TGFß1 single-nucleotide polymorphism C-509T alters mucosal cell function in pediatric eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic Th2 antigen-driven disorder associated with tissue remodeling. Inflammation and remodeling lead to esophageal rigidity, strictures, and dysphagia. TGFß1 drives esophageal remodeling including epithelial barrier dysfunction and subepithelial fibrosis. A functional SNP in the TGFß1 gene that increases its transcription (C-509T) is associated with elevated numbers of esophageal TGFß1-expressing cells. We utilized esophageal biopsies and fibroblasts from TT-genotype EoE children to understand if TGFß1 influenced fibroblast and epithelial cell function in vivo. Genotype TT EoE esophageal fibroblasts had higher baseline TGFß1, collagen1α1, periostin, and MMP2 (p < 0.05) gene expression and distinct contractile properties compared with CC genotype (n = 6 subjects per genotype). In vitro TGFß1 exposure caused greater induction of target gene expression in genotype CC fibroblasts (p < 0.05). Esophageal biopsies from TT-genotype subjects had significantly less epithelial membrane-bound E-cadherin (p < 0.01) and wider cluster distribution at nanometer resolution. TGFß1 treatment of stratified primary human esophageal epithelial cells and spheroids disrupted transepithelial resistance (p < 0.001) and E-cadherin localization (p < 0.0001). A TGFß1-receptor-I inhibitor improved TGFß1-mediated E-cadherin mislocalization. These data suggest that EoE severity can depend on genotypic differences that increase in vivo exposure to TGFß1. TGFß1 inhibition may be a useful therapy in subsets of EoE patients.

Formoterol and salmeterol induce a similar degree of ß2-adrenoceptor tolerance in human small airways but via different mechanisms.

BACKGROUND AND PURPOSE: Steroids prevent and reverse salbutamol-induced ß(2)-adrenoceptor tolerance in human small airways. This study examines the effects of the long-acting ß(2) agonists (LABAs) formoterol and salmeterol, and the ability of budesonide to prevent desensitization. EXPERIMENTAL APPROACH: Long-acting ß(2) agonists in the presence and absence of budesonide were incubated with human precision-cut lung slices containing small airways. Tolerance was deduced from measurements of reduced bronchodilator responses to isoprenaline and correlated with ß(2)-adrenoceptor trafficking using a virally transduced, fluorescent-tagged receptor. The ability of the LABAs to protect airways against muscarinic-induced contraction was also assessed. KEY RESULTS: Following a 12 h incubation, both formoterol and salmeterol attenuated isoprenaline-induced bronchodilatation to a similar degree and these effects were not reversible by washing. Pre-incubation with budesonide prevented the desensitization induced by formoterol, but not that induced by salmeterol. Formoterol also protected the airways from carbachol-induced bronchoconstriction to a greater extent than salmeterol. In the epithelial cells of small airways, incubation with formoterol promoted receptor internalization but this did not appear to occur following incubation with salmeterol. Budesonide inhibited the formoterol-induced reduction in plasma membrane ß(2)-adrenoceptor fluorescence. CONCLUSIONS AND IMPLICATIONS: Although both formoterol and salmeterol attenuate isoprenaline-induced bronchodilatation, they appear to induce ß(2)-adrenoceptor tolerance via different mechanisms; formoterol, but not salmeterol, enhances receptor internalization. Budesonide protection against ß(2)-adrenoceptor tolerance was correlated with the retention of receptor fluorescence on the plasma membrane, thereby suggesting a mechanism by which steroids alter ß(2)-adrenoceptor function.

Structural and functional characterization of the human gene for sorting nexin 1 (SNX1).

