Evolutionary conservation of the chromosomal configuration and regulation of amylase genes among eight species of the Drosophila melanogaster species subgroup

Nuclear DNA was extracted from each of the eight species comprising the Drosophila melanogaster species subgroup. Southern hybridization of this DNA by using a molecular probe specific for the alpha-amylase coding region showed that the duplicated structure of the amylase locus, first found in D. melanogaster, is conserved among all species of the melanogaster subgroup. Evidence is also presented for the concerted evolution of the duplicated genes within each species. In addition, it is shown that the glucose repression of amylase gene expression, which has been extensively studied in D. melanogaster, is not confined to this species but occurs in all eight members of the species subgroup. Thus, both the duplicated gene structure and the glucose repression of Drosophila amylase gene activity are stable over extended periods of evolutionary time.      

Hepatic tissue enzymes: study in cultures of rat liver epithelial cells. Control and analysis of cells by cytogenetic and mass spectrometric methods. Pharmacological applications]

Extensive studies of parameters conditioning selection and high plating efficiency of epithelial liver cells at primary seeding allowed us to set up a technique for the routine culture of liver cells from rats of various ages (18 day-old pc to 7 month-old) in Ham F10 medium supplemented with 10 p. cent fetal calf serum and 10 p. cent human serum. Cultures, after several passages, or sometimes at primary seeding were free of fibroblasts. The quality of water for culture medium preparation was found to be a very important parameter. G-banding caryotype showed that cells in culture were diploid until 15-20 passages. Various metabolic pathways have been studied in primary culture and in cell lines: enzymes of the anaerobic metabolism of hexoses and metabolism of steroid hormones and xenobiotics. Activity of glucose-6-phosphatase was nearly lost in all cultures. Activity of glucose-6-phosphatase was nearly lost in all cultures. Aldolase showed a specific liver activity with a cleavage ratio of phosphofructoses (F-1,6-diP/F-1-P) equal to 1 or about 1 in several primary cultures and cell lines. Many metabolites arising from incubation of cell lines with 14C-labelled corticosterone, corticosterone-21-sulfate, testosterone and progesterone have been isolated and quantitated by gas liquid chromatography (GC) and mass fragmentography coupled to GC, using 14C/12C isotope ratio measurements. These metabolites indicate the presence in cultured cells of 3 alpha/beta-steroid-reductases, 4-ene steroid reductases and hydroxylases at various positions: 2 alpha, 2 beta, 6 alpha, 6 beta, 7 alpha, 17 beta and 16 alpha. These cell lines were able to activate carcinogens through the epoxide-diol pathway and are suitable for drug metabolism study.     

Aldosterone biosynthesis induced by ACTH and angiotensin II in newborn rat adrenocortical cells transfected by c-EJ-Ha-ras oncogene.

Adrenocortical cells were obtained by fractionated trypsination of newborn rat adrenal glands and transfected with a plasmid containing the EJ/T24-Ha-ras oncogene. Isolation of adhesive cells led to a proliferative cell line with an overexpression of 21 kDa ras protein. These cells incubated with corticosterone or deoxycorticosterone as the precursor produced a high level of 18-hydroxycorticosterone and aldosterone as identified by gas chromatography- mass spectrometry. ACTH and angiotensin II increased the basal production of aldosterone nineteen-fold and six-fold respectively. Under ACTH stimulation the ratio between aldosterone and 18-hydroxycorticosterone production was 1:3. The transformation of corticosterone under angiotensin II stimulation yielded up to 41% of 18-hydroxycorticosterone (4.7 micrograms/mg of cell protein per 24h) and 4.4% of aldosterone (0.5 microgram/mg of cell protein per 24h) in a low potassium concentration medium (6 mmol/l). To our knowledge this is the first report of continuous proliferative adrenocortical cells producing aldosterone.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Fatty acids bind to the fungal elicitor cryptogein and compete with sterols

Cryptogein is a proteinaceous elicitor of plant defense reactions which also exhibits sterol carrier properties. In this study, we report that this protein binds fatty acids. The stoichiometry of the fatty acid-cryptogein complex is 1:1. Linoleic acid and dehydroergosterol compete for the same site, but elicitin affinity is 27 times lower for fatty acid than for sterol. We show that C7 to C12 saturated and C16 to C22 unsaturated fatty acids are the best ligands. The presence of double bonds markedly increases the affinity of cryptogein for fatty acids. A comparison between elicitins and known lipid transfer proteins is discussed.     

