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Human breast epithelium in organ culture: effect of hormones on growth and morphology.

Normal breast tissue from a 17-year-old girl was grown in organ culture for 3 weeks. A comparison was made between the effects on the epithelium of a defined culture medium containing various combinations of hormones and serum-supplemented medium that has been used to successfully maintain other human tissues for 4 months routinely, and in some cases for up to 1 year. After culture for 3 weeks the explants were exposed to [3H]thymidine and autoradiographs were prepared and evaluated in order to determine labeling indexes. The only serum-free defined medium that permitted any significant survival or labeling of the cells contained insulin + hydroxycortisone + prolactin. However, serum-supplemented medium along gave an even higher labeling index, and this was elevated more in media containing either progesterone or other combinations of hormones. Our study indicates that normal human breast (removed at the early postovulatory stage of the menstrual cycle) can be maintained in a differentiated state for 12 days in serum-supplemented media. By 2 weeks the cells had begun to migrate onto the surface of the explant. They then began to accumulate tonofilaments so that after 3 weeks in culture nearly all of the cells contained tonofilaments. The one exception was found in breast tissue cultured in the presence of human chorionic gonadotropin, where the cells maintained differentiated characteristics, despite the fact that they contained many lysosomes.

Adolescent↗

Activated RSC-nucleosome complex and persistently altered form of the nucleosome.

RSC, an abundant, essential chromatin-remodeling complex, related to SWI/SNF complex, binds nucleosomes and naked DNA with comparable affinities, as shown by gel shift analysis. The RSC-nucleosome complex is converted in the presence of ATP to a slower migrating form. This activated complex exhibits greatly increased susceptibility to endo- and exonucleases but retains a full complement of histones. Activation persists in the absence of ATP, and on removal of RSC, the nucleosome is released in an altered form, with a diminished electrophoretic mobility, greater sedimentation rate, and marked instability at elevated ionic strength. The reaction is reversible in the presence of RSC and ATP, with conversion of the altered form back to the nucleosome.

Adenosine Triphosphate↗

A role for alpha4-integrin in the pathology following Semliki Forest virus infection.

Migration of cells into the central nervous system (CNS) is a pivotal step in the pathogenesis of immune-mediated diseases such as multiple sclerosis (MS), experimental allergic encephalomyelitis (EAE) and virus-induced demyelinating diseases. Such migration is dependent on expression of adhesion molecules. The expression of adhesion molecules in the CNS was studied in Biozzi ABH mice infected with Semliki Forest virus (SFV) A7(74) - an important demyelinating model of MS. Expression of LFA-1alpha/CD11a, LFA-1beta/CD18 and ICAM-1/CD56 were rapidly elevated and remained high whereas MAC-1, CD44 and VCAM-1/CD106 were less widely expressed. The alpha4-integrin VLA-4/CD49d was more specifically associated with CNS lesions. To identify the importance of VLA-4, CD44, ICAM-1 and MAC-1 in the pathogenesis of SFV infection, monoclonal antibodies that block these adhesion molecules were administered in vivo during infection. Anti-VLA-4 treatment dramatically reduced the cellular infiltrates and demyelination within the CNS but did not affect the clearance of virus while antibodies to CD44, ICAM and MAC-1 antibody treatment had no effect. This study demonstrates that SFV infection induces the expression of adhesion molecules within the CNS and that VLA-4 plays an important role in the development of inflammation and demyelination in the CNS following SFV infection.

Alphavirus Infections↗

Mechanism and role of localized activation of Rho-family GTPases in growth factor-stimulated fibroblasts and neuronal cells.

Rho-family GTPases regulate various aspects of cell function by controlling cytoskeletal changes; however, their spatial regulation within the cells remains largely unknown. To understand this regulation, we have studied the spatiotemporal activity of Rho-family GTPases in migrating cells and growth factor-stimulated cells by using probes based on the principle of fluorescence resonance energy transfer. In migrating fibroblasts and epithelial cells, the level of RhoA activity is high both at the contractile tail and at the leading edge, whereas Rac1 and Cdc42 activities are high only at the leading edge. In cells stimulated with epidermal growth factor or nerve growth factor, activities of Rac1 and Cdc42 were transiently elevated in a broad area of the plasma membrane, followed by a localized activation at nascent lamellipodia. In contrast, on epidermal growth factor stimulation, RhoA activity decreased diffusely at the plasma membrane. Notably, RhoA activity persisted at the tip of growth factor-induced membrane ruffles and, in agreement with this finding, RhoA is required for membrane ruffling. These observations suggest that the activities of Rho-family GTPases are elaborately regulated in a time- and space-dependent manner to control cytoskeletal changes and that the basic mechanism of controlling cell shape via Rho-family GTPases is common to various cell types.