The aim of the present study was to identify the gene for sorting nexin 1 (SNX1) to evaluate the potential for tissue-specific alternative splicing and to analyze the activity of the SNX1 promoter. The coding DNA for SNX1 was divided between 15 exons in 43 kb of genomic DNA located on human chromosome 15q22. Although SNX1 mRNA expression was widespread in human tissues, alternative splicing is thought to generate skipped exons in SNX1 cDNAs. By determination of the SNX1 gene structure and an analysis of the mRNAs in a variety of tissues using RT-PCR, we demonstrated that SNX1 mRNAs are alternatively spliced. Exon-skipped products were less abundant than full-length SNX1 mRNA species, but the ratio of skipped to full-length mRNA indicated that alternative splicing may be developmentally regulated in the liver. Consistent with widespread mRNA expression, the SNX1 promoter was GC rich and lacked a TATA box, features characteristic of housekeeping promoters. The promoter activity was dependent on the presence of proximal sequences that contained initiator elements and predicted binding sites for the transcription factors Sp1 and E2F. These findings indicate that regulation of SNX1 gene expression at the transcriptional level is likely minor. Rather, developmentally specific exon skipping provides a potential mechanism for regulating the activity of SNX1.

Self-assembly and binding of a sorting nexin to sorting endosomes.

The fate of endocytosed membrane proteins and luminal contents is determined by a materials processing system in sorting endosomes. Endosomal retention is a mechanism that traps specific proteins within this compartment, and thereby prevents their recycling. We report that a sorting nexin SNX1, a candidate endosomal retention protein, self-assembles in vitro and in vivo, and has this property in common with its yeast homologue Vps5p. A comparison of SNX1 expressed in bacterial and in mammalian systems and analyzed by size-exclusion chromatography indicates that in cytosol SNX1 tetramers are part of a larger complex with additional proteins. An endosomal retention function would require that SNX1 bind to endosomal membranes, yet the complexes that we analyzed were largely soluble and little SNX1 was found in pellet fractions. Using green fluorescent protein fusions, endocytic compartment markers and fluorescence recovery after photobleaching, we found that there is an equilibrium between free cytoplasmic and early/sorting endosome-bound pools of green fluorescent protein-SNX1. Fluorescence resonance energy transfer indicated that spectral variants of green fluorescent protein-SNX1 were oligomeric in vivo. In cell extracts, these green fluorescent protein-SNX1 oligomers corresponded to tetrameric and larger complexes of green fluorescent protein-SNX1. Using video microscopy, we observed small vesicle docking and tubule budding from large green fluorescent protein-SNX1 coated endosomes, which are features consistent with their role as sorting endosomes. http://www.biologists.com/JCS/movies/jcs2058.html

Identification of a human orthologue of Sec34p as a component of the cis-Golgi vesicle tethering machinery.

The roles of the components of the Sec34p protein complex in intracellular membrane trafficking, first identified in the yeast Saccharomyces cerevisiae, have yet to be characterized in higher eukaryotes. We cloned a human cDNA whose predicted amino acid sequence showed 41% similarity to yeast Sec34p with homology throughout the entire coding region. Affinity-purified antibodies raised against the human SEC34 protein (hSec34p) recognized a cellular protein of 94 kDa in both soluble and membrane fractions. Like yeast Sec34p, cytosolic hSec34p migrated with an apparent molecular mass of 300 kDa on a glycerol velocity gradient, suggesting that it is part of a protein complex. Immunofluorescence microscopy localized hSec34p to the Golgi compartment in cells of all species examined, where it co-localized well with the cis/medial Golgi marker membrin and partially co-localized with cis-Golgi network marker p115 and trans-Golgi marker TGN38. The co-localization with membrin was maintained at 15 degrees C and after microtubule depolymerization with nocodazole. During transport of the tsO45 vesicular stomatitis virus G protein through the Golgi, there was significant overlap with the hSec34p compartment. Green fluorescent protein-hSec34 expressed in HeLa cells was restricted to Golgi cisternae, and its membrane association was sensitive to brefeldin A treatment. Taken together, our findings indicate that hSec34p is part of a peripheral membrane protein complex localized on cis/medial Golgi cisternae where it may participate in tethering intra-Golgi transport vesicles.

Acute effects on the human EEG after an external exposure to 200 ppm methanol.

