Thrombopoietin regulates proliferation, apoptosis, secretory activity and intracellular messengers in porcine ovarian follicular cells: involvement of protein kinase A.

Thrombopoietin (TPO) is known to be involved in megakariocytopoesis, but its role in the control of ovarian function is unknown. The aims of this study were to determine whether TPO can regulate the proliferation, apoptosis and secretory activity of ovarian cells, to identify possible intracellular mediators of TPO action, especially protein kinase A (PKA), and to define their interrelationships within ovarian cells. We investigated the effect of TPO treatment (0, 1, 10 or 100 ng/ml) on the following characteristics of cultured porcine ovarian follicles, determined using SDS-PAGE and Western blotting, immunocytochemistry, RIA and ELISA: the expression of intracellular peptides associated with proliferation (PCNA), apoptosis (Bax), tyrosine kinase (TK, phosphotyrosine), Cdc2/p34 kinase, PKA and the transcription factor CREB-1, and the secretion of progesterone, androstenedione, estradiol-17beta, oxytocin, inhibin A, inhibin B, IGF-I, transforming growth factor-2beta (TGF-2beta) and IGF-binding protein 3 (IGFBP-3). The involvement of PKA-dependent pathways was examined by evaluating the effect of a PKA blocker (KT5720, 1 microg/ml), either alone or in combination with TPO, on the parameters listed above. A TPO-induced increase in expression of PCNA, Bax, PKA, TK, Cdc2/p34 and CREB was observed. Furthermore, TPO was able to inhibit androstenedione, estradiol, TGF-2beta and IGFBP-3 secretion, and to stimulate oxytocin, inhibin A, inhibin B and IGF-I secretion. Progesterone secretion was not stimulated. The PKA blocker KT5720, when given alone, reduced the expression of Bax and TGF-2beta, augmented the expression of PKA, CREB and oxytocin, but did not influence the secretion of progesterone, androstenedione, estradiol, IGFBP-3, inhibins A and B or IGF-I. When given together with TPO, the PKA blocker prevented or reversed the action of TPO on PKA, CREB, androstenedione, estradiol, IGFBP-3, oxytocin, but not its effect on Bax, TGF-2beta or inhibin B. On the other hand, treatment with KT5720 augmented the effect of TPO on progesterone, inhibin A and IGF-I. These results provide the first evidence that TPO may be a potent regulator of ovarian function (e.g. proliferation, apoptosis and the secretion of peptide hormones, steroids, growth factors and growth factor-binding protein, as well as of the expression of some intracellular messengers). Furthermore, they demonstrated the importance of PKA in controlling these functions and in mediating the effects of TPO on ovarian cells. It remains possible that other (TK- and Cdc2/p34-dependent) intracellular mechanisms are also involved in mediating TPO action on the ovary.

EXG1p, a novel exo-beta1,3-glucanase from the fungus Cochliobolus carbonum, contains a repeated motif present in other proteins that interact with polysaccharides.

Genomic and cDNA copies of EXG1, a gene encoding an exo-beta1, 3-glucanase from the plant pathogenic fungus Cochliobolus carbonum, were isolated. The gene contains two introns of 50 and 53 bp, and the mRNA has a 5'-untranslated region of 90 nt and a 3'-untranslated region of 159 nt. The deduced protein product, EXG1p, has a predicted signal peptide of 17 amino acids, but based on the known N-terminus of the mature protein is further processed to remove an additional 25 amino acids. The sequence of EXG1p is not closely related to any other known protein, but has a low similarity (29% overall amino acid identity) to BGN13.1, an endo-beta1,3-glucanase from the mycoparasitic fungus Trichoderma harzianum. EXG1p contains two imperfect copies of a 23-amino acid motif that is found in several other proteins that interact with polysaccharides, including plant and bacterial polygalacturonases, phage neck appendage protein, phage endoneuramidase, and bacterial mannuronan epimerase.

MP1: a MEK binding partner that enhances enzymatic activation of the MAP kinase cascade.

