved in 55, 75, 70 and 95%, respectively (P = 0.037). Overall, series results demonstrated R0 resection in 75% of cases, with 23.7% requiring conversion to endoscopic piecemeal mucosal resection, and 1.25% incomplete resections. Complications included perforations (7.5%) and bleeding (3.7%). Multivariate analysis revealed factors more likely to result in association with non en bloc vs. En bloc resection, where polyp size ≥35 mm [70 vs. 23.4%; odds ratio (OR) 13.2 (1.7-100.9); P = 0. 013], severe fibrosis [40 vs. 11.7%; OR 10.2 (1.2-86.3); P = 0.033] and where carbon dioxide for insufflation was not used [65 vs. 30%; OR 0.09 (0.01-0.53); P = 0.008]. CONCLUSION: CR-ESD by hydrodissection has good safety and efficacy profile and offers well tolerated and effective treatment for complex polyps. As such, this technique may be useful in the West, in centers, where previous gastric ESD is not frequent or Japanese mentoring is not possible.

Marrow fat and the bone microenvironment: developmental, functional, and pathological implications.

Bone marrow adipogenesis is a normal physiologic process in all mammals. However, its function is unknown. The mesenchymal stem cell is the marrow precursor for adipocytes as well as osteoblasts, and PPARG is an essential differentiation factor for entrance into the fat lineage. Mouse models have provided significant insight into the molecular cues that define stromal cell fate. In humans, accelerated marrow adipogenesis has been associated with aging and several chronic conditions, including diabetes mellitus and osteoporosis. Newer imaging techniques have been used to determine the developmental time course of fat generation in bone marrow. However, more studies are needed to understand the interrelationship among hematopoietic, osteoblastic, and adipogenic cells within the marrow niche.

A cyclized peptide derived from alpha fetoprotein inhibits the proliferation of ER-positive canine mammary cancer cells.

The effects of estradiol (E2) and of an AFP-derived cyclized peptide (cP) on the proliferation of primary cultures of cancer cells isolated from spontaneous canine mammary tumors were studied. The cellular response to E2 and cP was related to the expression of estradiol receptor (isoforms alpha and beta). In ER-positive cells, 2 nM estradiol increased cell proliferation and the phosphorylation of ERK1/2; 2 microg/ml cP inhibited all these effects. Estradiol also increased HER2 immunoreactivity in ER-positive cells, an effect that was reverted to its basal values by cP. Estradiol stimulated in these cells the release of MMP2 and MMP9 and the shedding of HB-EGF, effects that the cP did not affect. ER-negative cells were refractory to estradiol or cP. All canine mammary tumor cells in culture responded to treatments analogously to human mammary cancer cells. Our results support the proposal of cP as a new, potentially effective therapeutic agent for the management of mammary cancer.

Is fatty acid composition of human bone marrow significant to bone health?

The bone marrow adipose tissue (BMAT) is a conserved component of the marrow microenvironment, providing storage and release of energy and stabilizing the marrow extent. Also, it is recognized both the amount and quality of BMAT are relevant to preserve the functional relationships between BMAT, bone, and blood cell production. In this article we ponder the information supporting the tenet that the quality of BMAT is relevant to bone health. In the human adult the distribution of BMAT is heterogeneous over the entire skeleton, and both BMAT accumulation and bone loss come about with aging in healthy populations. But some pathological conditions which increase BMAT formation lead to bone impairment and fragility. Analysis in vivo of the relative content of saturated and unsaturated fatty acids (FA) in BMAT indicates site-related bone marrow fat composition and an association between increased unsaturation index (UI) and bone health. With aging some impairment ensues in the regulation of bone marrow cells and systemic signals leading to local chronic inflammation. Most of the bone loss diseases which evolve altered BMAT composition have as common factors aging and/or chronic inflammation. Both saturated and unsaturated FAs originate lipid species which are active mediators in the inflammation process. Increased free saturated FAs may lead to lipotoxicity of bone marrow cells. The pro-inflammatory, anti-inflammatory or resolving actions of compounds derived from long chain poly unsaturated FAs (PUFA) on bone cells is varied, and depending on the metabolism of the parent n:3 or n:6 PUFAs series. Taking together the evidence substantiate that marrow adipocyte function is fundamental for an efficient link between systemic and marrow fatty acids to accomplish specific energy or regulatory needs of skeletal and marrow cells. Further, they reveal marrow requirements of PUFAs.

Increased adipogenesis of osteoporotic human-mesenchymal stem cells (MSCs) characterizes by impaired leptin action.