OBJECTIVES: Even low concentrations of organic solvents may cause acute effects on the human central nervous system. The German MAK (threshold limit value) of methanol is 200 ppm. The aim of this study was to investigate whether acute exposure to 200 ppm methanol causes adverse effects, measured by EEG, and moreover, whether it is possible to differentiate between sedative and excitatory effects with this method. METHODS: Twelve healthy subjects were exposed for 4 h to 200 ppm and to 20 ppm (control) in an exposure chamber in a cross-over design. The EEG was recorded before (reference) and at the end of each exposure with, the subject's eyes closed and opened and during a choice reaction test (color word stress test). Spectral power was calculated by fast Fourier transformation. Subjective symptoms and effects of blinding with 20 ppm methanol were assessed by questionnaires. RESULTS: The study was a single-blind one. During subjects' exposure to 200 ppm, their scores for prenarcotic and irritating symptoms were not different from controls. In the closed-eye condition of subjects, the spectral power of the theta-band and of some electrodes of the delta-band was significantly less at the end of exposure to 200 ppm, than that of controls. In the open-eye condition and during the color word stress test no significant changes were found. CONCLUSION: The changes in the theta-band suggest a slight excitatory effect of 200 ppm methanol. The effect was weak, as scores of acute symptoms did not change. With respect to our results, it is not necessary for the MAK value to be decreased.

Acute effects of 200 ppm 1,1,1-trichloroethane on the human EEG.

OBJECTIVES: Even low concentrations of organic solvents used at work may cause acute effects on the human central nervous system. We investigated the acute effects of 200 ppm 1,1,1-trichloroethane on the human EEG. METHODS: 12 healthy subjects were exposed for 4 hours to 200 ppm and to 20 ppm (control) in an exposure chamber in a cross-over design. EEG was recorded before (reference) and at the end of each exposure with eyes closed and open and during the Color Word Stress test. Spectral power was calculated by Fast Fourier transformation and related to reference values (per cent of baseline). Subjective symptoms and effects of blinding with 20 ppm 1, 1,1-trichloroethane were assessed by questionnaire. RESULTS: Blinding was not effective because of the strong smell of 1,1, 1-trichloroethane. The score for tiredness increased slightly during and after exposure to 200 ppm. In the closed eye condition, the median percentage of spectral power increased at all electrodes of the delta -band, significantly at temporo-occipital leads. In the theta-band, the percentage of the median spectral power was elevated at most of the electrodes but the parietal and some temporal ones. As to the alpha subset1-band, the percentage of the median spectral power was lower at the temporo-parieto-occipital electrodes, yielding significance at T subset4. In the alpha subset2-band, the percentage of the median spectral power was lower at all electrodes, significantly at T subset4 and T subset5. The percentage of the median spectral power of the temporo-parieto-occipital electrodes of the beta subset1 -band was lower during exposure to 200 ppm. There were no clear-cut changes in the beta subset2 -band, in the open eye condition and during the Color Word Stress test. CONCLUSION: The changes in EEG and the increased score for tiredness indicate a slight sedative effect of 200 ppm 1,1,1-trichloroethane.

The product of the MLD gene is a member of the membrane fatty acid desaturase family: overexpression of MLD inhibits EGF receptor biosynthesis.

Membrane fatty acid desaturases are responsible for inserting double bonds into specific positions in fatty acids. We have cloned a new member of the human membrane fatty acid (lipid) desaturase gene family, MLD. The derived amino acid sequence of MLD contains three consensus motifs, HX3H, HX2HH, and HX2HHXFP, that are characteristic of a group of membrane fatty acid desaturases. MLD is predicted to be a multiple membrane-spanning protein and is found to be extractable from particulate fractions with detergent but not salt or urea. MLD is widely expressed in human tissues and is localized to the endoplasmic reticulum. Cotransfection of MLD with the epidermal growth factor (EGF) receptor resulted in decreased expression of the receptor but did not affect platelet-derived growth factor receptor expression. MLD overexpression inhibited biosynthesis of the EGF receptor, suggesting a possible role of a fatty acid desaturase in regulating biosynthetic processing of the EGF receptor.