Transformation and mineralization of benzo[<Emphasis Type="Italic">a</Emphasis>]pyrene by microbial cultures enriched on mixtures of three- and four-ring polycyclic aromatic hydrocarbons

Microorganisms originating from a soil contaminated by low levels of polycyclic aromatic hydrocarbons (PAHs) were enriched with three- and four-ring PAHs as primary substrates in the presence of benzo[a]pyrene (BaP). Most enrichment cultures, isolated in the presence or absence of a sorptive matrix, significantly transformed BaP. Evidence of BaP mineralization was obtained with cultures enriched on phenanthrene and anthracene. Our findings supplement literature data suggesting the wide occurrence of microbial activity against BaP. Journal of Industrial Microbiology & Biotechnology (2002) 28, 70–73 DOI: 10.1038/sj/jim/7000211 Received 11 December 2000/ Accepted in revised form 04 September 2001     

Leptin activation of mTOR pathway in intestinal epithelial cell triggers lipid droplet formation,cytokine production and increased cell proliferation

Accumulating evidence suggests that obesity and enhanced inflammatory reactions are predisposing conditions for developing colon cancer. Obesity is associated with high levels of circulating leptin. Leptin is an adipocytokine that is secreted by adipose tissue and modulates immune response and inflammation. Lipid droplets (LD) are organelles involved in lipid metabolism and production of inflammatory mediators, and increased numbers of LD were observed in human colon cancer. Leptin induces the formation of LD in macrophages in a PI3K/mTOR pathway-dependent manner. Moreover, the mTOR is a serine/threonine kinase that plays a key role in cellular growth and is frequently altered in tumors. We therefore investigated the role of leptin in the modulation of mTOR pathway and regulation of lipid metabolism and inflammatory phenotype in intestinal epithelial cells (IEC-6 cells). We show that leptin promotes a dose- and time-dependent enhancement of LD formation. The biogenesis of LD was accompanied by enhanced CXCL1/CINC-1, CCL2/MCP-1 and TGF-β production and increased COX-2 expression in these cells. We demonstrated that leptin-induced increased phosphorylation of STAT3 and AKT and a dose and time-dependent mTORC activation with enhanced phosphorilation of the downstream protein P70S6K protein. Pre-treatment with rapamycin significantly inhibited leptin effects in LD formation, COX-2 and TGF-β production in IEC-6 cells. Moreover, leptin was able to stimulate the proliferation of epithelial cells on a mTOR-dependent manner. We conclude that leptin regulates lipid metabolism, cytokine production and proliferation of intestinal cells through a mechanism largely dependent on activation of the mTOR pathway, thus suggesting that leptin-induced mTOR activation may contribute to the obesity-related enhanced susceptibility to colon carcinoma.     

Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, <Emphasis Type="Italic">in vitro</Emphasis>effects on cartilage metabolism and intra-articular levels after injection

Introduction  

Intraarticular administration of autologous conditioned serum (ACS) recently demonstrated some clinical effectiveness in treatment of osteoarthritis (OA). The current study aims to evaluate the in vitro effects of ACS on cartilage proteoglycan (PG) metabolism, its composition and the effects on synovial fluid (SF) cytokine levels following intraarticular ACS administration.     

Gas chromatography-mass spectrometry of isobutyl ester trimethylsilyl ether derivatives of bile acids and application to the study of bile sterol and bile acid biosynthesis in rat liver epithelial cell lines

The derivatization of bile acids into trimethylsilyl ether isobutyl ester (IBTMS) and of neutral sterols into trimethylsilyl ether (TMS) allowed the separation on an OV-1 capillary gas chromatography column of 15 bile steroids as follows: cholesterol, 7 alpha-hydroxycholesterol, 6 beta-hydroxycholesterol, 6 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, lithocholate, deoxycholate, 25-hydroxycholesterol, chenodeoxycholate, cholate, murocholate, hyodeoxycholate, ursodeoxycholate, hyocholate, and beta-muricholate. Fragmentation data of the coupled gas chromatographic-mass spectrometric (GC-MS) analysis of these nine bile acids as IBTMS derivatives under electron impact and chemical ionizations (methane, isobutane, and ammonia) are given. The ammonia chemical ionization appears to be the best mode for compound identification and quantitation due to fragmentations into high mass ions. The comparison of methylene units of the five sterols as TMS derivatives and of each type of methyl, TMS, or isobutyl ester of the nine bile acids as TMS ethers showed that isobutyl esterification increased dramatically the retention time of the bile acids, allowing their separation after the neutral sterols. Different methods of GC-MS analysis were applied to the study of bile steroid secretion in long-term rat liver epithelial cell lines, either serum-supplemented cell lines or serum-free cell lines, growing in serum-free medium since the primary explanation or after adaptation of serum-supplemented lines to this medium. It is demonstrated for the first time that liver epithelial cell lines maintain the metabolic pathway leading from synthesized cholesterol to dioxygenated sterols and the two normal main primary bile acids of the liver, chenodeoxycholic acid and cholic acid, up to 32-47% of the in vivo daily rate, and in addition the production of alpha-muricholic acid, the bile acid marker of murine liver.