Animals↗

Nerve growth factor regulates both the phosphorylation and steady-state levels of microtubule-associated protein 1.2 (MAP1.2).

This study characterizes effects of nerve growth factor (NGF) on the steady-state level and phosphorylation of a high molecular mass microtubule-associated protein in PC12 rat pheochromocytoma cells. Past work showed that NGF significantly raises the relative levels of this phosphoprotein, designated MAP1.2, with a time course similar to that of neurite outgrowth. To study this in greater detail, MAP1.2 in PC12 cell lysates was resolved by SDS-PAGE in gels containing 3.25% acrylamide/4 M urea and identified by comigration with material immunoprecipitated from the lysates by MAP1 antibodies. Quantification by metabolic radiolabeling with [35S]methionine or by silver staining revealed a 3.0-3.5-fold increase in MAP1.2 levels relative to total cell protein after NGF treatment for 2 wk or longer. A partial increase was detectable after 3 d, but not after 2 h of NGF exposure. As measured by incorporation of [32P]phosphate, NGF had a dual effect on MAP1.2. Within 15 min to 2 h, NGF enhanced the incorporation of phosphate into MAP1.2 by two- to threefold relative to total cell phosphoproteins. This value slowly increased thereafter so that by 2 wk or more of NGF exposure, the average enhancement of phosphate incorporation per MAP1.2 molecule was over fourfold. The rapid action of NGF on MAP1.2 could not be mimicked by either epidermal growth factor, a permeant cAMP derivative, phorbol ester, or elevated K+, each of which alters phosphorylation of other PC12 cell proteins. SDS-PAGE revealed multiple forms of MAP1.2 which, based on the effects of alkaline phosphatase on their electrophoretic mobilities, differ, at least in part, in extent of phosphorylation. Before NGF treatment, most PC12 cell MAP1.2 is in more rapidly migrating, relatively poorly phosphorylated forms. After long-term NGF exposure, most is in more slowly migrating, more highly phosphorylated forms. The effects of NGF on the rapid phosphorylation of MAP1.2 and on the long-term large increase in highly phosphorylated MAP1.2 forms could play major functional roles in NGF-mediated neuronal differentiation. Such roles may include effects on microtubule assembly, stability, and cross-linking and, possibly for the rapid effects, nuclear signaling.

Alkaline Phosphatase↗

Cell-specific expression of helix-loop-helix transcription factors encoded by the E2A gene.

The E2A gene encodes transcription factors of the helix-loop-helix family that are implicated in cell-specific gene expression as part of dimeric complexes that interact with E box enhancer elements. It has previously been shown that transcripts of the E2A gene can be detected in a wide range of cell types. We have now examined expression of the mouse E2A gene at the protein level using polyclonal antisera directed against distinct portions of the E2A protein to probe blots of cellular extracts. A 73 kDa protein was identified by this analysis: this protein is highly enriched in cell lines of B lymphoid origin as compared to pancreatic beta-cells and fibroblast cells. The detection of this protein selectively in extracts of lymphoid cells correlates with the presence of the E box-binding activity LEF1/BCF1 in these cells; this binding activity was previously shown to be efficiently recognized by antiserum directed against E2A gene products. Transfection of cells with full length E2A cDNA leads to appearance of protein co-migrating with the 73 kDa protein on SDS gel electrophoresis and co-migrating with LEF1/BCF1 on mobility shift analysis. Our results are consistent with the view that the DNA-binding activity LEF1/BCF1 is a homodimer of E2A proteins; the selective appearance of this putative cell-specific transcription factor in B lymphoid cells seems to be attributable, at least in part, to the elevated E2A protein concentrations in these cells.