Characterization of the 5'-flanking region of the gene for the cAMP-inducible protein kinase A subunit, RIIbeta, in Sertoli cells.

Activation of cyclic AMP-dependent protein kinases (protein kinase A, PKA) by gonadotropins and cyclic AMP (cAMP) plays an important role in the regulation of testicular functions. A regulatory subunit, RIIbeta, of PKA is transcriptionally induced in rat Sertoli cells in response to treatment with cAMP. The present study addresses regulatory mechanisms leading to increased transcription of the rat RIIbeta gene. We have localized a footprint which overlaps one of the major transcription initiation sites in the basal promoter (-293 to -123). One of the proteins binding this sequence belongs to the NF-1 family of transcription factors. We also observed binding to a basic helix-loop-helix (bHLH) response element. Furthermore, transfection studies of various 5'-deletions of the rat RIIbeta gene in primary cultures of rat Sertoli cells and in peritubular cells revealed the presence of an upstream region (-723 to -395, cAMP-responsive region) inhibiting basal expression from the rat RIIbeta gene only in Sertoli cells. This region was found to enhance cAMP responsiveness in Sertoli cells but not in peritubular cells. Interactions with downstream elements seemed to be important for the function of the cAMP-responsive region. Although some short stretches reveal homology to the cAMP-responsive regions of other slowly cAMP-responding genes, and an AP-1-like element is present, no strong resemblance to any known regulatory element responsive to cAMP is found.

Enhanced degradation of EGF receptors by a sorting nexin, SNX1.

The vectorial movement of proteins requires specific recognition by components of the vesicular trafficking machinery. A protein, sorting nexin-1 (SNX1), was identified in a human cell line that bound to a region of the epidermal growth factor receptor (EGFR) containing the lysosomal targeting code. SNX1 contains a region of homology to a yeast vacuolar sorting protein, and overexpression of SNX1 decreased the amount of EGFR on the cell surface as a result of enhanced rates of constitutive and ligand-induced degradation. Thus, SNX1 is likely to play a role in sorting EGFR to lysosomes.

Association of epidermal growth factor receptors with coated pit adaptins via a tyrosine phosphorylation-regulated mechanism.

We investigated the mechanism by which ligand-activated epidermal growth factor receptors (EGFR) associate with coated pit adaptor protein (AP) complexes. In vivo association, assayed by coimmunoprecipitation of AP with mutant EGFR, required tyrosine kinase activity, intact autophosphorylation sites, and the regulatory carboxyl terminus of EGFR. The role of autophosphorylation of EGFR in interaction with AP was examined in vitro using a BIAcore instrument. Purified active EGFR, immobilized on the biosensor surface, was reversibly autophosphorylated or dephosphorylated by treatment with ATP or phosphatase. Autophosphorylation of EGFR significantly increased AP binding. Once formed, EGFR AP complexes were resistant to disassembly by dephosphorylation of EGFR or competition with phosphotyrosine, indicating that phosphorylated tyrosine residues do not directly participate in AP binding. Induction of conformational changes in EGFR by treatment with urea increased AP binding up to 10-fold in the absence of EGFR autophosphorylation. A recombinant EGFR carboxyl terminus specifically bound the AP complex and each of the isolated alpha- and beta-subunits of AP2. We conclude that tyrosine autophosphorylation of EGFR exposes structural motif(s) in the carboxyl terminus of EGFR that interact specifically with AP2.

Identification and characterization of the GC-rich and cyclic adenosine 3',5'-monophosphate (cAMP)-inducible promoter of the type II beta cAMP-dependent protein kinase regulatory subunit gene.