Signal transduction is controlled both by regulation of enzyme activation and by organization of enzymatic complexes with nonenzymatic adapters, scaffolds, and anchor proteins. The extracellular signal-regulated kinase (ERK) cascade is one of several evolutionarily conserved mitogen-activated protein (MAP) kinase cascades important in the regulation of growth, apoptosis, and differentiation. A two-hybrid screen was conducted to identify nonenzymatic components of this signaling cascade that might be important in regulating its activity. A protein called MP1 (MEK Partner 1) was identified that bound specifically to MEK1 and ERK1 and facilitated their activation. When overexpressed in cultured cells, MP1 enhanced activation of ERK1 and activation of a reporter driven by the transcription factor Elk-1. Expression of MP1 in cells increased binding of ERK1 to MEK1. MP1 apparently functions as an adapter to enhance the efficiency of the MAP kinase cascade.

Insulin-like growth factor binding protein-3 levels during early and late follow-up after surgery in acromegalic patients.

Disease activity in acromegaly is accurately reflected by growth hormone (GH) concentration during oral glucose tolerance test (OGTT) and insulin-like growth factor-I (IGF-I) levels, representing an integrated index of GH activity. This prospective study was performed to evaluate whether plasma IGF binding protein 3 (IGFBP-3) might also reflect the hormonal disease activity in pituitary acromegaly after operative treatment during early and late follow-up. Twenty-two acromegalic patients were studied. Data were obtained pre-, intra- and post-operatively in 13 cases. In 9 patients the acromegalic activity was studied only after treatment. The hormonal assessment included repeated blood samples for estimation of IGF-I, IGFBP-3 and repeated OGTTs. In each case 100 sigma g octreotide (Sandostatin lambda, Sandoz, Basel) was injected to test the acute response of GH, IGF-I and IGFBP-3. Intraoperatively, GH levels were estimated to examine acutely the influence of tumour reduction on GH levels. Patients were considered cured when GH levels (GH60min) were less than 2 ng/ml during OGTT 4 weeks after surgery. The data outlined that in patients with normalized GH60min levels, normalized IGFBP-3 levels were noticed 4 weeks and 12 months post-operatively. In non-cured patients normalized IGFBP-3 concentrations were found in 11 out of 15 cases in the late post-treatment phase. In contrast only 1 of 7 cured patients had persistently elevated IGF-I levels within the first month post-operatively, whereas no case of the non-cured patients had IGF-I values in the normal range. Despite these observations a strong correlation of IGF-I and IGFBP-3 did not exist before one year post-operatively -- either in the cured or in the non-cured patients. Serum IGFBP-3 in patients with pituitary acromegaly does not provide a predictive value of appreciable magnitude concerning cure or non-cure from the disease- whether examined early or late in the post-operative period. Absolute levels of IGFBP-3 may thus cause misinterpretation concerning cure of acromegalics after surgery.

Cloning and sequencing of a cDNA for firefly luciferase from Photuris pennsylvanica.

The first cDNA from the Photurinae subfamily of the Lampyridae encoding a firefly luciferase from lantern mRNA of Photuris pennsylvanica has been cloned, sequenced, the amino-acid sequence predicted and the sequence reported to GenBank. The cDNA was about 1.8 kb in length with the largest open reading frame coding for a 545-residue protein. The 5' noncoding region is 61 bp long and the 3' noncoding region is 135 bp in length. There is a 24-nucleotide poly(A) tail. When the amino-acid residues are aligned, P. pennsylvanica contains 154 (about 28% of the total residues) that are conserved in all 16 of the deduced luciferase sequences that are presently available. In this P. pennsylvanica luciferase, the amino acids at 276 of the positions are the same at corresponding positions of at least one of the other enzymes. There are two amino-acid differences between this luciferase and the unpublished sequence obtained by Dr. Keith Wood for a putative larval Photuris firefly luciferase cloned from a Maryland firefly. Signature amino-acid sequences and domains found in the deduced sequence are for adenylate kinase, the putative AMP-binding domain, luciferin 4-monooxygenase, 4-coumarate CoA ligase, long-chain fatty acid CoA ligase, 2-acylglycerophosphoethanolamine acyltransferase, the microbody-directing sequence, peptide-synthesizing complexes, and acyladenylate-synthesizing enzymes.

