Effect of cell density and growth factors on matrix GLA protein expression by normal rat kidney cells

The present studies demonstrate that the expression of the vitamin K-dependent matrix Gla protein (MGP) is critically dependent on cell density in culture. Subculture of confluent NRK cells to 1/30 of the confluent cell density causes a 50- to 100-fold decline in MGP expression per cell within two days. MGP expression subsequently increases with increasing cell density and eventually attains a level of expression per cell at five days post-confluence that is over 2,000-fold greater than was seen in the cells two days after the 1 to 30 subculture. These reversible, density-dependent changes in MGP expression are far larger than have been previously reported for other secreted proteins and suggest that the as yet unknown function of MGP requires its expression at high cell density but not at low. We have also observed that human epidermal growth factor (EGF) causes a 20-fold reduction in MGP expression in post-confluent, non-dividing cultures and suggest that the suppression of MGP function at high density may be a prelude to cell migration or division in response to appropriate signals. J. Cell. Physiol. 171:125–134, 1997. © 1997 Wiley-Liss, Inc.     

Modulation of the estrogen-regulated proteins cathepsin D and pS2 by opioid agonists in hormone-sensitive breast cancer cell lines (MCF7 and T47D): Evidence for an interaction between the two systems

In many cancer cell lines, including breast, prostate, lung, brain, head and neck, retina, and the gastrointestinal tract, opioids decrease cell proliferation in a dose-dependent and reversible manner. Opioid and/ or other neuropeptide receptors mediate this decrease. We report that only the steroid-hormone-sensitive cell lines MCF7 and T47D respond to opioid growth inhibition in a dose-dependent manner. Therefore, an interaction of the opioid and steroid receptor system might exist, as is the case with insulin. To investigate this interaction, we have assayed two estrogen-inducible proteins (pS2 and the lysosomal enzyme cathepsin D) in MCF7 and T47D cells. When cells were grown in the presence of FBS (in which case a minimal quantity of estrogens and/ or opioids is provided by the serum), we observed either no effect of etorphine or ethylketocyclazocine (EKC) or an increase of secretion and/ or production of pS2 and cathepsin D. However, when cells were cultured in charcoal-stripped serum and in the absence of phenol red, the effect of the two opioids is different: EKC decreased the production and/ or secretion of pS2 and cathepsin D, whereas etorphine increased their synthesis and/ or secretion. The differential effect of the two general opioids was attributed to their different receptor selectivity. Furthermore, the variations of the ratio of secreted/ produced protein and the use of cycloheximide indicate that opioids selectively modify the regulatory pathway of each protein discretely. In conclusion, through the interaction with opioid and perhaps other membrane-receptor sites, opioid agonists modify in a dose-dependent manner the production and the secretion of two estrogen-regulated proteins. Opioids may therefore disturb hormonal signals mediated by the estrogen receptors. Hence, these chemicals may have potential endocrine disrupting activities. J. Cell. Biochem. 71:416–428, 1998. © 1998 Wiley-Liss, Inc.     

Mitogenic effect of erythropoietin on neonatal rat cardiomyocytes: Signal transduction pathways

The mitogenic effect of recombinant human erythropoietin (rHuEpo) on primary cultures of neonatal rat cardiac myocytes was observed. rHuEpo triggered a dose-dependent increase in myocyte proliferation. The hormone effect over optimally grown control culture 1 day after addition was maximum with 0.5 U/ml and was inhibited with anti-rHuEpo. Inhibitors of enzymatic pathways known to be involved in the cytokines intracellular mechanism such as genistein (tyrosine kinase inhibitor), 2-nitro-4-carboxiphenyl-N,N-diphenylcarbamate (phospholipase C [PLC] inhibitor), and 1-(5-isoquinolinylsufonyl)-2-methyl-piperazine (protein kinase C [PKC] inhibitor) prevented the mitogenic action of rHuEpo. Also the inhibition of Na⁺-K⁺-ATPase activity by ouabain blunted the stimulatory action of rHuEpo on cell proliferation. The mitogenic action of the hormone was correlated with cardiac membrane paranitrophenilphosphatase (pNPPase) and PKC activity, since concentrations of rHuEpo that stimulate DNA synthesis increased pNPPase and PKC activity. Moreover, the enzymatic inhibition of tyrosine kinase, PLC, and PKC attenuated the stimulatory action of rHuEpo upon cardiac pNPPase activity. In this paper we demonstrate a non-hematopoietic action of rHuEpo showing both mitogenic and enzymatic effect upon primary myocyte cell culture and on pNPPase activity of neonatal rat heart. These effects are related to the capacity of rHuEpo to stimulate Na⁺-K⁺-ATPase activity and appear to be secondary to the activation of tyrosine kinase and PKC, indicating that in the rHuEpo mediated mitogenic action on cardiomyocytes involves the activation of the same enzymatic pathways that have been described by other cytokines in different tissues. © 1996 Wiley-Liss, Inc.     

