Tetracycline up-regulates COX-2 expression and prostaglandin E2 production independent of its effect on nitric oxide

Tetracyclines (doxycycline and minocycline) augmented (one- to twofold) the PGE2 production in human osteoarthritis-affected cartilage (in the presence or absence of cytokines and endotoxin) in ex vivo conditions. Similarly, bovine chondrocytes stimulated with LPS showed (one- to fivefold) an increase in PGE2 accumulation in the presence of doxycycline. This effect was observed at drug concentrations that did not affect nitric oxide (NO) production. In murine macrophages (RAW 264.7) stimulated with LPS, tetracyclines inhibited NO release and increased PGE2 production. Tetracycline(s) and L-N-monomethylarginine (L-NMMA) (NO synthase inhibitor) showed an additive effect on inhibition of NO and PGE2 accumulation, thereby uncoupling the effects of tetracyclines on NO and PGE2 production. The enhancement of PGE2 production in RAW 264.7 cells by tetracyclines was accompanied by the accumulation of both cyclooxygenase (COX)-2 mRNA and cytosolic COX-2 protein. In contrast to tetracyclines, L-NMMA at low concentrations (< or = 100 microM) inhibited the spontaneous release of No in osteoarthritis-affected explants and LPS-stimulated macrophages but had no significant effect on the PGE2 production. At higher concentrations, L-NMMA (500 microM) inhibited NO release but augmented PGE2 production. This study indicates a novel mechanism of action of tetracyclines to augment the expression of COX-2 and PGE2 production, an effect that is independent of endogenous concentration of NO.     

Steric restrictions on the binding of large metal ions to serum transferrin

Apotransferrin in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid at 25 degrees C and pH 7.4 was titrated with acidic solutions of Lu3+, Tb3+, and Eu3+. Metal binding at the two specific metal-binding sites of transferrin was followed from changes in the difference UV spectra at 245 nm. The binding of Tb3+ was also followed from changes in the fluorescence emission spectrum at 549 nm. Apotransferrin was titrated with solutions containing varying ratios of the metal ion and the competitive chelating agent nitrilotriacetic acid, and metal-transferrin binding constants were calculated by nonlinear least-squares fits of the absorbance as a function of titrant added. The sequential carbonate-independent equilibrium constants for the binding of two metal ions are log KM1 = 11.08 and log KM2 = 7.93 for Lu3+, log KM1 = 11.20 and log KM2 = 7.61 for Tb3+, and log KM1 = 9.66 and log KM2 = 7.27 for Eu3+. Titrations of both C-terminal and N-terminal monoferric transferrins indicate that all of these metal ions bind more strongly to the C-terminal binding site. The trend in log K values as a function of the lanthanide ionic radius has been evaluated both by plots of log K versus the metal ion charge/radius ratio and by linear free-energy relationships in which binding constants for complexes of the larger lanthanides are plotted versus the binding constants for complexes with the smallest lanthanide, Lu3+. Both methods indicate that there is a sharp drop in the binding constants for the C-terminal binding site for metals larger than Tb3+. This decrease is attributed to a steric hindrance to the binding of the larger cations. The steric effect is not as strong for metal binding at the N-terminal site. As a result, the selectivity for binding to the C-terminal site, which is quite high for the smaller lanthanides, drops sharply on going from Tb3+ to Nd3+.     

Milking of microalgae

The low productivity of algal cultures in the production of high-value compounds is the most significant bottleneck for commercialization of this technology. Cultures in which cell mass is reused for continuous production are proposed as a solution to overcome this problem. Recently, a method was developed in which beta-carotene was harvested from the microalga Dunaliella salina grown in a two-phase bioreactor. This raises the question of whether this technique could also be used in the mass production of secondary metabolites. Understanding the mechanism of the milking process and its relationship to the product formation pathway should reveal whether other products can be milked from various species of microalgae.     

