Corioamnionitis histológica y morbimortalidad neonatal: aproximación al síndrome de respuesta inflamatoria fetal / Chorioamnionitis neonatal morbidity: approach to fetal inflammatory response syndrome

Antecedentes: El síndrome de respuesta inflamatoria fetal (SRIF) es una entidad relacionada con la presencia de inflamación intrauterina y suele asociarse a infección intraamniótica. Su consecuencia más grave es la lesión cerebral y posterior desarrollo de parálisis cerebral. Objetivo: Evaluar la relación entre el síndrome de respuesta inflamatoria fetal y el desarrollo de complicaciones neonatales. Método: Estudio descriptivo y retrospectivo, realizado en el Hospital Universitario La Paz de Madrid, buscando una aproximación al SRIF desde la corioamnionitis histológica/funiculitis. El grupo de estudio constituido por 35 gestaciones simples pretérmino recogidas durante el primer semestre de 2008 y en las que la anatomía patológica de la placenta y anexos ovulares demostró la presencia de una corioamnionitis histológica y/o funiculitis. Resultados: Siete casos (20 por ciento) presentaban clínica sospechosa de infección intraamniótica, si bien en 28 gestantes (80 por ciento) existían factores de riesgo asociados al síndrome de respuesta inflamatoria fetal. Mortalidad perinatal en el grupo estudiado fue de 11,4 por ciento (4 casos). Sólo en 2 pacientes (5,7 por ciento) se pudo relacionar la muerte con el SRIF. En 28 recién nacidos (80 por ciento) se encontró algún tipo de patología, siendo la misma inherente a dicho síndrome en 17 casos (48,6 por ciento), destacando sepsis neonatal (40 por ciento), leucomalacia periventricular (14,3 por ciento) y displasia broncopulmonar (5,7 por ciento). Conclusión: Se comprueba el alto riesgo neonatal del SRIF. El conocimiento de esta condición, abre una serie de controversias diagnósticas y terapéuticas que obliga a una reevaluación de los protocolos actuales de manejo de la amenaza de parto pretérmino y la rotura prematura de membranas de pretérmino.

Background: The fetal inflammatory response syndrome (FIRS) is an entity related to intrauterine inflammation which is commonly associated with intraamniotic infection. The most serious consequence is the neurologic damage and the subsequent development of cerebral palsy. Aims: To evaluate the relationship between the fetal inflammatory response syndrome and the development of neonatal complications. Method: Descriptive and retrospective study realized in "La Paz" University Hospital of Madrid, looking for an approximation to the FIRS from histologic chorioamnionitis/funisitis. Group of study constituted by 35 single preterm gestations collected during the first semester of 2008 and in which the pathologic anatomy study of the placenta and annexes showed the presence of histological corioamnionitis and / or funisitis. Results: Suspicious clinic was found in 7 cases (20 percent) but in 28 cases (80 percent) risk factors associated to FIRS were present. Perinatal mortality found was 11.4 percent (4 cases). Only in 2 cases (5.7 percent) the cause was relationated with FIRS. Pathology associated was found in 28 newborn (80 percent), being 17 cases (48.6 percent) pathology associated with the fetal inflammatory response syndrome, enhancing neonatal sepsis (40 percent), periventricular leukomalacia (14.3 percent), and bronchopulmonar dysplasia (5.7 percent). Conclusion: It is verified that FIRS enteals a high neonatal risk. The knowledge of this entity opens some diagnostic and therapeutic controversies. Current management protocols of preterm labor and preterm premature rupture of membranes should be revised.

P38 MAPK protects against TNF-alpha-provoked apoptosis in LNCaP prostatic cancer cells.