Melatonin and serotonin regulate the release of insulin-like growth factor-I, oxytocin and progesterone by cultured human granulosa cells.

The direct influence of indoleamines on ovarian peptide hormones and growth factor secretion, in contrast to steroidogenesis, is yet to be thoroughly investigated. The aim of our in vitro experiments was to investigate the influence of melatonin and serotonin (5-hydroxy-tryptamine) (0.01-10 micrograms/ml) on the release of insulin-like growth factor-I (IGF-I), oxytocin and progesterone by cultured human granulosa cells. It was observed that both melatonin and serotonin stimulate IGF-I release. Melatonin also stimulated oxytocin output. Serotonin increased oxytocin secretion only at the highest dose (10 micrograms/ml). Both melatonin and serotonin were potent inhibitors of progesterone release. The present results suggest a possible involvement of the indoleamines melatonin and serotonin in the direct regulation of growth factor, nonapeptide and steroid hormone secretion by human ovarian cells.

Elevated levels of insulin-like growth factor (IGF) binding protein-3 in rheumatoid arthritis synovial fluid inhibit stimulation by IGF-I of articular chondrocyte proteoglycan synthesis.

The objective of this study was to quantify insulin-like growth factor (IGF) binding proteins (IGFBPs) in the synovial fluid (SF) and plasma of patients with rheumatic diseases and to study the role of these proteins in the regulation of cartilage proteoglycan (PG) synthesis. Immunological determination of IGFBP-2, IGFBP-3, IGF-I, IGF-II, interleukin-1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF alpha) was undertaken in the SF and plasma of 115 patients with rheumatoid arthritis (RA; n = 53), osteoarthritis (OA; n = 44) and other rheumatic disorders. We also determined the effects of SF on bovine cartilage PG synthesis in culture. IGFBP-2 and IGFBP-3 were elevated in the plasma (by 38% and 28%, respectively) and SF (by 56% and 59%, respectively) of patients with RA compared to age- and sex-matched OA controls (determined by RIA and confirmed by Western ligand blot). IGF-I and IGF-II did not differ significantly between the two groups. OA SF, and, to a lesser extent, RA SF stimulated cartilage PG synthesis in culture, and more than 60% of this activity was neutralised by a specific monoclonal anti-IGF-I antibody. Human IGFBP-3 dose-dependently inhibited the stimulation of cartilage PG synthesis effected by SF or human IGF-I. In RA patients, the SF concentration of IGFBP-3 was positively correlated with SF levels of IL-1 beta and TNF alpha, with the serum levels of C-reactive protein and with the erythrocyte sedimentation rate. We concluded that IGF-I is, under the conditions studied, the most important anabolic factor in human SF with respect to articular cartilage PG synthesis. The bioactivity of IGF-I in joints is modulated by IGFBP-3, which is elevated in RA SF compared to OA SF. Elevated IGFBP-3 in RA SF may reduce the availability of IGF-I to articular chondrocytes, thus interfering with cartilage PG synthesis in RA.

Oxytocin affects the release of steroids, insulin-like growth factor-I, prostaglandin F2alpha and cyclic nucleotides by human granulosa cells in vitro.

The aim of the present experiments was to examine the effects of oxytocin (1-10 000 ng/ml) on hormone and cyclic nucleotide secretion by human granulosa cells cultured in a serum-supplemented medium. The release of progesterone, oestradiol, insulin-like growth factor-I, prostaglandin F2alpha, cAMP and cGMP into the incubation medium was analysed by radioimmunoassay. An inhibition of progesterone, but not of oestradiol release was observed. Oxytocin also stimulated insulin-like growth factor-I, prostaglandin F2alpha, cAMP and cGMP output. The results suggest an involvement of oxytocin in the autocrine/paracrine regulation of steroid, insulin-like growth factor, prostaglandin and cyclic nucleotide release by human ovarian cells.

High-dose progesterone infusion in healthy males: evidence against antiglucocorticoid activity of progesterone.