Animals↗

Urokinase plasminogen activator enhances neointima growth and reduces lumen size in injured carotid arteries.

OBJECTIVES: Increases in urokinase plasminogen activator (uPA) have been reported in tissues undergoing remodelling, but its effects on the vessel intima formation are not known. We investigated its effects on carotid artery intima, media and lumen size, as well as smooth muscle cell (SMC) proliferation and migration in vivo. DESIGN AND METHODS: Carotid arteries of rats were distended with an inflated balloon catheter and uPA, or uPA-neutralizing antibodies were applied perivascularly in pluronic gel; control rats received vehicle. Carotid artery structure, cell migration and proliferation were assessed after 4 days by quantitative morphometry and immunohistochemistry. RESULTS: Four days after increasing vessel uPA, the intima/media ratio was double compared to that in control rats (both P < 0.05). The size of the lumen reduced by 75%, compared to the vehicle-treated vessels (P < 0.05). The elevation in uPA also increased SMC numbers in the intima and media, compared to the vehicle-treated vessels (both P < 0.05). Antibody neutralizing endogenous uPA attenuated the growth responses in the distended arteries, reduced neointimal SMC numbers by approximately 50% and prevented much of the reduction in lumen size. CONCLUSIONS: Thus, local increases in uPA in distended, injured arteries augment SMC migratory and proliferative responses, leading to increases in the thickness of the carotid artery intima and media and a reduction in lumen size; effects at least partially attributable to its proteolytic properties.

Animals↗

A comparison of variability and population structure for major histocompatibility complex and microsatellite loci in California coastal steelhead (Oncorhynchus mykiss Walbaum).

The major histocompatibility complex (MHC) contains genes integral to immune response in vertebrates. MHC genes have been shown to be under selection in a number of vertebrate taxa, making them intriguing for population genetic studies. We have conducted a survey of genetic variation in an MHC class II gene for steelhead trout from 24 sites in coastal California and compared this variation to that observed at 16 presumably neutral microsatellite loci. A high amount of allelic variation was observed at the MHC when compared to previously published studies on other Pacific salmonids. Elevated nonsynonymous substitutions, relative to synonymous substitutions, were detected at the MHC gene, indicating the signature of historical balancing selection. The MHC data were tested for correlations to and deviations from the patterns found with the microsatellite data. Estimates of allelic richness for the MHC gene and for the microsatellites were positively correlated, as were estimates of population differentiation (F(ST)). An analysis for F(ST) outliers indicates that the MHC locus has an elevated F(ST) relative to the neutral expectation, although a significant result was found for only one particular geographical subgroup. Relatively uniform allele frequency distributions were detected in four populations, although this finding may be partially due to recent population bottlenecks. These results indicate that, at the scale studied here, drift and migration play a major role in the observed geographical variability of MHC genes in steelhead, and that contemporary selection is relatively weak and difficult to detect.

Alleles↗

Fibroblast chemotactic response elicited by native bronchoalveolar lavage fluid from patients with fibrosing alveolitis.

BACKGROUND: In fibrosing alveolitis activation of lung fibroblasts is the decisive event in the pathogenetic sequence leading to pulmonary fibrosis. Fibroblast stimulating activity was measured in bronchoalveolar lavage (BAL) fluid to assess its relationship to the activity of fibrosing alveolitis. METHODS: Nine control subjects and 40 patients with fibrosing alveolitis caused by idiopathic pulmonary fibrosis (n = 22) or pulmonary involvement in systemic sclerosis (n = 18) were studied. All patients were followed up by lung function testing for a minimum of six months (mean (SE) 13.3 (1.4) months). Twenty five patients received immunosuppressive therapy and 15 refused. At the beginning of follow up BAL was performed and, as a possible indicator of fibroblast stimulating mediators within the lungs, chemotactic migration of cultured human fibroblasts elicited by native BAL fluid was measured in Boyden-type chambers and expressed as a percentage of the chemoattractant effect of 25 ng/ml platelet derived growth factor. The procollagen III peptide level in BAL fluid served as a marker for collagen synthesis. RESULTS: Chemoattractant activity was elevated in the patients with idiopathic pulmonary fibrosis and systemic sclerosis compared with the control group, (mean (SE) 56.4% (8.5%)) and 72.3% (16.3%) v 12.6% (4.0%). Chemoattractant activity was inversely correlated with total lung capacity (TLC) (r = -0.45) and with vital capacity (VC) (r = -0.33). Procollagen III peptide concentrations in BAL fluid and chemoattractant activity were not significantly correlated. For further evaluation chemoattractant activity of 36% (mean value of controls +2 SD) was used to separate normal (< 36%) from elevated (> or = 36%) activity. At the end of follow up, untreated patients with high chemoattractant activity (> or = 36%) showed a significant reduction of VC, TLC, and exercise arterial oxygen tension (PaO2) and a small decrease in carbon monoxide transfer factor (TLCO), whereas a significant improvement in VC, TLC, and TLCO and a small increase of exercise PaO2 occurred in treated patients with high chemoattractant activity. Patients with low chemoattractant activity (< 36%) showed no consistent change in lung function measurements, irrespective of treatment. In contrast, lung function results and differential cell counts in BAL fluid failed to identify progressive disease. CONCLUSIONS: In patients with fibrosing alveolitis the chemoattractant activity of BAL fluid seems to be an independent indicator of lung fibroblast stimulating activity providing relevant information about disease activity, and may help to improve the clinical management of these patients.