PURPOSE: One of the most relevant aspects in cell death regulation is the signalling of apoptosis by the serine/threonine kinases MAPKs. The aim of this study was to investigate the effects of TNF-alpha stimulation on MAPK activation, and the pro- or anti-apoptotic role of these kinases in LNCaP and PC3 cells. MATERIAL AND METHODS: Treatments were carried out using several TNF-alpha concentrations, as well as specific pharmacological inhibitors of MAPKs. Apoptosis rates were evaluated by DAPI staining and flow cytometry. MAPK phosphorylation/activation was measured by Western blot. RESULTS: TNF-alpha induced apoptosis in a dose-dependent manner in LNCaP but not in PC3 cells. The MAPK inhibitors revealed that the apoptotic rate in LNCaP cells increased significantly following p38 inhibition. The kinase inhibitors failed to cause changes in apoptosis in PC3 cells. CONCLUSIONS: The potentiation of apoptosis by p38 inhibition points to this kinase as a possible target for the treatment of androgen-dependent prostatic cancer.

Cell cycle control related proteins (p53, p21, and Rb) and transforming growth factor beta (TGFbeta) in benign and carcinomatous (in situ and infiltrating) human breast: implications in malignant transformations.

A comparative study of the products of the cell cycle control genes p53 (mutated form), p21, Rb (nonphosphorylated and phosphorylated form) and TGFbeta was performed by immunohistochemistry and Western blot, in benign breast disorders and breast cancer (in situ and infiltrating tumors). For the five proteins studied, the relative numbers of positively stained cells were higher in in situ carcinoma than in benign breast diseases. In infiltrating breast tumors, the relative numbers of positively stained cells were even higher than in in situ tumors except for the percentage of pRb immunostained cells, which decreased slightly in infiltrative tumors. For the other four proteins, the percentages of positively stained cases were similar to those found in in situ tumors. In the three groups of patients, TGFbeta immunoreaction appeared in the cytoplasm while immunoreactions to p53, p21, Rb, and pRb were found always in the nucleus except for p21 in in situ tumors, which showed cytoplasmic immunoreaction. Present results suggest that accumulation of mutated p53, cytoplasmic p21, and pRb in breast gland epithelium might be a crucial point in the development of in situ adenocarcinoma. In the infiltrating tumors, the expression of p21 in the nuclei and the decrease in pRb expression suggest an insufficient attempt to hinder cell proliferation.

The p38 transduction pathway in prostatic neoplasia.

It has been proposed that, among other cellular responses, TNF-alpha induces not only cell death, but also cell proliferation by activation of p38. It has also been reported that IL-1-alpha favours cell proliferation by p38 activation. The aim of the present study was to evaluate upstream (alpha-PAK, MEK-6) and downstream (Elk-1 and ATF-2) components of the p38 transduction pathway in normal prostate, benign prostatic hyperplasia (BPH), and prostate carcinoma (PC). Immunohistochemical and western blot analyses were performed in 20 samples of normal prostate, 47 samples of BPH, and 27 samples of PC. In all normal prostates, immunoreactivity for p-Elk-1 and p-ATF-2 was observed in epithelial cell nuclei, but no expression of alpha-PAK or MEK-6. In BPH, there was expression of alpha-PAK (cytoplasm) and MEK-6 (cytoplasm), while the proportions of lesions that were immunoreactive for p-Elk-1 (nucleus and cytoplasm) and p-ATF-2 (nucleus) decreased. In PC, the percentages of cells that were immunoreactive for alpha-PAK (cytoplasm) or MEK-6 (cytoplasm) rose slightly in comparison with BPH, while the percentages of cells that were immunoreactive for p-Elk-1 (nucleus and cytoplasm) or p-ATF-2 (nucleus and cytoplasm) were much higher than in BPH. It is concluded that overexpression of alpha-PAK, MEK-6, p38, p-Elk-1, and p-ATF-2 in BPH, and more intensely in PC, enhances cell proliferation. In BPH, such proliferation is triggered by IL-1 and in part counteracted by the TNF-alpha/AP-1 pathway, which promotes apoptosis. In PC, proliferation is triggered by IL-1 and TNF-alpha (the TNF-alpha/AP-1 pathway is diverted towards p38 activation). Since in a study of the same patients immunoexpression of IL-1alpha and IL-1RI was previously observed to be increased in PC, inhibition of p38 is a possible target for PC treatment, as this inhibition would both decrease IL-1-induced cell proliferation and increase TNF-alpha-induced cell death.

