Repressor and int synthesis of bacteriophage lambda in the E. coli host mutant ER437.

Summary Analysis of phage infection of the host mutant ER437 by SDS polyacrylamide gel electrophoresis and autoradiography has revealed altered expression of repressor and integration function (Int). We show that in this host Int as well as repressor synthesis is not dependent upon the cIII gene product in the usual manner, nor is their synthesis turned off in the normal way.     

Continuous production of gibberellic acid in a fixed-bed reactor by immobilized mycelia of Gibberella fujikuroi in calcium alginate beads

Summary The continuous production of gibberellic acid with immobilized mycelia of Gibberella fujikuroi was maintained over a hundred days in a tubular fixed-bed reactor. Free mycelium at the beginning of the storage phase was harvested from G. fujikuroi shake-flask culture and was immobilized by ionotropic gelation in calcium alginate beads.The continuous recycle production system consisted of a fixed-bed reactor, a container in which the culture medium was heated, stirred and aerated, and valves for sample withdrawal or reactant addition during the first 1320 h (55 days). A two-phase continuous extractor was then added for the last 960 hours (40 days). Free and immobilized mycelium shake-flask cultures with the same strain used in the continuous culture system were also realized to compare growth, maintenance and production parameters. The results show about the same gibberellic acid productivity in both free and immobilized mycelium shakeflask cultures: 0.384 and 0.408 mgGA₃·gBiomass^-1 ·day^-1, respectively, whereas in the continuous system the gibberellic acid production is about twice as large for a similar biomass: 0.768 mgGA₃·gBiomass^-1·day^-1. Several factors affecting the overall productivity of the immobilized systems were found to be: the quality and the quantity of mycelia in the biocatalyst beads and the immobilization conditions.     

Gonadotropins at menopause: the influence of obesity, insulin resistance, and estrogens

Obese, postmenopausal women have lower FSH levels. To determine whether this is due to higher estrogen exposure, we compared feedback gonadotropin sensitivity and its relation to insulin resistance in four groups of obese and lean, postmenopausal women. Group one was treated with 400 mg troglitazone (TG) daily for two weeks; 150 clomiphene citrate (CC) was added daily for the second week. Group two received 150 mg CC daily for a week. Group three received 1000 mg metformin (MET) daily for two weeks, with 120 mg raloxifene (RAL) added during the second week. Group four received 120 mg RAL for a week. Before and after each period, a serum pool was obtained from samples taken every minute during a 10 ml interval. The women recruited for this study were categorized as obese or lean based on BMI >/= 29 or BMI < 29, respectively. Obese, menopausal women had lower FSH (45.5 IU/l) and LH (16.2 IU/l) values than those of lean (64.1 IU/l and 23.0 IU/l), but the obese menopausal women had higher leptin, DHEAS, glucose, insulin, and HOMA-IR levels. Log [FSH] was associated with BMI (r = -0.53, P < 0.000001) and number of pregnancies (r = -0.37, P = 0.0009). TG treatment did not change HOMA-IR or gonadotropin levels, but DHEAS and androstenedione levels decreased significantly. CC alone or together with TG, diminished FSH (-7.9 and -9.2) and LH (-2.5 and -3.6) concentrations, with a greater reduction in lean women. MET reduced glucose and the HOMA-IR index without affecting gonadotropin or steroid levels. Conclusions: obese, menopausal women have lower FSH levels due to greater estrogen exposure, by mechanisms unrelated to insulin resistance.     

