Hum Cell, 2004 Mar, 17(1), 59 - 66 Establishment and characterization of a nerve cell line (NC-HIMT) from HIMT cells derived from a human ovarian immature teratoma with special reference to the induction of neuron differentiation by retinoic acid; Yokose T et al.; A nerve cell line designated NC-HIMT was established from a HIMT cell line derived from a benign ovarian, three germ layer immature teratoma removed from a 21-year-old Japanese female . The HIMT cells were elongated, ellipsoid or spherical in shape, whose karyotype was on the high side of normal diploidy . Small amounts of retinoic acid enhanced differentiation and maturation of the HIMT cells into nervous tissue, and the NC-HIMT cell line was established by the colony isolating technique when the HIMT cell line was cultured in the presence of retinoic acid-supplemented medium . After establishment, the NC-HIMT cell line was cultured and maintained in retinoic acid-free growth medium . Even though these cells were cultured without retinoic acid, the phenotype of nerve cells remained and the cells were also maintained in a state of high normal diploidy . The nerve cells contacted each other with their long cell projections and formed networks . Immunocytochemical observations using anti-bovine NSE, alpha-internexin, neurofilament 200kD, peripherin and GFAP confirmed that the cells were either nerve cells or glia cells . These results assume that HIMT cells, which were derived from an immature teratoma, have progenitor and/or stem cells which can differentiate into nerve and/or glial cells. Hum Cell, 2004 Mar, 17(1), 49 - 57 New method for forming large embryoid bodies using the wall of the culture dish along with an analysis of their structural characteristics; Yamamoto M et al.; Early embryonic stem (EES) cells, which were established from 2 cell stage embryos obtained from ddY mice, had similar characteristics as embryonic stem (ES) cells . These cells were maintained in an undifferentiated stage in growth media supplemented with leukemia inhibitory factor (LIF) and were capable of differentiating into triploblastic tissues under various growth factors . It has been known that normal sized embryoid bodies (EBs) are formed by removing LIF . In this study, large EBs gradually formed along the side wall of a culture dish, particularly at the boundary between the air and the growth medium when cells were cultured for a considerable period of time and without subculturing . We call this method the "wall adhesion culture" procedure . The method itself is simple and do not need any instruments except plastic dishes because only the side walls of the dishes were utilized . The mean thickness of the large EBs was about 1.5 mm 3 months after establishing the static culture . Their surface was covered with a monolayer of cells and they contained an eosinophilic cell matrix . By electron microscopy, some characteristic structures was observed, such as intracisternal A particles which were present inside the swelling of the rough endoplasmic reticulum . Since many tissues derived from ES cells are obtained through EBs, it is expected that efficient acquisition of sufficient quantities of these structures using the wall adhesion culture procedure will be a shortcut for using ES cells in regenerative medicine. J Biol Chem, 2004 Nov 12, 279(46), 47890 - 7 Epub 2004 Sep 13. TreT, a novel trehalose glycosyltransferring synthase of the hyperthermophilic archaeon Thermococcus litoralis; Qu Q et al.; The gene cluster in Thermococcus litoralis encoding a multicomponent and binding protein-dependent ABC transporter for trehalose and maltose contains an open reading frame of unknown function . We cloned this gene (now called treT), expressed it in Escherichia coli, purified the encoded protein, and identified it as an enzyme forming trehalose and ADP from ADP-glucose and glucose . The enzyme can also use UDP- and GDP-glucose but with less efficiency . The reaction is reversible, and ADP-glucose plus glucose can also be formed from trehalose and ADP . The rate of reaction and the equilibrium favor the formation of trehalose . At 90 degrees C, the optimal temperature for the enzymatic reaction, the half-maximal concentration of ADP-glucose at saturating glucose concentrations is 1.14 mm and the V(max) is 160 units/mg protein . In the reverse reaction, the half-maximal concentration of trehalose at saturating ADP concentrations is 11.5 mm and the V(max) was estimated to be 17 units/mg protein . Under non-denaturating in vitro conditions the enzyme behaves as a dimer of identical subunits of 48 kDa . As the transporter encoded in the same gene cluster, TreT is induced by trehalose and maltose in the growth medium. J Appl Microbiol, 2004, 97(4), 867 - 75 Repression of reserve lipid turnover in Cunninghamella echinulata and Mortierella isabellina cultivated in multiple-limited media; Papanikolaou S et al.; AIMS: To study patterns of reserve lipid biosynthesis and turnover (degradation) in two oleaginous Zygomycetes, namely Cunninghamella echinulata and Mortierella isabellina under various growth conditions . Fatty acid composition of the reserve lipid of both strains was also studied in all growth steps . METHODS AND RESULTS: Cunninghamella echinulata and Mortierella isabellina were grown in carbon-excess