The bone marrow contains mesenchymal stem cells (MSCs) that differentiate to the osteogenic and adipogenic lineages. The fact that the decrease in bone volume of age-related osteoporosis is accompanied by an increase in marrow adipose tissue implies the importance that the adipogenic process may have in bone loss. We previously observed that MSCs from control and osteoporotic women showed differences in their capacity to differentiate into the osteogenic and adipogenic pathways. In vitro studies indicate that bone marrow stromal cells are responsive to leptin, which increases their proliferation, differentiation to osteoblasts, and the number of mineralized nodules, but inhibits their differentiation to adipocytes. The aim of the present report was to study the direct effect of leptin on control and osteoporotic MSCs analyzing whether the protective effect of leptin against osteoporosis could be expressed by inhibition of adipocyte differentiation. MSCs from control, and osteoporotic donors were subjected to adipogenic conditions, in the absence or in the presence of 62.5 nM leptin. The number of adipocytes, the content of PPARgamma protein, and mRNA, and leptin mRNA were measured by flow cytometry, Western blot, and RT-PCR, respectively. Results indicate that control and osteoporotic MSCs differ in their adipogenic potential as shown by expression of active PPARgamma protein. Leptin exerted an antiadipogenic effect only on control MSCs increasing the proportion of inactive phosphorylated PPARgamma protein. Finally, results obtained during adipogenesis of osteoporotic cells suggest that this process is abnormal not only because of increased adipocyte number, but because of impaired leptin cells response.

A cyclic peptide derived from alpha-fetoprotein inhibits the proliferative effects of the epidermal growth factor and estradiol in MCF7 cells.

A cyclic peptide derived from the active domain of alpha-fetoprotein (AFP) significantly inhibited the proliferation of MCF7 cells stimulated with the epidermal growth factor (EGF) or estradiol (E2). The action of these three agents on cell growth was independent of the presence of calf serum in the culture medium. Our results demonstrated that the cyclic peptide interfered markedly with the regulation of MAPK by activated c-erbB2. The cyclic peptide showed no effect on the E2-stimulated release of matrix metalloproteinases 2 and 9 nor on the shedding of heparin-binding EGF into the culture medium. We propose that the AFP-derived cyclic peptide represents a valuable novel antiproliferative agent for treating breast cancer.

A peptide derived from alpha-fetoprotein inhibits the proliferation induced by estradiol in mammary tumor cells in culture.

This study was aimed to obtain additional information on the activity of a cyclized 9-amino acid peptide (cP) containing the active site of alpha fetoprotein, which inhibits the estrogen-stimulated proliferation of tumor cells in culture and of xenografts in immunodeficient mice. Breast cancer cells cultured in the presence of 2 nM estradiol were exposed to cP for different periods and their proliferation, estradiol binding parameters, clustering tendency and expression of E-cadherin and p21Cip1 were analyzed by biochemical and cell biology methods. The proliferation of MCF7 cells was significantly decreased by the addition of 2 microg/ml cP to the medium. cP did not increase cell death rate nor alter the number of binding sites for estradiol nor the endogenous aromatase activity of MCF7 cells. cP also decreased the proliferation of estrogen-dependent ZR75-1 cells but had no effect on estrogen-independent MDA-MB-231 cells. An increased nuclear p21Cip1 expression detected after cP treatment suggests that cP slows MCF7 cell proliferation via this regulator. We propose that cP could represent a novel breast cancer therapeutic agent whose mechanism of action is different from that of tamoxifen or of inhibitors of aromatase.

A synthetic peptide derived from alpha-fetoprotein inhibits the estradiol-induced proliferation of mammary tumor cells in culture through the modulation of p21.

A stable cyclized 9-mer peptide (cP) containing the active site of alpha-alpha fetoprotein (alphaFP) has been shown to be effective for prevention of estrogen-stimulated tumor cell proliferation in culture or of xenographt growth in immunodeficient mice. cP does not block 17beta-estradiol (E2) binding to its receptors, but rather appears to interfere with intracellular processing of the signal that supports growth. To obtain insight on that mechanism we studied the effect of cP on the proliferation of MCF-7 cells in culture. Proliferation in the presence of 2 microM E2 is decreased up to 40% upon addition of 2 microg ml(-1) cP to the medium; the presence of cP did not increase cell death, cP reduced also the proliferation of estrogen-dependent ZR75-1 cells but had no effect on autonomous MDA-MB-231 cells, cP did not modify the number of binding sites for labeled E2 or affected cell death. We detected increased nuclear p21Cip1 immunoreactivity after cP treatment. Our results suggest that cP acts via p21Cip1 to slow the process of MCF-7 cells through the cycle.