Proteolytic cleavage confers nitric oxide synthase inducing activity upon prolactin

Prolactin (PRL), originally associated with milk secretion, is now known to possess a wide variety of biological actions and diverse sites of production beyond the pituitary. Proteolytic cleavage is a common post-translational modification that can either activate precursor proteins or confer upon the peptide fragment unique biological actions not exerted by the parent molecule. Recent studies have demonstrated that the 16-kDa N-terminal proteolytic cleavage product of PRL (16K-PRL) acts as a potent inhibitor of angiogenesis. Despite previous demonstrations of 16K-PRL production in vivo, biological functions beyond its antiangiogenic actions remain unknown. Here we show that 16K-PRL, but not full-length PRL, acts to promote the expression of the inducible isoform of nitric oxide synthase (iNOS) and nitric oxide (*NO) production by pulmonary fibroblasts and alveolar type II cells with potency comparable with the proinflammatory cytokines interleukin-1beta, interferon gamma, and tumor necrosis factor alpha. The differential effect of 16K-PRL versus PRL occurs through a receptor distinct from known PRL receptors. Additionally, pulmonary fibroblasts express the PRL gene and endogenously produce 16K-PRL, suggesting that this pathway may serve both autocrine and paracrine roles in the regulation of *NO production. These results reveal that proteolytic cleavage of PRL confers upon this classical hormone potent iNOS inducing activity, suggesting its role in inflammatory/immune processes.     

Recombinant extracellular domain of rabbit growth hormone receptor and biological activity of somatogenic hormones

The cDNA of the extracellular domain of rabbit growth hormone receptor (rbGHR-ECD) was cloned in the prokaryotic expression vector pMON, to enable its expression in Escherichia coli after induction with nalidixic acid. The bacterially expressed rbPRLR-ECD protein, contained within the refractile-body pellet, was solubilized in 4.5 M urea, refolded, and purified on a Q-Sepharose column, pH 8, by stepwise elution with NaCl. The bioactive monomeric 28-kDa fraction was eluted in 0.15 M NaCl, yielding 50 mg/2.5 l of induced culture. The purified protein was over 98% homogeneous, as shown by SDS-PAGE in the presence or absence of reducing agent, and by chromatography on a Superdex column. Gel filtration was used to determine the stoichiometry of rbGHR-ECD's interaction with human (h), ovine (o), chicken (ch) and common carp (cc) GHs and with bovine (b) and caprine (c) placental lactogens (PLs). The formation of 2:1 complexes was indicated in all cases. Binding experiments using radiolabelled oGH as a ligand revealed it to be the most effective competitor, followed by bPL, cPL, hGH chGH and ccGH, with respective IC50 values of 0.27, 0.94, 1.55, 2.13, 41.9 and 51.2 nM. Rabbit GHR-ECD inhibited the bPL-inducible proliferation of FDC-P1 cells stably transfected with rbGHR and Nb2 cells possessing rat PRLR. The biological activity of oGH, hGH, cPL, bPL, chGH and ccGH was tested in the FDC-P1 cells stably transfected with rbGHR and yielded the respective EC50 values (in nM) of 0.024, 0.023, 0.021, 0.24, 4.71 and 0.49. These results indicate remarkable discrepancies between the binding capacities and biological activities: the possible reasons for these findings are discussed.     

Synthetic genetic array analysis of the PtdIns 4-kinase Pik1p identifies components in a Golgi-specific Ypt31/rab-GTPase signaling pathway

Phosphorylated derivatives of phosphatidylinositol are essential regulators of both endocytic and exocytic trafficking in eukaryotic cells. In Saccharomyces cerevisiae, the phosphatidylinositol 4-kinase, Pik1p generates a distinct pool of PtdIns(4)P that is required for normal Golgi structure and secretory function. Here, we utilize a synthetic genetic array analysis of a conditional pik1 mutant to identify candidate components of the Pik1p/PtdIns(4)P signaling pathway at the Golgi. Our data suggest a mechanistic involvement for Pik1p with a specific subset of Golgi-associated proteins, including the Ypt31p rab-GTPase and the TRAPPII protein complex, to regulate protein trafficking through the secretory pathway. We further demonstrate that TRAPPII specifically functions in a Ypt31p-dependent pathway and identify Gyp2p as the first biologically relevant GTPase activating protein for Ypt31p. We propose that multiple stage-specific signals, which may include Pik1p/PtdIns(4)P, TRAPPII and Gyp2p, impinge upon Ypt31 signaling to regulate Golgi secretory function.