High concentrations of unbound cortisol in late pregnancy have been explained by the antiglucocorticoid activity of high progesterone levels. To further test this hypothesis we studied the effect of high-dose progesterone on baseline and corticotrophin-releasing hormone (CRH)-induced hormone secretion in humans. In a double-blind crossover study eight healthy male volunteers received either progesterone (0.714 mg.kg-1.h-1 for 60 min followed by a dose of 0.45 mg.kg-1.h-1 over a total infusion time of 315 min) or vehicle as a continuous intravenous infusion. At 210 min a CRH test (0.1 microgram/kg body weight as bolus iv) was performed. Within 30 min after the start of progesterone administration the serum progesterone level increased to 454 +/- 31 nmol/l and remained in the range of third trimester pregnancy concentrations throughout the infusion period. During vehicle infusion the progesterone level remained in the normal range for healthy males and demonstrated a small but significant increase after CRH (1.52 +/- 0.23 vs 0.74 +/- 0.14 mmol/l; p < 0.01). However, baseline and CRH-stimulated serum cortisol and plasma adrenocorticotrophic hormone remained unaffected by high-dose progesterone. Moreover, unbound salivary cortisol also was not affected by progesterone, suggesting that there is no significant competition for transcortin binding sites. In conclusion, no antiglucorticoid activity was found after short-term administration of progesterone in males. These findings cast doubts on the concept that the alterations of the pituitary-adrenal axis in late pregnancy are induced by the antiglucocorticoid activity of high progesterone concentrations.

A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

Identification of enhancer and silencer regions involved in salt-responsive expression of Crassulacean acid metabolism (CAM) genes in the facultative halophyte Mesembryanthemum crystallinum.

In response to salinity or drought stress, the facultative halophyte Mesembryanthemum crystallinum will switch from C3 photosynthesis to Crassulacean acid metabolism (CAM). During this switch, the transcription rates of many genes encoding glycolytic, gluconeoagenic, and malate metabolism enzymes are increased. In particular, transcription of the Ppc1 and Gap1 genes encoding a CAM-specific isozyme of phosphoenolpyruvate carboxylase and NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, respectively, is increased by salinity stress. To investigate the molecular basis of salt-induced gene regulation, we examined the Ppc1 and Gap1 promoters for cis-elements and trans-acting factors that may participate in their expression. Ppc1 or Gap1 promoter-beta-glucuronidase chimeric gene constructs containing various deletions were introduced into intact, detached M. crystallinum leaves by microprojectile bombardmen. The Ppc1 5'-flanking region contains several salt-responsive enhancer regions and one silencer region reflecting the complex regulation patterns exhibited by this promoter in vivo. A region localized between nucleotides -977 and -487 relative to the transcriptional start site appears to regulate the magnitude of salt-inducibility. In contrast, the Gap1 promoter contains a single region from -735 to -549 that confers salt-responsive gene expression. Alignment of these 5'-flanking regions reveals several common sequence motifs that resemble consensus binding sites for the Myb class of transcription factors. Electrophoretic gel mobility shift assays indicate that both the -877 to -679 region of Ppc1 and the -735 to -549 region of Gap1 form a DNA-protein complex unique to nuclear extracts from salt-stressed plants. The appearance of this DNA-protein complex upon salt stress suggests that it may participate in salt-induced transcriptional activation of Ppc1 and Gap1.

The release of insulin-like growth factor-I by luteinized human granulosa cells in vitro: regulation by growth hormone, oxytocin, steroids and cAMP-dependent intracellular mechanisms.