Inhibition of quinolinate phosphoribosyl transferase by pyridine analogs of quinolinic acid

The enzyme quinolinate phosphoribosyl transferase was purified from ATCC strain 23269. An HPLC method was developed for the analysis of the product of the enzyme reaction, nicotinate mononucleotide. Steady state kinetics in the forward reaction demonstrated a sequential mechanism for the enzyme. In order to gain more information on the mechanism of the enzyme reaction, a series of 2 substituted nicotinic acids and 2 substituted 3-nitropyridines were investigated as inhibitors of the reaction. The results indicate that potent inhibition results when the quinolinic acid analogs possessed a negatively charged group at the 2 position of the pyridine ring.     

Raptor distribution in relation to landscape composition in semi-arid Mediterranean habitats

1. Breeding sites of raptors were studied in relation to land-use and edge habitat using two different scales in semi-arid Mediterranean landscapes in south-eastern Spain. Habitat relationships were analysed using Generalized Linear Models.
2. The proportion of forest cover at a small scale was the best predictor for all species. At a larger scale, the proportion of forest cover was also a good predictor, and the amount of edge habitat between forest and extensive agriculture was a very good predictor of booted and short-toed eagle densities.
3. Models for sedentary species of raptor were similar using both scales whereas trans-Saharan migrant raptors seemed to be more sensitive to larger landscape features that included longer edges between forest and extensive agriculture.
4. Habitat mosaics created by forestry and traditional farming were especially important for Mediterranean raptors. Strengthening of the Agri-environmental Regulation (2078/92) will be necessary to compensate for agricultural intensification proposals promoted under the Common Agricultural Policy (CAP).     

Actin of Histriculus cavicola: characteristics of the highly divergent hypotrich ciliate actins.

A macronuclear gene-sized molecule carrying an actin gene from the hypotrich ciliate, Histriculus cavicola, was characterized. Southern blot analysis using a coding region probe suggested that actin in H. cavicola is encoded by a single gene. A comparison of the promoter regions indicated that the H. cavicola actin gene has a TATA box in the 5' flanking region in a position identical to those in other oxytrich ciliates. The coding sequence of this gene is not interrupted by any introns, and codes for a protein of 375 amino acid residues. This protein shares a high degree of similarity with other oxytrichid actins, and a relatively low similarity with actins from other eukaryotes. Comparative analyses of sequences indicated that most of the amino acid substitutions in hypotrich actins are found in surface loops, while the core structures are well-conserved. The sites that interact with DNase I and several regions involved in actin-actin contact have diverged considerably in hypotrich actins, while nucleotide-binding sites are the best-conserved interaction motif.     

Simple and sensitive determination of 5-fluorouracil in plasma by high-performance liquid chromatography: Application to clinical pharmacokinetic studies

5-Fluorouracil (5-FU) is an antineoplastic agent widely employed in the treatment of many types of cancer. Recent studies have proved the need for individual adjustment of 5-FU dosage based on pharmacokinetics. A simple and sensitive high-performance liquid chromatographic method for the determination of 5-FU in plasma and their preliminary clinical pharmacokinetics is described. After sample acidification with 20 μl of orthophosphoric acid (5%), the drug is extracted from plasma using n-propanol–diethyl ether (16:84). The organic layer is evaporated to dryness, the residue dissolved in 100 μl of mobile phase and 20 μl of this mixture is injected into a LiChrospher 100RP-18 (5 μm, 250×4.0 mm) analytical column. Mobile phase consisted of potassium dihydrogenphosphate (0.05 M, adjusted to pH 3). The limit of quantitation was 2 ng/ml. The method showed good precision: the within-day relative standard deviation (RSD) for 5-FU (10–20 000 ng/ml) was 3.75% (2.57–5.93); the between-day RSD for 5-FU, in the previously described range, was 5.74% (4.35–7.20). The method presented here is accurate, precise and sensitive and it has been successfully applied for 5-FU pharmacokinetic investigation and therapeutic drug monitoring.