IL-6, its receptors and its relationship with bcl-2 and bax proteins in infiltrating and in situ human breast carcinoma.

AIMS: To characterize the expression pattern of IL-6 and its receptors (IL-6R(alpha) and gp130), to relate this pattern to bcl-2 and bax expression and to elucidate the effects on the proliferation/apoptosis equilibrium in benign conditions and in situ and infiltrating breast cancer. METHODS AND RESULTS: The immunoexpression of IL-6 and its receptors (IL-6R(alpha) and gp130), and their relationship with bcl-2 and bax proteins, were studied in in situ and infiltrating tumours and in benign breast lesions by means of Western blotting and immunohistochemistry. The percentages of samples positive for IL-6, bcl-2 and bax and their immunoreaction densities were higher in in situ carcinomas and infiltrating tumours than in benign lesions; although in in situ lesions were not so high as in infiltrating tumours, except for bax, whose immunoexpression was as weak as in benign conditions, resulting in a bcl-2/bax ratio higher than in infiltrating tumours. CONCLUSIONS: The high expression of IL-6 and its receptors in tumours might be related to the enhanced cell proliferation occurring in breast cancer. IL-6 could act by increasing bcl-2 expression and thus altering the proliferation/apoptosis balance toward neoplastic cell proliferation. The increased bax immunoreactivity observed only in infiltrating tumours, which was not so high as the increase in bcl-2 immunoreactivity, might be interpreted as an attempt to hinder cell proliferation.

Immunohistochemical detection of the retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) in the marbled newt along the annual cycle.

Retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) expression in the testis of the marbled newt were investigated with special attention to the changes during the annual testicular cycle, using light microscopy immunohistochemistry and Western blot analysis. The annual testicular cycle of the marbled newt (Triturus marmoratus marmoratus) comprises three periods: (a) proliferative period (germ cell proliferation from primordial germ cells to round spermatids, April-June); (b) spermiogenesis period (July-September); and (c) quiescence period (interstitial and follicular cells form the glandular tissue, October-April). In the proliferative period, primordial germ cells and primary spermatogonia immunostained intensely to the three types of RXRs and also to FXR. In the other periods, immunostaining to these antibodies was weak or absent. Secondary spermatogonia stained weakly to the four antibodies in the proliferative period, and only to FXR, also weakly, in the spermiogenesis period. Immunoreactive primary spermatocytes were weakly labeled with the RXR antibodies in the proliferative period. Spermatids and spermatozoa did not stain to any antibody in any period. Follicular cells only immunostained to RXR-gamma and only in the quiescence period when they are forming the glandular tissue, together with the interstitial cells. As follicular cells, interstitial cells only immunostained in the quiescence period; however, they immunoreacted to the three types of RXRs. These findings suggest that in the newt, RXRs and FXR are involved in spermatogenesis control by regulating the proliferation of primordial germ cells and spermatogonia. In addition, RXR-gamma seems to be also involved in the development of the glandular (steroidogenic) tissue.

Ligand-dependent interactions of coactivators steroid receptor coactivator-1 and peroxisome proliferator-activated receptor binding protein with nuclear hormone receptors can be imaged in live cells and are required for transcription.

Members of the nuclear receptor superfamily are thought to activate transcription by recruitment of one or more recently identified coactivator complexes. Here we demonstrate that both peroxisome proliferator-activated receptor binding protein (PBP) and steroid receptor coactivator-1 (SRC-1) are required for ligand-dependent transcription of transiently transfected and chromosomally integrated reporter genes by the estrogen receptor (ER) and retinoic acid receptor (RAR). To examine ligand-dependent interactions between nuclear receptors and specific coactivators in living cells, these proteins were tagged with cyan (CFP) and yellow (YFP) mutants of the green fluorescent protein. Fluorescence resonance energy transfer (FRET) from the CFP to the YFP indicated interaction between the receptor and coactivator. CFP fusions to RAR or its ligand-binding domain exhibited rapid ligand-dependent FRET to YFP-tagged nuclear receptor interaction domains of the coactivators SRC-1 and PBP. The ER-ligand-binding domain, unlike RAR, also exhibited some basal interaction with coactivators in unstimulated cells that was abolished by the receptor antagonists tamoxifen or ICI182,780. Inhibition of FRET by tamoxifen but not ICI182,780 could be reversed by estradiol, whereas estradiol-enhanced FRET could not be inhibited by either antagonist, indicating that ligand effects can show varying degrees of hysteresis. These findings suggest that ligand-dependent transcriptional activities of the RAR and ER require concurrent or sequential recruitment of SRC-1 and PBP-containing coactivator complexes.