Adult↗

Prolonged ambulatory canine colonic motility.

Canine gastrointestinal motility is studied at present in animals confined to a small cage or sling. The aims of this study were to record colonic activity over a 24-h period in eight dogs by an ambulatory method. Motility signals from implanted strain gauges were processed and stored via a portable battery-operated amplifier and digital recorder housed in a jacket. Ambulant interdigestive activity was the same as observed in laboratory experiments, with migrating colonic motor complexes (CMCs) and infrequent giant contractions (GCs). Feeding caused a multiphasic alteration in motility for 582.1 +/- 18.1 min (mean +/- SE). There were four distinct phases. During the "early" (0-2 h) postprandial period, phase 1 (mean duration: 55.1 +/- 4.0 min), which was distinguished by CMCs of high frequency and elevated amplitude in the proximal colon, and phase 2 (78.2 +/- 6.2 min), which had CMC characteristics similar to those in the interdigestive period, occurred. Phase 3 (218.8 +/- 13.6 min), a further period of increased motility, and phase 4 (339.1 +/- 14.0 min), characterized by low-amplitude long-duration CMCs, occurred during the "late" (2 h onward) postprandial response. With the exception of phase 3, postprandial phases were not always present following food intake, and their expression was markedly influenced by variations in meal time and by defecation immediately following feeding. Spontaneous defecation was characterized by a variety of motor profiles, with a GC accompanying two-thirds of episodes. We conclude that a more complete picture of canine colonic motility has been documented because of the development of the ambulatory system.

Animals↗

Oral epithelial overexpression of IL-1alpha causes periodontal disease.

Periodontal disease is a bacterial infection that results in inflammatory destruction of tissues that support the teeth, including connective tissue and bone. In this study, we report that transgenic mice that overexpress the 17-kDa form of IL-1alpha in the basal layer of oral mucosal epithelium develop a syndrome that possesses all of the cardinal features of periodontal disease, including epithelial proliferation and apical migration, loss of attachment, and destruction of cementum and alveolar bone. In this model, bacterial colonization and infection were not required, since levels of periodontal bacteria were equivalent in transgenic and wild-type mice, and continuous treatment with antibiotics from birth did not ameliorate the disease. Our findings therefore indicate that elevated levels of IL-1alpha in the oral micro-environment can mediate all of the clinical features of periodontal disease.

Alveolar Bone Loss↗

Proteins in the luminal fluid from the bovine oviduct.

Oviducal fluid was collected by cannulation from four cows and by irrigation from fifteen slaughtered cows. The proteins in the fluid were examined by polyacrylamide gel electrophoresis at pH 4-5 and pH 8-9, isoelectric focusing on polyacrylamide, immunodiffusion, immunoelectrophoresis, affinity chromatography and gel filtration. The macromolecular components found were mainly serum proteins but small amounts of other proteins were detected in oestrous and dioestrous samples by electrophoresis at pH 8-9 following fractionation of the fluid by gel filtration or affinity chromatography. Small amounts of cathodically migrating proteins were detected directly by electrophoresis at pH 4-5 in dioestrous samples but not in oestrous samples. Determination of glycosidase activities revealed that the levels at oestrus were similar to the levels detected in serum. At dioestrus, the activities of B-N-acetylgalactosaminidase and beta-N-acetylglucosaminidase were elevated.

