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Biomedical subjects

H Fleming

Publications and source records attributed to H Fleming.

At least 19 recordsLinked to original sources
PubMed1995

Differentiation in human endometrial cells in monolayer culture: dependence on a factor in fetal bovine serum.

Human epithelial cells of the Ishikawa endometrial line can be stimulated to differentiate and form multicellular structures in 4-5 day-old monolayer cultures by the addition of a protein factor from fetal bovine serum. Multicellular structures become obvious over an 18-30-h period as the cells enlarge, separate from the dish, and form domes. These structures are similar to those that result from polarization in other epithelial cell lines. Ishikawa dome formation appears to be a multistage process. The appearance of enlarged differentiated cells is detected within hours of adding fetal bovine serum; these enlarged cells lift off the surface of the dish within 6-8 more hours. Domes are observed about 24 h after the addition of fetal bovine serum. Sometimes dome cells migrate into a "bud-like" structure that extends out from the dome. Differentiation of the domes is dependent on a factor from fetal calf serum that behaves similarly to a very large protein or complex of proteins, greater than 300 kd. Progesterone appears to enhance the formation of domes but does not elicit dome formation in the absence of serum factor.

Animals↗
PubMed1995

Induction of heat labile alkaline phosphatase by butyrate in differentiating endometrial cells.

The addition of 2 mM sodium butyrate to monolayers enhances differentiation of Ishikawa endometrial cells. Cells from this cell line have been shown to enlarge and lift off the dish into dome structures over a period of 24-48 h in response to a factor in fetal bovine serum (FBS) [Fleming, 1995 J Cell Biochem in press]. When butyrate is added to monolayers, together with FBS, three- to fourfold higher numbers of differentiated structures, domes and predomes, can be counted. It had previously been shown [Holinka et al., 1986b] that estradiol induces heat stable placental alkaline phosphatase in Ishikawa cells. The addition of butyrate, on the other hand, results in a significant increase in levels of a heat labile alkaline phosphatase isozyme. The heat labile isozyme is also increased to some extent in cells stimulated to differentiate in response to FBS in the absence of butyrate. Differential inhibition by homoarginine and phenylalanine indicates that butyrate is inducing the liver-bone kidney isozyme that is found in endometrial glands in vivo.

Alkaline Phosphatase↗
PubMed1991

Posterior fossa epithelial cyst of ectodermal origin.

The case of a large ventral cyst of the posterior fossa of probable ectodermal origin in a 33-year-old patient is presented. Intracranial epithelial cysts are rare, and their histogenesis is a subject of controversy. Previous reports have hypothesized either a neuroepithelial or endodermal origin for the majority of these cysts. This case is unusual because it is the first case, to our knowledge, where melanocytes and melanin have been demonstrated within the cyst wall. These findings suggest an ectodermal origin for this cyst.

Adult↗
PubMed1991

Sodium salicylate centrally augments ventilation through cholinergic mechanisms.

Several different stimuli, including hydrogen ions, may exert their effect on central ventilatory control through cholinergic mechanisms. Salicylates are known to be central respiratory stimulants. Therefore this study explored whether the ventilatory effect of sodium salicylate (SAL) is through cholinergic mechanisms. Ventriculocisternal perfusion was used in spontaneously breathing anesthetized (pentobarbital sodium, 30 mg/kg) mongrel dogs to study the effects of SAL (50 mM), atropine (ATR, 4.8 mM), and SAL-ATR on ventilation. After 15 min of perfusion with mock cerebrospinal fluid, each test agent was perfused for 15 min at a rate of 1 ml/min. Cardiovascular and ventilatory parameters were monitored. Values at 15 min of test agent perfusion were compared with values at 15 min of mock cerebrospinal fluid perfusion, with each animal used as its own control. Body temperature was kept between 37.5 and 39.0 degrees C. Perfusion with SAL increased minute ventilation (VE) by 54% (P less than 0.005) and respiratory frequency by 50% (P less than 0.005). Tidal volume was not changed, but mean inspiratory flow rate increased (P less than 0.05). Perfusion with ATR decreased VE by 22% (P less than 0.1), and perfusion with SAL-ATR decreased VE by 20% (P = 0.01). No significant cardiovascular changes were noted in any group. We conclude that SAL increases VE centrally, primarily by increasing respiratory frequency. Because ATR blocked this effect, cholinergic mechanisms are probably involved in the salicylates' central stimulation of ventilation.