A rat genomic clone containing 4.5 kilobases of 5'-flanking DNA and the first exon of the type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase was isolated, restriction mapped, and sequenced. The proximal 400-basepair promoter region was GC rich, lacked TATA/CAAT box motifs, and initiated transcription at multiple sites. Bandshifting and DNase-I footprinting experiments using this region of the RII beta promoter detected several related specific DNA-protein complexes formed using crude and fractionated nuclear extracts from rat ovary, brain, adrenal gland, and liver. All binding in these experiments mapped to a domain within the same region found to confer cAMP inducibility to a chloramphenicol acetyltransferase (CAT) reporter gene when transfected into primary cultures of rat granulosa cells. Although GC boxes (putative SP1-binding sites) and activator protein-2 (AP-2) elements were present in this functional region, and although expression vectors containing AP-2 sites conferred high levels of cAMP regulation of the CAT gene in cultured ovarian cells, neither the GC boxes nor the AP-2 sites were protected by footprint analyses or required for band shift activity of nuclear extract protein. These known regulatory elements, therefore, may be involved in functional activity of the RII beta promoter, but additional cis-acting DNA and trans-acting factors (yet to be characterized) also appear to interact with the functional promoter of the RII beta gene and regulate the hormone-specific expression of the A-kinase subunit in ovarian and neuronal cells.

Radiation therapy as local treatment in Ewing's sarcoma. Results of the Cooperative Ewing's Sarcoma Studies CESS 81 and CESS 86.

The Cooperative Ewing's Sarcoma Studies, CESS 81 and CESS 86, are multiinstitutional trials with more than 80 participating institutions from Germany, the Netherlands, Austria, and Switzerland. Treatment consists of four courses of multiagent chemotherapy and local therapy. Local therapy was not randomized and was either radical surgery or resection plus postoperative irradiation or definitive radiation therapy. Here results according to local therapy have been analyzed for 93 protocol patients with localized Ewing's sarcoma (ES) who have been recruited in CESS 81 from January 1981 to February 1985 and 122 protocol patients recruited in CESS 86 from January 1986 to November 1989. The 3-year relapse-free survival (RFS) was 55% in CESS 81 and 62% in CESS 86. In CESS 81, the RFS was better for surgically treated than for irradiated patients. In this study there was an extremely high incidence of local failures (50%) after definitive irradiation. In CESS 86, however, the results after radiation therapy have been improved markedly (3-year RFS 67% after radiation therapy, 65% after surgery, and 62% after resection plus irradiation). Possible explanations for the improvement of radiotherapeutic results are as follows: selections for patients for radiation therapy, start of local therapy, and quality of radiation therapy. In CESS 86, irradiated patients were randomized to receive either conventionally fractionated irradiation with less intense chemotherapy or hyperfractionated irradiation with simultaneous chemotherapy. There was no difference in treatment results at the time of analysis. The authors conclude that selection of patients for local treatment modalities and quality of treatment performance has an impact on local and overall treatment results in ES.

An adenosine 3'5'-monophosphate-responsive deoxyribonucleic acid element confers forskolin sensitivity on gene expression by primary rat granulosa cells.

The following studies were conducted with the goal of understanding some of the molecular mechanisms by which cAMP alters granulosa cell function. In this regard, we characterized the expression of messages for the cholesterol side-chain cleavage cytochrome P450 (P450scc) enzyme and a type II cAMP-dependent protein kinase subunit (RII beta) in granulosa cells isolated from small antral follicles and cultured in 1% fetal bovine serum-containing medium. Forskolin (FSK) stimulated cAMP production followed by accumulation of RII beta mRNAs, induction of P450scc mRNA, and, finally, progesterone biosynthesis. The regulation of each mRNA displayed a different sensitivity to actinomycin-D treatment. To determine if the modulation of RII beta or the induction of P450scc could be mediated by the enhancer activity of a cAMP-responsive DNA element (CRE), plasmid DNA containing the CRE and TATA box of the human glycoprotein alpha-subunit (alpha G) gene fused to the chloramphenicol acetyl transferase (CAT) gene was introduced into cultured granulosa or interstitial cells, and the ability of FSK to stimulate the expression of the CAT enzyme was measured. When granulosa cells were isolated from preantral/small antral follicles and maintained for at least 5 days in culture, 5 or 10 microM FSK reversibly stimulated expression of the CAT enzyme within 18 h posttransfection. Under similar conditions, interstitial ovarian cells (prepared from the residual ovarian tissue remaining after granulosa cell isolation) were unable to express the template unless the cells were pretreated for 24 h with FSK before transfection. Thereafter, CAT gene expression by interstitial cells was maintained in a FSK-insensitive manner. To determine if cAMP-dependent transcription of the reporter gene required the same sequences that had been characterized for placenta-derived cells, a truncated plasmid lacking the CRE of the alpha G gene was transfected. Under no condition was expression of the CAT gene observed from a CRE-deficient template. The acute transcriptional activation of a CRE/TATA box-containing gene by cAMP in granulosa cells indicated that transcription-activating proteins interacting with the CRE were either present in these cells or were rapidly synthesized after stimulation. To distinguish between these two possibilities, protein synthesis was transiently inhibited after transfection, before the addition of FSK, by incubating the cells for 2 h with 25 micrograms/ml cycloheximide. Cycloheximide treatment alone did not stimulate transcription from the CRE-containing molecule. Incubation with cycloheximide, followed by treatment with FSK, increased CAT activity 2-fold compared to t