Acetylglucosaminidase↗

[Hereditary hemoglobin H disease in Japanese siblings diagnosed by human parvovirus B19 infection].

Hereditary hemoglobin H (HbH) disease was diagnosed in 2 Japanese sisters who presented with aplastic crisis following acute human parvovirus B19 (HPV B19) infection. The proband, an 8-year-old girl, developed persistent fever and pallor, and samples of her peripheral blood showed hypochromic microcytic anemia. Other laboratory data were consistent with hemolytic anemia. Fever and signs of hypochromic microcytic anemia also developed in her sister 9 days later. Cation exchange HPLC analysis of their hemoglobin revealed abnormal hemoglobin migrating faster than HbF, a finding consistent with HbH. Although they presented neither arthralgia nor skin rash, we concluded that their aplastic crisis was induced by HPV B19, because HPV B19 DNA was detected in samples of their peripheral blood by PCR analysis, and HPV B19 IgM and IgG antibody titers were elevated. A genetic analysis of the alpha-globin gene in both sisters and their parents disclosed that the father was heterozygous for alpha-Thal-2, the mother, heterozygous for alpha-Thal-1, and the proband and her sister, double heterozygous for alpha-Thal-1 and alpha-Thal-2. alpha-Thal-2 is a 3.7 kb-deletion allele.

Child↗

Pdef expression in human breast cancer is correlated with invasive potential and altered gene expression.

Ets transcription factors control multiple biological processes, including cell proliferation, differentiation, apoptosis, angiogenesis, transformation, and invasion. Pdef is an Ets transcription factor originally identified in prostate tissue. We demonstrate that human Pdef is expressed at high levels primarily in tissues with high epithelial cell content, including prostate, colon, and breast. We also determined that Pdef protein is reduced in human invasive breast cancer and is absent in invasive breast cancer cell lines. We next assessed the functional consequences of these observations. Significantly, expression of Pdef in breast cancer cells leads to inhibition of invasion, migration, and growth. Expression of Pdef also results in the down-regulation of urokinase-type plasminogen activator and activation of the promoter of the tumor suppressor gene, MASPIN: Growth-suppressive effects of Pdef expression are mediated in part by a G(0)-G(1) cell cycle arrest associated with elevated p21 levels. Collectively, these results indicate that Pdef loss may alter the expression of genes controlling progression to invasive breast cancer.

Breast Neoplasms↗

Modulated expression of plasminogen activator system components in cultured cells from dissociated mouse dorsal root ganglia.

The development and regeneration of the peripheral nervous system (PNS) is highly dependent on the migration of Schwann cells and the extension of axons toward their distant targets. Plasminogen activators (PAs) are associated with the surface of several cell types of neural origin where they are believed to mediate localized degradation of extracellular matrix, thus facilitating cell motility. In this study, we characterize the expression of tissue-type (tPA) and urokinase (uPA) PAs, as well as the urokinase cell surface receptor (uPAR) during differentiation of cultured cells from mouse dorsal root ganglia. During the first day in culture, the mRNA levels of all three components increase from 75- to 163-fold, as shown using a quantitative PCR method. By 72 hr, the mRNA levels decrease and approach basal levels. This transient increase is in direct correlation with the differentiation of neurons and Schwann cells and the formation of a neuritic network in these regenerating cultures. Densitometric analysis of gel zymographs demonstrates that the elevation in mRNA levels is accompanied by similar increases in the activity levels of tPA and uPA. Interestingly, in situ hybridization studies of the cultures show that tPA mRNA is restricted to small sensory neurons, whereas uPA mRNA is localized predominantly in large sensory neurons. uPAR mRNA is expressed by both neuronal subpopulations and, to a lesser extent, by Schwann cells and fibroblasts. Taken together, these results further support a role for the PA system in facilitating axon extension and cell migration during development and regeneration of the PNS.