Animals↗
PubMed1989

Arteriovenous malformations of the uterus.

Six cases of the rare condition of arteriovenous malformation of the uterus are reported. Uterine bleeding was the most common presentation, with the site of the lesion and the caliber of the vessels determining its degree. A preoperative diagnosis was made by angiography in two patients, and was aided by ultrasonography in one.

Adult↗
PubMed1988

Gallstones at autopsy and cholecystectomy: a comparative study.

An examination of 613 post-mortems gave a prevalence of biliary disease at autopsy of 36.5%, higher than reported previously in Australia. This consisted of an asymptomatic gallstone prevalence of 18.9%, with a further 5.7% of the autopsies having granular biliary sludge and 11.9% having had a previous cholecystectomy. Although the rate of occurrence of cholesterol gallstones was approximately half that of the pigment gallstones and pigment biliary sludge combined, no significant association between the sex of the postmortems and stone type was observed at autopsy (chi 2(1) = 0.1: P greater than 0.05). The ratio of biliary disease between females and males was approximately 2:1. Gallstones and biliary sludge from 310 cholecystectomy patients showed that cholesterol gallstones were approximately twice as common in men, and approximately six times as common in women than pigment gallstones. In this group of patients there was a significant association between the sex of the patient and the rate of occurrence of stone type. The rate of occurrence of cholesterol gallstones was significantly higher than pigment gallstones in both the males and females at cholecystectomy (chi 1(2) = 18.97; P less than 0.0001). A female to male ratio of approximately 2:1 was also observed. A statistically significant higher rate of pigmented biliary disease was observed at autopsy than at cholecystectomy. (chi 2 = 101.0; P less than 0.0001). Analyses on biliary sludge, a filterable, fine granular pigmented material in bile, suggest that it may be the direct precursor for a number of different gallstone types.

Adult↗
PubMed1984

Coagulation factor XIII: a useful polymorphic genetic marker.

The plasmas of two groups of subjects were examined for blood coagulation Factor XIII-A (FXIII-A, F13A) by electrophoresis in agarose using a Tris-EDTA-borate buffer to separate the common variants, F13A*1, F13A*2, and F13A*3. Dimeric subunits were visualized in UV light as monodansyl cadaverine bound to casein at the position of the transglutaminase activity representing F13A. One test group consisted of 307 members of three large Caucasian families. The other consisted of 148 consecutive patients whose plasmas had been sent to the clinical laboratory for determination of prothrombin time. Segregation analysis and father-to-son transmission confirmed that F13A is inherited as an autosomal co-dominant trait. The allelic frequencies in the random sample were F13A*1 = 0.82 and F13A*2 = 0.18. This sample included both blacks and whites, and the gene frequencies were not significantly different in the two races. The gene frequencies among the unrelated spouses of the three white families were A*1 = 0.75, A*2 = 0.24, A*3 = 0.01. Genetic equilibrium was present in both groups. The degree of polymorphism, the availability of blood, the ease of assessment, the absence of selective pressure, and the uniformity of gene frequencies in two major American ethnic groups make F13A a very useful marker for linkage studies and paternity testing. F13A has been provisionally assigned to chromosome 6. Linkage analysis of our family data did not provide evidence of linkage to two chromosome 6 markers, properdin factor B (BF) and glyoxalase 1 (GLO). The highest lod score (Z) was between F13A and the Kidd (Jk) blood group (theta = 0.68 at = 0.24).

Alleles↗
PubMed1984

Characteristics of cyclic nucleotide dependent regulation of cytoplasmic E2 binders in cultured endometrial and breast cells.

Estradiol binding levels have previously been shown to be increased or decreased by the addition of of cGMP or cAMP respectively to cellular homogenates prepared from human endometrial tissue or from the endometrial cell line HEC 1. Similar cyclic nucleotide dependent effects have now been demonstrated in homogenates prepared from a second endometrial cell line, HEC 50, and from two breast cancer cell lines, CG-5 and T47D. It was previously shown that ATP and Mg2+ are necessary for the generation or inactivation of E2 binding sites. The present study demonstrates that the concentration of K+ and of dithiothreitol can also affect these reactions.

Breast Neoplasms↗
PubMed1984

Regulation of estrogen receptor levels in endometrial cancer cells.