15-deoxy-delta 12,14-prostaglandin J2 inhibits multiple steps in the NF-kappa B signaling pathway.

Prostaglandin J(2) (PGJ(2)) and its metabolites Delta(12)-PGJ(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) are naturally occurring derivatives of prostaglandin D(2) that have been suggested to exert antiinflammatory effects in vivo. 15d-PGJ(2) is a high-affinity ligand for the peroxisome proliferator-activated receptor gamma (PPARgamma) and has been demonstrated to inhibit the induction of inflammatory response genes, including inducible NO synthase and tumor necrosis factor alpha, in a PPARgamma-dependent manner. We report here that 15d-PGJ(2) potently inhibits NF-kappaB-dependent transcription by two additional PPARgamma-independent mechanisms. Several lines of evidence suggest that 15d-PGJ(2) directly inhibits NF-kappaB-dependent gene expression through covalent modifications of critical cysteine residues in IkappaB kinase and the DNA-binding domains of NF-kappaB subunits. These mechanisms act in combination to inhibit transactivation of the NF-kappaB target gene cyclooxygenase 2. Direct inhibition of NF-kappaB signaling by 15d-PGJ(2) may contribute to negative regulation of prostaglandin biosynthesis and inflammation, suggesting additional approaches to the development of antiinflammatory drugs.

Regulation of macrophage gene expression by the peroxisome proliferator-activated receptor-gamma.

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma), which is a member of the nuclear hormone receptor superfamily and was originally shown to play an important role in adipocyte differentiation and glucose homeostasis, is now known to regulate cellular proliferation and inflammatory responses. A range of synthetic and naturally occurring substances activates PPAR-gamma, however the identities of endogenous ligands for PPAR-gamma and their means of production in vivo have not been well established. In monocytes and macrophages, interleukin-4 (IL-4) increases the expression of 12/15-lipoxygenase and thus 13-HODE and 15-HETE production. We show that IL-4 induces the expression of PPAR-gamma and provide evidence that the coordinate induction of PPAR-gamma and 12/15-lipoxygenase mediates IL-4 dependent transcription of the CD36 gene and down-regulation of iNOS in macrophages. These findings suggest that PPAR-gamma activity may play an important role in mediating macrophage gene expression signaled by IL-4.

The peroxisome proliferator-activated receptor(PPARgamma) as a regulator of monocyte/macrophage function.

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors of the nuclear hormone receptor super-family, which includes the steroid, retinoid, and thyroid hormone receptors. The PPARs can be activated by fatty acids and their eicosanoid metabolites, and have until recently been considered primarily to regulate genes involved in glucose and lipid homeostasis. In the past year there has been an explosive increase in research implicating PPARgamma in macrophage biology, cell cycle regulation, and atherosclerosis. This review describes recent insights into the role of PPARgamma in the macrophage lineage, and its potential function in the regulation of inflammatory responses and atherosclerosis.

Interleukin-4-dependent production of PPAR-gamma ligands in macrophages by 12/15-lipoxygenase.