Animals↗

Elevated production of interleukin 6 by hepatic MNC correlates with ICAM-1 expression on the hepatic sinusoidal endothelial cells in autoimmune MRL/lpr mice.

MRL/lpr mice, which are a model of SLE and rheumatoid arthritis in humans, develop profound lymphadenopathy resulting from the accumulation of CD3+ 4-8- double-negative (DN) alpha beta T cells in peripheral lymphoid tissues. We previously indicated that these DN alpha beta T cells preferentially proliferate in the liver and migrate to the periphery. In this study, we analyzed whether any kind of cytokine was produced by hepatic mononuclear cells (MNC) in MRL/lpr mice. The evidence obtained indicates that interleukin 6 (IL-6) was vigorously produced by hepatic MNC in diseased MRL/lpr mice under unstimulated conditions. MNC in the spleen of these mice produced small amounts of IL-6, while those in the lymph nodes did not produce any appreciable amounts of IL-6. These activities of hepatic MNC in diseased MRL/lpr mice were almost completely neutralized by anti-mouse IL-6 monoclonal antibody (mAb). On the other hand, immunohistochemical staining of light- and electron-microscopic analyses revealed that the intracellular cell adhesion molecule 1 (ICAM-1) was expressed on the hepatic sinusoidal endothelial cells of diseased MRL/lpr mice. Moreover, ICAM-1 was newly induced in the hepatic sinusoids of control C3H/He mice by an intravenous injection of 50 units of recombinant mouse IL-6. These data suggest that ICAM-1 expressed on the hepatic sinusoidal endothelial cells in MRL/lpr mice is induced by IL-6, which is produced by hepatic MNC, and that such ICAM-1 may be responsible for the saturation of inflammatory cells and the proliferation of lymphocytes in the liver of MRL/lpr mice.

Animals↗

Comparison of two lymphokines (macrophage migration inhibition, leukocyte adherence inhibition factors) and carcinoembryonic antigen, in colorectal cancer and colonic premalignant lesions.

Previous studies in our laboratory on 92 patients with colonic cancer have suggested a promising degree of specificity and sensitivity for a macrophage migration inhibition factor (MIF) test using patient's lymphocytes incubated with a human colon cancer extract. This study compares the results of the MIF technique with serum carcinoembryonic antigen (CEA) levels and with the lymphocyte adherence inhibition (LAI) test in 18 colon cancer patients and 27 patients with conditions considered to predispose to colon cancer (colonic adenomas, ulcerative colitis, and Crohn's disease). Among colonic cancer patients, MIF and LAI were positive in 17 out of 18, but CEA was elevated in eight. MIF and CEA were negative in all 16 normal control subjects; LAI was negative in 13. Among patients with colonic adenomas, MIF and LAI were positive in three of five; CEA was negative in all. In the ulcerative colitis and Crohn's disease group, MIF was positive in seven of 22, LAI was positive in 11, and CEA was negative in all 22. Thus, MIF and LAI appear to be sensitive marker's for human colonic cancer. More extensive studies and precise characterization of these groups are warranted.

Carcinoembryonic Antigen↗

Regulation of plasminogen activator inhibitor-1 levels in human monocytes.

The regulation of PAI-1 synthesis by elutriation-purified human monocytes was studied in vitro and compared to that for PAI-2. PAI-1 formation, as measured by ELISA, was upregulated by TGF-beta (> or = 1 ng/ml) and surprisingly down-regulated by LPS (100 ng/ml), particularly in the presence of TGF-beta; LPS elevated PAI-2 levels (ELISA) while TGF-beta reduced its basal levels and those in LPS-treated cultures. Concomitant changes in mRNA expression occurred. The glucocorticoid dexamethasone (10(-7) M) elevated PAI-1 and acted in concert with TGF-beta in this regard at both the antigen and mRNA levels; interleukin-4 (IL-4) (250 pM) failed to mimic the steroid in its regulation of PAI-1 formation. Since monocyte/macrophage PA activity is likely to be important in tissue remodeling and cell migration at sites of inflammation and in fibrinolysis, it is proposed from these studies that PAI-1, as well as the usually considered PAI-2, may be involved in the negative control of PA activity in this cell type. The synthesis of each PAI appears to be independently regulated.

Cells, Cultured↗