Recent experimental results from our laboratories revealed the following facts: Addition of GMP to homogenates or cytosol prepared from endometrial tissue or cultured endometrial adenocarcinoma cells during the assay for specific estrogen binders markedly increases specific binding levels. The effect is completed in about 15 min at 4 C (Fleming et al, 1983). Cyclic AMP has the opposite effect and in many cases lowers the number of binding sites to undetectable levels. ATP, a nucleotide that stimulates a particulate form of guanylate cyclase, Na2MoO4, a compound that can elevate cGMP levels (Fleming and Blumenthal, unpublished) and GTP, a metabolic precursor of cGMP, increase specific estradiol binding in the presence of plasma membranes and soluble factors. Cyclic AMP reduces the levels of estrogen binding when added to cell homogenates or to cytosol and counteracts the effects of cGMP, MoO4, ATP and GTP. ATP is required for the expression of cGMP and cAMP effects on estradiol binding. It is therefore likely that phosphorylations are involved in the generation and inactivation of estrogen binding sites. Divalent cation requirements for these effects also suggest participation of protein kinases in these processes. The reported effects of nucleotides and molybdate have been observed in specimens of histologically normal endometrium, in specimens of endometrial carcinoma, in two endometrial adenocarcinoma cell lines, HEC-1 and HEC-50 (Suzuki et al, 1980), and in two breast cancer cell lines, CG-5, a variant of MCF-7 obtained in Iacobelli's laboratory (Natoli et al, 1983), and in T47D) (Fleming et al, in press) Rapid changes in the levels of estrogen binding capacity observed in endometrial cells in culture can be associated with changes in cGMP/cAMP ratios shown, to vary during the cell cycle. Although it has not yet been demonstrated that cGMP-induced increases in specific estrogen binding can enhance responses to available estrogens, such possibility is of potential importance. Reduction of estrogen receptor levels in patients with cancers of estrogen sensitive tissues may inhibit tumor growth promoted by endogenous estrogen. Cho-Chung et al have recently reported that cholera toxin causes a reduction in estrogen receptor levels and arrests hormone dependent growth of DMBA-induced mammary carcinoma in rats (Cho-Chung et al, 1983). They postulated that the effect of cholera toxin is mediated by a cAMP effect on the estrogen receptor, an hypothesis supported by the observation that only tumors containing receptor responded to treatment. Conversely, cGMP-induced increases in specific estrogen binders may be useful in promoting a response of tumors to estr

Adenocarcinoma↗
PubMed1983

Rapid changes in specific estrogen binding elicited by cGMP or cAMP in cytosol from human endometrial cells.

Addition of cGMP to cytosol of human endometrium or to cells of the endometrial cancer line HEC-1 produced severalfold increases in specific estrogen binding (EB) levels. This effect was maximal with 1 microM cGMP in the presence of 0.1 mM isobutylmethylxanthine (a phosphodiesterase inhibitor) during incubations with [3H]estradiol. In contrast, cAMP decreased EB levels under similar conditions. The effects of cyclic nucleotides on EB levels were complete in less than 15 min in the presence of Mg2+, Mn2+, or Ca2+. The EB sites generated by the addition of cGMP during labeling of cytosol with 10 nM [3H]estradiol were found to sediment in the 8S and 4S regions of low-salt glycerol gradients. No changes in EB levels were observed when cyclic nucleotides were added to cytosol depleted of ATP by preincubation at 4 degrees C for 3 hr, but responsiveness was restored by addition of exogenous ATP. The ATP requirement and the pattern of dependence of cyclic nucleotide actions on divalent cation concentrations suggest that cGMP and cAMP effects may be mediated by kinases and may involve phosphorylations. Another possibility is that the cyclic nucleotides interact allosterically with the binder in the presence of ATP. Addition of sodium molybdate, ATP, and GTP to homogenates of endometrial tissue or HEC-1 cells produces increases in EB levels similar to those obtained by the addition of cGMP. However, these compounds are much less active when added to cytoplasm or cytosol. On the basis of these and other observations, it is hypothesized that molybdate, ATP, and GTP affect EB levels primarily by increasing cGMP concentrations through processes involving a plasma membrane-bound guanylate cyclase.

Adenosine Triphosphate↗
PubMed1982

Growth characteristics of primary cultures of stromal cells from human endometrium.