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-dependent nuclear receptor that has been implicated in the modulation of critical aspects of development and homeostasis, including adipocyte differentiation, glucose metabolism and macrophage development and function. PPAR-gamma is activated by a range of synthetic and naturally occurring substances, including antidiabetic thiazolidinediones, polyunsaturated fatty acids, 15-deoxy-delta prostaglandin J2 and components of oxidized low-density lipoprotein, such as 13-hydroxyoctadecadienoic acid (13-HODE) and 15-hydroxyeicosatetraenoic acid (15-HETE). However, the identities of endogenous ligands for PPAR-gamma and their means of production in vivo have not been established. In monocytes and macrophages, 13-HODE and 15-HETE can be generated from linoleic and arachidonic acids, respectively, by a 12/15-lipoxygenase that is upregulated by the TH2-derived cytokine interleukin-4. Here we show that interleukin-4 also induces the expression of PPAR-gamma and provide evidence that the coordinate induction of PPAR-gamma and 12/15-lipoxygenase mediates interleukin-4-dependent transcription of the CD36 gene in macrophages. These findings reveal a physiological role of 12/15-lipoxygenase in the generation of endogenous ligands for PPAR-gamma, and suggest a paradigm for the regulation of nuclear receptor function by cytokines.

Expression of the peroxisome proliferator-activated receptor gamma (PPARgamma) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-dependent transcription factor that has been demonstrated to regulate fat cell development and glucose homeostasis. PPARgamma is also expressed in a subset of macrophages and negatively regulates the expression of several proinflammatory genes in response to natural and synthetic ligands. We here demonstrate that PPARgamma is expressed in macrophage foam cells of human atherosclerotic lesions, in a pattern that is highly correlated with that of oxidation-specific epitopes. Oxidized low density lipoprotein (oxLDL) and macrophage colony-stimulating factor, which are known to be present in atherosclerotic lesions, stimulated PPARgamma expression in primary macrophages and monocytic cell lines. PPARgamma mRNA expression was also induced in primary macrophages and THP-1 monocytic leukemia cells by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Inhibition of protein kinase C blocked the induction of PPARgamma expression by TPA, but not by oxLDL, suggesting that more than one signaling pathway regulates PPARgamma expression in macrophages. TPA induced the expression of PPARgamma in RAW 264.7 macrophages by increasing transcription from the PPARgamma1 and PPARgamma3 promoters. In concert, these observations provide insights into the regulation of PPARgamma expression in activated macrophages and raise the possibility that PPARgamma ligands may influence the progression of atherosclerosis.

Diverse signaling pathways modulate nuclear receptor recruitment of N-CoR and SMRT complexes.

Several lines of evidence indicate that the nuclear receptor corepressor (N-CoR) complex imposes ligand dependence on transcriptional activation by the retinoic acid receptor and mediates the inhibitory effects of estrogen receptor antagonists, such as tamoxifen, suppressing a constitutive N-terminal, Creb-binding protein/coactivator complex-dependent activation domain. Functional interactions between specific receptors and N-CoR or SMRT corepressor complexes are regulated, positively or negatively, by diverse signal transduction pathways. Decreased levels of N-CoR correlate with the acquisition of tamoxifen resistance in a mouse model system for human breast cancer. Our data suggest that N-CoR- and SMRT-containing complexes act as rate-limiting components in the actions of specific nuclear receptors, and that their actions are regulated by multiple signal transduction pathways.

The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation.

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors that is predominantly expressed in adipose tissue, adrenal gland and spleen. PPAR-gamma has been demonstrated to regulate adipocyte differentiation and glucose homeostasis in response to several structurally distinct compounds, including thiazolidinediones and fibrates. Naturally occurring compounds such as fatty acids and the prostaglandin D2 metabolite 15-deoxy-delta prostaglandin J2 (15d-PGJ2) bind to PPAR-gamma and stimulate transcription of target genes. Prostaglandin D2 metabolites have not yet been identified in adipose tissue, but are major products of arachidonic-acid metabolism in macrophages, raising the possibility that they might serve as endogenous PPAR-gamma ligands in this cell type. Here we show that PPAR-gamma is markedly upregulated in activated macrophages and inhibits the expression of the inducible nitric oxide synthase, gelatinase B and scavenger receptor A genes in response to 15d-PGJ2 and synthetic PPAR-gamma ligands. PPAR-gamma inhibits gene expression in part by antagonizing the activities of the transcription factors AP-1, STAT and NF-kappaB. These observations suggest that PPAR-gamma and locally produced prostaglandin D2 metabolites are involved in the regulation of inflammatory responses, and raise the possibility that synthetic PPAR-gamma ligands may be of therapeutic value in human diseases such as atherosclerosis and rheumatoid arthritis in which activated macrophages exert pathogenic effects.