Induction of adenosine 3',5'-monophosphate-dependent protein kinase subunits during adipogenesis in vitro.

Fatty acid metabolism in adipocytes is known to be regulated by the intracellular transducer cAMP. This study was undertaken to determine the temporal and hormonal regulation of cAMP-dependent protein kinase during the differentiation of preadipocyte mesenchymal cells to adipocytes. For this we have used a stable cell line (TA1) in which the undifferentiated preadipocyte acquires adipocyte functions and morphology after growth to confluence. We observed that synthesis of type I and II cAMP-dependent protein kinases was induced during the adipogenic conversion of growth-arrested TA1 cells. In preconfluent cells, neither mRNAs encoding regulatory subunits (RI, RII beta) and catalytic subunit (C alpha) nor the peptides themselves were detectable. Within several days of growth arrest at high cell density, mRNAs for RI, RII beta, and C alpha were detectable in total RNA extracted from cell populations. The subunits themselves were detectable in some, but not all, of the cells by indirect immunofluorescence. Immunoblotting of cytosolic extracts indicated that RI and the beta-isoform of RII (mol wt = 52,000) were expressed in these cells. Analysis of subunit presence or absence in single cells by immunofluorescence also indicated that kinase subunit expression preceded the accumulation of lipid droplets within the cells. Further, the subunits were predominantly associated with a reticular cytoplasmic structure (Golgi apparatus?) abutting the nucleus. Conversion of TA1 cells to adipocytes can be accelerated by indomethacin (125 microM) or dexamethasone (1 microM) treatment, compounds that also enhanced the accumulation of RII beta and C alpha mRNAs. Within 2-3 days of addition of indomethacin to confluent cultures, RII beta message content is increased about 20-fold, and protein content is increased about 5-fold relative to those in untreated cultures. C alpha mRNA content is increased about 5-fold relative to that in untreated cells. The response to dexamethasone requires 6-7 days, and changes in RII beta message levels were the most pronounced. We also observed the induction of mRNAs for the functionally relevant mRNA lipoprotein lipase in indomethacin-treated cells. In addition to this apparent transcriptional regulation of kinase subunit expression, we provide evidence for regulation at the posttranscriptional level. Within a differentiated culture, there exist stem cells that can be selected, will repopulate the dish, and will again differentiate into adipocytes upon growth arrest at high cell density. In preconfluent populations of these stems cells, unlike the preconfluent TA1 cells originally plated, both RII beta and C alpha messages were present.(ABSTRACT TRUNCATED AT 400 WORDS)

[Radiotherapy in Ewing sarcoma: current results of the German Society of Pediatric Oncology studies CESS 81 and CESS 86]. / Radiotherapie beim Ewing-Sarkom: Aktuelle Ergebnisse der GPO-Studien CESS 81 und CESS 86.