Polycystin-1 activates and stabilizes the polycystin-2 channel

Autosomal dominant polycystic kidney disease (ADPKD) is a prevalent genetic disorder largely caused by mutations in the PKD1 and PKD2 genes that encode the transmembrane proteins polycystin-1 and -2, respectively. Both proteins appear to be involved in the regulation of cell growth and maturation, but the precise mechanisms are not yet well defined. Polycystin-2 has recently been shown to function as a Ca(2+)-permeable, non-selective cation channel. Polycystin-2 interacts through its cytoplasmic carboxyl-terminal region with a coiled-coil motif in the cytoplasmic tail of polycystin-1 (P1CC). The functional consequences of this interaction on its channel activity, however, are unknown. In this report, we show that P1CC enhanced the channel activity of polycystin-2. R742X, a disease-causing polycystin-2 mutant lacking the polycystin-1 interacting region, fails to respond to P1CC. Also, P1CC containing a disease-causing mutation in its coiled-coil motif loses its stimulatory effect on wild-type polycystin-2 channel activity. The modulation of polycystin-2 channel activity by polycystin-1 may be important for the various biological processes mediated by this molecular complex.     

Identification of epidermal growth factor-responsive genes in normal rat ovarian surface epithelial cells

Alteration in epidermal growth factor receptor (EGFR) family signaling is among the most frequently implicated effectors of human oncogenesis. Overexpression of members of this family of receptors has often been detected in many epithelial tumors and is believed to be associated with an overall poor prognosis in patients with cancer. Therefore, we hypothesized that identification of potential EGF target genes in normal cells will provide a basis for unbiased genetic analysis of this signaling pathway in cancer. We utilized Atlas Rat 1.2 nylon cDNA arrays (Clontech) to determine gene expression changes in normal rat ovarian surface epithelial (ROSE) cells following EGF treatment. The results indicate activation of genes involved in a wide variety of cellular mechanisms, including regulation of cell cycle and proliferation, apoptosis, and protein turnover. In addition, using an in vitro model of ovarian cancer, we demonstrated that malignant transformation of ROSE cells resulted in alteration of downstream effectors of the EGFR pathway, as exemplified by aberrant expression of p66Shc, c-Jun, c-Myc, c-Fos, Lot1, p21Cip/Waf, and cdc25A. These data suggest that knowledge of the downstream genetic lesions, which may result in loss of growth factor requirement of the affected cells, will be crucial for the selection of the EGFR pathway as an effective target for cancer therapy.     

Effect of phosphocreatine on H+ extrusion,pHi and dimorphism in Candida albicans

Candida albicans is an opportunistic pathogen. Its proliferation in human hosts is believed to be controlled by immunologic mechanisms. The plasma membrane of the fungus possesses an H(+)-ATPase (PM-ATPase) which actively extrudes protons to generate an electrochemical gradient which is used in co-transport of nutrients. This ATPase is associated with the growth, dimorphism and pathogenicity of the fungus. The physiological concentration of phosphocreatine (PCr) is 20-35 mM in skeletal muscles. H(+)-extrusion in Candida cells was strongly inhibited by PCr; 44% at 20 mM and 69% at 40 mM. H(+)-extrusion was stimulated 6.2-fold in the presence of 10 mM glucose. This glucose stimulated extrusion was inhibited significantly by PCr; 36% at 20 mM and 53% at 40 mM. The intracellular pH pattern of cells destined to differentiate was greatly altered in the presence of PCr. Evagination time for control cells was between 90-120 min. PCr, delayed dimorphism, reduced the population of cells differentiating to hyphae and also reduced the length of hyphae after each time interval. Only 60% differentiation was observed with 10 mM PCr and 40% for higher PCr concentration even after 210 min. Direct interaction of PM-ATPase and PCr has been demonstrated by difference spectrum measurement employing stopped flow spectrophotometer. It can be concluded that PCr may be playing a significant role in checking growth and pathogenesis of C. albicans.