Hypothalamic hypophyseal inhibitory factor (HHIF) increases intrasynaptosomal free calcium concentration.

We have isolated from bovine hypothalamic and pituitary tissues a sodium pump inhibitor that is structurally different from ouabain. By mass spectrometric analysis, this purified factor revealed a single unique molecular ion with an accurate mass of 412.277 and a mass spectra different from that of ouabain. It has been previously shown that this factor inhibits the Ca2+, Mg(2+)-ATPase of the plasma membrane of synaptosomes. Because Ca2+ plays a major role in cellular excitability, we carried out a systematic study of the effects of this inhibitor on the Ca2+ transport processes across the plasma membrane of synaptosomes: We measured ATP-dependent calcium uptake, Na(+)-Ca2+ exchange, and passive permeability using 45Ca2+ and Millipore filtration, chlortetracycline fluorescence, and light-scattering, respectively. This factor inhibits the Na+, K(+)-ATPase activity of the synaptosomal plasma membrane vesicles in the same range of concentrations that produced an increase of intrasynaptosomal free calcium, with nearly the same K0.5 value. In addition, in this concentration range, this factor stimulated 10- to 11-fold the passive flux of Ca2+ and 2.5- to 3-fold the Ca2+ influx via the Na(+)-Ca2+ exchange in these membranes with respect to control values. Measurements of fluorescence anisotropy showed that in this concentration range, the inhibitor did not significantly change the order parameter (fluidity) of these membranes. These results suggest that besides its known inhibition of the sodium pump, this factor could play a role in the control of Ca2+ homeostasis by direct modulation of transport systems implicated in the control of intracellular calcium.

Peroxisome proliferator-activated receptors and retinoic acid receptors differentially control the interactions of retinoid X receptor heterodimers with ligands, coactivators, and corepressors.

As the obligate member of most nuclear receptor heterodimers, retinoid X receptors (RXRs) can potentially perform two functions: cooperative binding to hormone response elements and coordinate regulation of target genes by RXR ligands. In this paper we describe allosteric interactions between RXR and two heterodimeric partners, retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs); RARs and PPARs prevent and permit activation by RXR-specific ligands, respectively. By competing for dimerization with RXR on response elements consisting of direct-repeat half-sites spaced by 1 bp (DR1 elements), the relative abundance of RAR and PPAR determines whether the RXR signaling pathway will be functional. In contrast to RAR, which prevents the binding of RXR ligands and recruits the nuclear receptor corepressor N-CoR, PPAR permits the binding of SRC-1 in response to both RXR and PPAR ligands. Overexpression of SRC-1 markedly potentiates ligand-dependent transcription by PPARgamma, suggesting that SRC-1 serves as a coactivator in vivo. Remarkably, the ability of RAR to both block the binding of ligands to RXR and interact with corepressors requires the CoR box, a structural motif residing in the N-terminal region of the RAR ligand binding domain. Mutations in the CoR box convert RAR from a nonpermissive to a permissive partner of RXR signaling on DR1 elements. We suggest that the differential recruitment of coactivators and corepressors by RAR-RXR and PPAR-RXR heterodimers provides the basis for a transcriptional switch that may be important in controlling complex programs of gene expression, such as adipocyte differentiation.

Frequent alterations in gene expression in colon tumor cells of the microsatellite mutator phenotype.