In CESS 81 the rate of local recurrences was high particularly in patients with radiation for local control. To improve the safety of local control, in the follow-up study CESS 86 chemotherapy for high risk patients was intensified. The combination of surgery with postoperative radiation was favoured when possible, local control was brought forward from week 18 to week 9, the doses of postoperative radiotherapy was increased from 36 to 46 Gy, and a radiation planning center was established for centralized planning of radiotherapy on the basis of tumor extension at diagnosis. In addition patients with radiation were randomized for conventional fractionation or a scheme of accelerated split course hyperfractionation with simultaneous chemotherapy. Preliminary results of 76 CESS 86 patients (incl. pilot phase), show a lowered rate of local recurrences compared to CESS 81: 6% local recurrences and 15% local recurrences in patients with radiation. With selection of patients with small and chemoresponsive tumors for radiotherapy no longer a disadvantage was seen for patients with radiotherapy concerning the safety of local control.

Hormonal regulation of estradiol biosynthesis, aromatase activity, and aromatase mRNA in rat ovarian follicles and corpora lutea.

To determine the molecular basis for changes in aromatase (P450arom) activity in rat ovarian follicles and corpora lutea, seven clones for rat P450arom cDNA have been identified and isolated from a rat granulosa cell lambda gt11 cDNA expression library using a 62 mer deoxyoligonucleotide probe (derived from an amino acid sequence of purified human placental aromatase) and a human placental P450arom cDNA probe. One of the rat P450arom cDNA clones contained an insert 1.2 kb in size. Both the human 1.8 kb cDNA and the rat 1.2 kb cDNA probes hybridized to a single species of P450arom mRNA that was 2.6 kb in size. Northern blot analysis revealed that corpora lutea isolated on day 15 of pregnancy contained high amounts of P450arom mRNA, whereas granulosa cells of antral follicles of hormonally primed, hypophysectomized rats (i.e., those from which mRNA was isolated to construct the cDNA library) contained only low amounts of P450arom mRNA. The lower amounts of P450arom in granulosa cells of preovulatory follicles in the estradiol-follicle-stimulating hormone primed hypophysectomized rats were unexpected because follicles incubated in medium containing testosterone substrate produce more estradiol than do corpora lutea isolated on day 15 of pregnancy and incubated under similar conditions. Additional studies will determine the hormonal events responsible for the elevated amounts and constitutive maintenance of P450arom mRNA and aromatase activity in luteal cells in vivo and in vitro.

Prostaglandin endoperoxide synthase in rat ovarian follicles: content, cellular distribution, and evidence for hormonal induction preceding ovulation.

These studies were undertaken to determine if the content of prostaglandin endoperoxide (PGH) synthase (PGS) was regulated in rat ovarian follicles in a time-, tissue-, and dose-dependent manner. For quantitation and localization of the enzyme by immunoblot and immunofluorescent analyses, respectively, a polyclonal antibody to highly purified (95%) ovine seminal vesicle PGS [mol wt, (Mr), 72,000] was generated in a rabbit, and an immunoglobulin G fraction of the antiserum was purified by elution from a PGS affinity resin. Soluble cell extracts were prepared from: 1) small antral (SA) and preovulatory (PO) rat follicles before and at selected time intervals after exposure to increasing doses (0.25-10.0 IU) of hCG, 2) granulosa and thecal cells of these same follicles, and 3) granulosa cells of hypophysectomized (H) rats before and after treatment with estradiol (HE), estradiol and FSH (HEF), and 10 IU hCG. Immunoblot analyses demonstrated that the content of PGS (Mr, 72,000) was low (negligible) in granulosa and thecal cells of SA and PO follicles, was induced preferentially (approximately 15-fold) in granulosa cells between 1 and 7 h after hCG treatment (0.5, 2.0, or 10 IU), and declined between 12-24 h after administration of 10 IU hCG, reaching undetectable amounts in corpora lutea isolated 48 h after hCG treatment. PGS (Mr, 72,000) was also induced by 10 IU hCG in granulosa cells of HEF rats, but was low in granulosa cells of H, HE, and HEF rats. In contrast, prostacyclin synthase was present in granulosa cells of preantral (H and HE rats), SA, and PO follicles, was not induced by hCG, and was highest in thecal cells. Immunofluorescent analyses confirmed both the tissue-specific localization and induction of PGS in granulosa cells of rat PO follicles treated with 10 IU hCG and the subcellular fractionation analyses showing that the PGS that was induced by hCG was localized primarily to a membrane (plasma membrane?) fraction of granulosa cells. Immunofluorescent data also demonstrated immunoreactive PGS in the vascular tissue of the rat corpus luteum but not in the luteal cells. Results of these studies document unequivocally that the synthesis of prostaglandins that is increased by LH/hCG in rats preceding ovulation is associated with an increased PGS content, that induction of the induced enzyme is transient, and that the enzyme is primarily localized to granulosa cell membrane fractions.