The aim of the present experiments was to demonstrate the release of insulin-like growth factor-I (IGF-I) by human granulosa cells, and to examine the role of growth hormone (GH), oxytocin, steroids and cAMP-dependent intracellular mechanism in its control. A significant accumulation of IGF-I in a serum-supplemented medium in which the human granulosa cells were cultured for 4 days was observed. The concentration of IGF-I in the medium was particularly high at 3 and 4 days of culture. The addition of GH (1-10,000 ng/ml) to the medium increased IGF-I secretion by the cells. A higher GH dose (100,000 ng/ml) was inhibitory. Oxytocin stimulated IGF-I release at doses of 10-10,000 ng/ml. Dibutyryl-cAMP, isobutyl-methyl-xanthine (inhibitor of cAMP catabolism) or forskolin (stimulator of cAMP production) inhibited IGF-I output at these doses. Additions of progesterone (1-1,000 ng/ml) did not affect IGF-I release, whilst adrostenedione and estradiol were stimulatory at doses of 1, 10, 100, 1,000 ng/ml and 10, 100 and 1,000 ng/ml respectively. Testosterone inhibited IGF-I at a dose of 1,000 ng/ml but not at lower doses (1, 10 or 100 ng/ml). Blockade of estradiol (but not of testosterone) in the medium by specific antisera (1 or 10%) significantly reduced IGF-I output. The same effect was observed with an antiserum to progesterone when added at 0.1%, whilst higher doses (1 or 10%) stimulated IGF-I secretion. The present observations demonstrate the involvement of peptide, steroid hormones and cAMP in the regulation of IGF-I secretion by luteinized human granulosa cells. In particular, both GH and oxytocin are stimulators of IGF-I release. Estradiol and androstenedione, but not testosterone, may also be stimulators of IGF-I output. The involvement of progesterone in this process can also not be excluded. A cAMP-dependent intracellular mechanism appears to play an inhibitory role in the regulation of IGF-I secretion by luteinized human granulosa cells.

[The prognostic significance of cathepsin D in primary breast cancer]. / Prognostische Bedeutung von Kathepsin D beim primären Mammakarzinom.

The prognostic significance of the lysosomal protease cathepsin D in breast cancer was evaluated in a retrospective study. Cathepsin D was measured in 346 deep-frozen (-70 degrees C) cytosol specimens of primary breast carcinomas (1982-1990). Among the established prognostic factors, only axillary lymph node involvement correlated with the expression of cathepsin D (> 40/60 pmol/mg) (p = 0.04-0.05). Univariate analyses of disease-free survival (DFS) and overall survival (OS) showed, that the expression of cathepsin D had no effect on the prognosis either in the whole population of breast cancer patients during long-term follow-up (n = 302; median observation time 51 months) or in the group of women with positive lymph nodes (n = 157; 46 months). However, within the group of N0 patients (n = 145; 57 months), high cathepsin D levels were associated with an unfavourable OS, but this relationship was statistically insignificant (p = 0.08-0.13). A similar influence of cathepsin D on DFS could not be demonstrated. Compared to tumour size, grading and receptor status in multivariate analysis, cathepsin D was a more indicative, but finally insignificant prognostic factor for OS. According to these results, cathepsin D may contribute only in combination with other prognostic factors to identify those 20-30% of node-negative patients with unfavourable prognosis, who may benefit from adjuvant therapy.

Cloning and targeted gene disruption of EXG1, encoding exo-beta 1, 3-glucanase, in the phytopathogenic fungus Cochliobolus carbonum.

The phytopathogenic fungus Cochliobolus carbonum produces an extracellular enzyme capable of degrading beta 1,3-glucan in an exolytic manner. On the basis of partial amino acid sequences of the purified enzyme, two degenerate oligonucleotides were synthesized and used as PCR primers to amplify a 1.1-kb fragment of corresponding genomic DNA. The PCR product was used to isolate the genomic copy of the gene, called EXG1. Partial sequencing of the genomic DNA confirmed that the PCR product corresponded to EXG1. A strain of the fungus specifically mutated in the EXG1 gene was constructed by homologous integration of an internal fragment of EXG1. In the mutant, enzymatic activity and the corresponding peak of UV absorption during high-pressure liquid chromatography purification were reduced by at least 98%. However, crude culture filtrates of the mutant retained 44% of the wild-type beta 1,3-glucanase activity. This residual activity was due to two additional activities which were chromatographically separable from the product of EXG1 and which were coeluted with beta 1,3-beta 1,4-glucanase activity. Growth of the EXG1 mutant was normal on sucrose and oat bran but was reduced by 65% on pure beta 1,3-glucan. The EXG1 mutant was still pathogenic to maize.