The growth rate of stromal cells derived from 23 specimens of human endometrial tissue taken at various phases of the cycle was measured during the first seven days of primary culture. The stromal cells were found to differ radically in their ability to grow in culture according to the phase of the cycle at which the tissue was obtained. Early and mid proliferative stromal cells were capable of proliferation but that capacity was lost in late proliferative tissue. Growth at significant rates reappeared at days 21--22 and persisted in cells from late secretory tissue. Following plating, a 2--4 day lag period preceded proliferation of all but the most rapidly growing cultures. Subcultures showed a shorter lag period and faster maximal growth rate than the primary cultures from which they were derived. Estradiol, 10(-9) M. inhibition stromal cell growth in primary culture and this effect was prevented by 10(-8) M progesterone. These observations indicate that proliferative characteristics of stromal cells in primary culture are strongly influenced by the hormonal environment of the endometrium at the time of biopsy.

Cell Division↗
PubMed1982

Variability of levels of specific estrogen binding in a human endometrial adenocarcinoma cell line.

Previous reports from this laboratory have described marked changes in the levels of specific estradiol (E2) binders in cultured endometrial adenocarcinoma cells (HEC-1B) that occur during the first 30 h after replating. In the present study, binding levels were measured daily during a period of 8 days. HEC-1B cells were incubated with 100 nM [3H]-estradiol at 4 and 30 degrees C, in the presence or absence of 10 microM diethylstilbestrol and concentrations of bound ligand were determined in the nuclear and cytoplasmic fractions. It was found that concentrations of specific estradiol binders increased the first day after plating and declined thereafter. Saturation analysis of estrogen binding sites in cytosol of HEC-1B cells labeled with [3H]-E2 or [3H]-estriol (e3), at concentrations ranging from 0.1-100 mM, showed two plateaus for E2 binding (at about 20 and 60 nM) but only one (at about 40 nM) for E3, when dextran-coated charcoal was used to separate free and bound ligand. The constants of dissociation of E2 and E3 for the high affinity binder were about 4 and 20 nM, respectively.

Adenocarcinoma↗
PubMed1982

Effects of cyclic nucleotides on estradiol binding in human endometrium.

The addition of molybdate to intact or homogenized cells of the endometrial adenocarcinoma line HEC-1 during incubation with [3H]estradiol ([3H]E2) at 4 C causes substantial increases in cytoplasmic E2 binding levels. A similar effect can be observed in homogenates of normal human endometrium. These effects of molybdate appear to involve activation of E2-binding sites. Fractionation of the homogenates and recombination of different fractions revealed that activation of specific E2-binding sites by by MoO4= requires cytosolic factors as well as factors associated with the cell membrane. In homogenates of neoplastic cells (HEC-1) and normal endometrium, the addition of ATP, GTP, or cGMP was also found to increase E2 binding to levels as high as those obtained by the addition of MoO4=. In contrast, the addition of cAMP was found to lower specific E2 binding levels and to counteract the effects of MoO4=, ATP, GTP, and cGMP. Levels of intracellular cAMP and cGMP can change rapidly in cells in culture. Since cGMP causes E2 binding levels to increase while cAMP causes them to decrease, changes in the levels of these two cyclic nucleotides may explain the fluctuation in concentrations of specific estrogen binders that we have previously reported to occur in cultured endometrial cells.

Adenocarcinoma↗
PubMed1981

Receptors, enzymes, and hormonal responses of endometrial cells.

Studies of hormone-related biochemical parameters of human endometrium are facilitated by the availability of a variety of types of endometrial tissue resulting from changes in hormonal environment during the menstrual cycle or from decidual, hyperplastic, and neoplastic transformations. Estrogen and progesterone receptors levels, rates of metabolism of ovarian hormones, enzymatic activities regulated by hormones or affecting intracellular concentrations of steroids, rates of production of prolactin and prostaglandins, and endogenous levels of hormones have been measured in samples of endometrial tissue and in separated glands and stroma. These studies have also included an evaluation of effects of estrogens and progestins administered to patients or added to the medium during in vito incubations of endometrium under organ culture conditions. Epithelial cells derived from isolated glands and stromal cells have been grown in primary cultures and also subcultured. These studies have revealed two mechanisms by which progestins exert an antiestrogenic effect on the endometrial tissue: viz, they were found to provoke a decline in estrogen receptor levels and to increase the metabolism of E2 through stimulation of estradiol 17 beta-dehydrogenase activity. In addition, studies of cells in culture have led to the unexpected finding of wide and rapid fluctuations in the levels of estrogen receptors, thus providing an opportunity to investigate factors involved in the regulation of concentrations of specific E2-binding sites.

Cells, Cultured↗