Gap junction modulation in rat uterus. III. Structure-activity relationships of estrogen receptor-binding ligands on myometrial and serosal cells.

A number of steroidal and nonsteroidal estrogen receptor-binding ligands were tested for their ability to affect the formation and internalization of gap junctions in hypophysectomized rat uterine myometrial and serosal cells. Potent estrogen, including diethylstilbestrol, estradiol benzoate (EB), estradiol-17 beta, and the weak estrogens, estriol and estrone, stimulate formation of macular and annular gap junctions in myometrium in a dose-dependent fashion when administered in daily injections over 5 days. Induction of annular gap junctions in the uterine serosal epithelium follows a similar dose-dependent pattern of estrogen stimulation but requires lower levels of hormone to initiate the response. In myometrium, differential stimulation of circular and longitudinal myometrial cell layers was observed, with 3 to 5 times more gap junctions detected in the circular than in the longitudinal layer. Progesterone, estriol, or estrone suppress the myometrial gap junction response to EB when administered concurrently with EB. However, the EB-stimulated appearance of myometrial cell gap junctions was blocked when the progesterone-to-estrogen ratio exceeded 100:1. The estrogen receptor-binding androgens, 5 alpha-androstane-3 beta,17 beta-diol (Adiol) and delta 5-androstene-3 beta,17 beta-diol failed to induce myometrial gap junctions at doses up to 5 mg/day for 5 days, whereas Adiol did induce annular gap junctions in the serosal cells at the highest dosage tested. Of the triphenylethylene derivatives and related compounds evaluated, including mixed isomers of tamoxifen and CI 628, the cis (zuclomiphene, ZUC) and trans (enclomiphene) isomers of clomiphene citrate, and a fixed-ring antiestrogen, nafoxidine, only ZUC was able to induce gap junctions in myometrial and serosal cells. These studies indicate that induction of gap junctions in rat uterine myometrial cells is an estrogen-dependent response that requires higher levels of estrogen than other estrogen-dependent target cell responses in the rodent uterus.

[X-ray image and differential diagnosis of pulmonary changes in Hodgkin's disease]. / Röntgenologisches Erscheinungsbild und Differentialdiagnose der Lungenveränderungen bei Morbus Hodgkin.

Of 291 patients with histologically confirmed lymphogranulomatosis, 45 (15.5%) with lung involvement were assessed. 22 (49%) of the patients with affected lung had the nodular sclerosing type of Hodgkin's disease. Radiologically, 15 patients showed a focal solitary manifestation of lymphogranulomatosis, whereas in 19 patients multiple, plane or circumscript areas of shadow were seen in one or both lobes of the lung. 11 further patients had unilateral or bilateral pleural effusions. Nodular (35%) and pneumonic (22%) opacities are the most frequent x-ray manifestations of pulmonary lymphogranulomatosis. The variable appearance of the lesions makes it difficult to differentiate against pulmonary infections, especially inflammatory complications, the incidence of which may be higher with these patients. In view of the far-reaching consequences, therefore, the nature of the pulmonary lesions should be clarified by biopsy in case of doubt, as early as possible (transbronchial biopsy and, if necessary, open lung biopsy).