Tumors of the microsatellite mutator phenotype (MMP) are characterized by the accumulation of many thousands of somatic mutations in tracts of simple repeated sequences or microsatellites. Using arbitrarily primed PCR fingerprinting of RNA (RAP-PCR), we have comparatively analyzed the overall gene expression patterns of several colorectal tumor cell lines with and without the MMP. A reproducible pattern of 30-40 main products was obtained for each fingerprint with a total of about 200 cell transcripts analyzed. Differences in RAP-PCR fingerprints were detected between these tumor cell lines. Some of these expression polymorphisms appeared to be specific for tumor cells of the MMP because they were present in two or more different MMP+ cell lines but absent in all MMP- cell lines analyzed. We also analyzed RNAs prepared from single cell clones isolated from these tumor cell lines. Reproducible differences in the fingerprints were detected between single cell clones from each of the cell lines analyzed. Examples of single cell clone-specific fingerprint differences from one of the MMP tumor cell lines were cloned and sequenced. Differential expression of some of these sequences was confirmed by Northern analysis using the cloned fragments as probes. Similar fingerprint alterations were also observed among single cell clones derived from single cell clones from mutator tumor cell lines, which appeared to exhibit higher clonal variation in gene expression compared with MMP- cells. The detection of inter- and intra-tumor alterations in gene expression by unbiased RAP-PCR show that these fluctuations occur with high frequency in tumor cells of the MMP. These results indicate that the profound genomic instability of tumor cells of the MMP is also reflected in a high incidence of alterations in their patterns of gene expression.

Modulation of the Ca2+,Mg(2+)-ATPase of sarcoplasmic reticulum by the hypothalamic hypophyseal inhibitory factor.

We have studied the effect of the endogenous inhibitor of the Na+ and Ca2+ pumps, HHIF, on sarcoplasmic reticulum (SR) vesicles. The effect of HHIF on the SR Ca2+,Mg(2+)-ATPase activity shows a biphasic pattern. Low HHIF concentrations activate the Ca2+,Mg(2+)-ATPase by dissipation of Ca2+ gradient across the SR membrane. Higher concentrations irreversibly inhibit this activity following a slow kinetic process both in intact SR membranes and in purified Ca2+,Mg(2+)-ATPase. Differential scanning calorimetry shows that the Ca2+,Mg(2+)-ATPase is denatured after incubation with HHIF concentrations which produced full inhibition of its activity. Micromolar Ca2+ and millimolar Mg2+ ADP protect against the irreversible inhibition of the Ca2+,Mg(2+)-ATPase by HHIF. The concentration of HHIF which produces 50% inhibition depends upon SR membrane concentration and upon the lipid:protein ratio in purified Ca2+,Mg(2+)-ATPase. From this we have obtained a partition coefficient for binding of HHIF to SR membranes of 0.6 (microgram SR protein/ml)-1.

Effect of hypothalamic-hypophysary inhibitory factor on mesangial cell activation.

We examined the effect of a sodium pump inhibitor isolated from bovine hypothalamus and pituitary tissues on contraction, proliferation, and calcium mobilization in primary cultures of rat mesangial cells. Hypothalamic-hypophysary inhibitory factor (HHIF) inhibited rubidium uptake in a concentration-dependent manner (0.2 U/mL: 56.8 +/- 6.3% inhibition). It also induced a concentration- and time-dependent decrease in planar cell surface area. Maximal contraction (25 +/- 5% reduction in cell size) was reached at 60 minutes with a concentration of 0.2 U/mL. This effect was inhibited by both verapamil and TMB-8 (10(-5) mol/L). HHIF was also observed to increase DNA synthesis (0.2 U/mL: 4361 +/- 168 versus 2129 +/- 162 cpm per well under control conditions) and cell proliferation (0.2 U/mL: 52,290 +/- 1931 versus 10,512 +/- 121 cells per well under control conditions). Both effects were also inhibited by verapamil and TMB-8. Moreover, HHIF induced the expression of immediate early genes c-fos and c-jun mRNA. HHIF-induced effects were accompanied by an increase in cytosolic free calcium (203 +/- 58 versus 101 +/- 2 nmol/L under control conditions), which was inhibited by verapamil and TMB-8. In summary, HHIF induces mesangial cell contraction and proliferation; these effects seem to be mediated by an increase in cytosolic free calcium levels.