batch cultures . In the investigated strains, accumulation of reserve lipid occurred only when the activity of both NAD(+)-isocitrate dehydrogenase (ICDH) and NADP(+)-ICDH were not detectable in the cell-free extract . Specifically, in C . echinulata, NAD(+)-ICDH activity was detected even after depletion of ammonium nitrogen in the medium, resulting in a delay of the initiation of lipid accumulation period . On the contrary, in M . isabellina, lipid accumulation occurred simultaneously with ammonium nitrogen exhaustion in the growth medium, as the activity of both NAD(+)- and NADP(+)-ICDH were not detectable after nitrogen depletion . In C . echinulata reserve lipid was not degraded after glucose had been exhausted . Supplementations of the medium with Fe(3+), yeast extract or Mg(2+) induced, however, reserve lipid breakdown and formation of lipid-free material . In M . isabellina after glucose exhaustion, notable lipid degradation occurred, accompanied by a significant lipid-free material biosynthesis . Nevertheless, in multiple-limited media, in which Mg(2+) or yeast extract, besides carbon and nitrogen, were limiting nutrients, reserve lipid breakdown was repressed . In both strains, the quantity of gamma-linolenic acid (GLA) in the reserve lipids {varying between 9 and 16% (w/w) in C . echinulata and 1.5-4.5% (w/w) in M . isabellina} was proportional to lipid-free biomass . CONCLUSIONS: Lipid accumulation period in Zygomycetes is initiated by the attenuation of ICDH activity in the mycelium while the regulation of ICDH from ammonium nitrogen is strain specific . While a single nitrogen limitation was enough to induce lipid accumulation, however, multiple limitations were needed in order to repress lipid turnover in oleaginous Zygomycetes . As for GLA, its biosynthesis in the mycelium seemed proportional to lipid-free biomass synthesis . SIGNIFICANCE AND IMPACT OF THE STUDY: Several nutrients are indispensable for functioning the mechanisms involved in the mobilization of reserve lipid in oleaginous moulds . Therefore, reserve lipid turnover in oleaginous moulds could be repressed in multiple-limited media. Plant Cell Physiol, 2004 Aug, 45(8), 1053 - 62 AtIPT3 is a key determinant of nitrate-dependent cytokinin biosynthesis in Arabidopsis; Takei K et al.; We analyzed the spatial expression pattern of Arabidopsis thaliana adenosine phosphates-isopentenyltransferase genes (AtIPT1, AtIPT3 to AtIPT8) and the effect of inorganic nitrogen sources on their regulation . In mature plants, the AtIPTs were differentially expressed in various tissues including the roots, leaves, stems, flowers and siliques . In transgenic seedlings expressing a gene for green fluorescent protein (GFP) driven by the AtIPT promoters, AtIPT1::GFP was predominantly expressed in the vascular stele of the roots, AtIPT3::GFP was in the phloem companion cells, AtIPT5::GFP was in the lateral root primordium and pericycle, and AtIPT7::GFP was in both the vascular stele and the phloem companion cells of the roots . In a long-term treatment, the accumulation level of AtIPT5 transcript was correlated with the concentrations of NO(3)(-) and NH(4)(+) in the growth medium . However, under nitrogen-limited conditions, AtIPT3 expression was rapidly induced by NO(3)(-) in the seedlings accompanying the accumulation of cytokinins, whereas AtIPT5 expression was little affected . The NO(3)(-)-dependent accumulation of both the AtIPT3 transcript and the cytokinins was markedly reduced in a Ds transposon-insertion mutant of AtIPT3 . These results suggest that nitrogen availability differentially regulates expression of AtIPT3 and AtIPT5, and that AtIPT3 is a key determinant of cytokinin biosynthesis in response to rapid changes in the availability of NO(3)(-). Ann Vasc Surg, 2004 May, 18(3), 302 - 7 Nicotine induces mononuclear leukocyte adhesion and expression of adhesion molecules, VCAM and ICAM, in endothelial cells in vitro; Albaugh G et al.; The pathology of atherosclerotic cardiovascular disease (ASCVD) has been characterized as an inflammatory response to vessel injury . The initial steps of this response involve mononuclear leukocyte (MNL) attachment and infiltration into the vessel wall . Leukocyte adhesion is potentiated by expression of cellular adhesion molecules . Vascular cell adhesion molecule-1 (VCAM) and intracellular adhesion molecule-1 (ICAM) are markers of cellular activation and have the ability to attach leukocytes to the endothelium, which is an initial event in the inflammatory response in the vessel wall . Human umbilical vein endothelial cells (HUVEC) were plated in endothelial growth medium (EGM) on plastic coverslips and grown until cells were 75% confluent . Free base nicotine (FBN) was diluted in EGM to a concentration of 10(-8) M and added to experimental cells . At 3 hr, coverslips were removed and fixed . Immunohistochemical staining (IHCS) was performed using a monoclonal antibody to human ICAM and VCAM . Digital image analysis (DIA) was performed to quantify the expression of ICAM and VCAM . An intensity stain index (ISI) measuring area and intensity of stain/total cellular area was determined . Additional HUVEC grown in a similar manner were either exposed to 10(-8) M FBN in EGM or EGM control for 4 hr, then were exposed to MNL suspension for 10 min . Coverslips were removed, rinsed, and fixed . Hematoxylin and eosin staining was performed and cells examined under light microscopy . Leukocyte number per high power field (HPF) was counted and compared to controls . Data were analyzed using analysis of variants (ANOVA) and Student's t-test . Differences were considered significant if p < 0.05 . ICAM and VCAM expression was absent in control cells . Nicotine exposure at 3 hr induced expression of VCAM (ISI = 30.85+/-0.77) and to a lesser extent ICAM (ISI = 16.6+/-1.39) (p < 0.001) . MNL adhesion was markedly increased in cells exposed to nicotine (79.4+/-16.9/HPF) when compared to control cells (1.8+/-0.91/HPF) exposed to MNL (p < 0.01) . These data show nicotine's ability to activate HUVEC as evidenced by induction of ICAM and VCAM expression in vitro . The biological effects of these adhesion molecules are demonstrated by a marked increase in MNL adhesion to HUVEC as demonstrated by leukocyte adhesion assay (LAA) . MNL adhesion and subsequent migration into the intima, if occurring in vivo, may be a vital step in the pathogenesis of ASCVD associated with nicotine exposure. Biosystems, 2004 Aug-Oct, 76(1-3), 89 - 100 The epitheliome: agent-based modelling of the social behaviour of cells; Walker DC et al.; We have developed a new computational modelling paradigm for predicting the emergent behaviour resulting from the interaction of cells in epithelial tissue . As proof-of-concept, an agent-based model, in which there is a one-to-one correspondence between biological cells and software agents, has been coupled to a simple physical model . Behaviour of the computational model is compared with the growth characteristics of epithelial cells in monolayer culture, using growth media with low and physiological calcium concentrations . Results show a qualitative fit between the growth characteristics produced by the simulation and the in vitro cell models. J Endocrinol, 2004 Sep, 182(3), 485 - 99 Functional characterization of HUVEC-CS: Ca2+ signaling, ERK 1/2 activation, mitogenesis and vasodilator production; Gifford SM et al.; While many endothelial cell lines exist, few are of human origin with characteristics close to the parent endothelial cell . We derived a subline (HUVEC-CS) of immortalized human umbilical vein endothelial cells (HUVEC-C) that proliferate in standard growth media and exhibit positive acetylated low-density lipoprotein (AcLDL) uptake, express eNOS, CD31 and ve-cadherin, and spontaneously form capillary-like structures when grown on Matrigel . HUVEC-CS also maintain endothelial cell characteristics at the level of mitogenesis, kinase activation and vasodilator production . Like primary HUVEC cells, HUVEC-CS express many of the key proteins necessary for vasodilator production, including epithelial nitric oxide synthase (eNOS), HSP 90, cav-1 and -2, cPLA2, and COX-1 and -2 . Prostaglandin I synthase (PGIS) was not detectable by Western blot analysis, consistent with primary HUVEC in which PGI2 production is minimal . Receptors were detected for angiotensin II (AII), bradykinin, ATP and growth factors . ATP induced a dose- and time-dependent rise in the intracellular free Ca2+ concentration ({Ca2+}i) . Initially, ATP stimulates P2Y receptors rather than P2X receptors, as demonstrated by the inability of ATP to initiate a Ca2+ response subsequent to emptying of the internal Ca2+ stores by thapsigargin . AII, bradykinin, epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) also caused a rise in {Ca2+}i in a subset of the cells . ATP, basic fibroblastic growth factor (bFGF), EGF and VEGF induced mitogenesis and caused a rise in ERK 2 activation within 10 min . L-Arginine to L-citrulline conversion assays showed that ATP, EGF and VEGF induced a significant rise in eNOS activity, and this correlates with an ability to induce Ca2+ mobilization and ERK 2 activation . In conclusion, HUVEC-CS are indeed endothelial cells and appear to be functionally very similar to primary HUVEC . These cells will prove a valuable tool for future studies in both basic and therapeutic sciences. Stroke, 2004 Oct, 35(10), 2378 - 84 Epub 2004 Sep 02. Transplantation of circulating endothelial progenitor cells restores endothelial function of denuded rabbit carotid arteries; He T et al.; BACKGROUND AND PURPOSE: Circulating endothelial progenitor cells (EPCs) play an important role in repair of injured vascular endothelium and neovascularization . The present study was designed to determine the effect of EPCs transplantation on the regeneration of endothelium and recovery of endothelial function in denuded carotid arteries . METHODS: Isolated mononuclear cells from rabbit peripheral blood were cultured in endothelial growth medium for 7 days, yielding EPCs . A rabbit model of common carotid artery denudation by passage of a deflated balloon catheter was used to evaluate the effects of EPCs on endothelial regeneration and vasomotor function . Immediately after denudation, autologous EPCs (10(5) cells in 200 microL saline) or 200 microL saline alone (control) were administered into the lumen of injured artery . RESULTS: Four weeks after transplantation, fluorescence-labeled colonies of EPCs were found in the vessel wall . Local transplantation of EPCs as compared with saline administration accelerated endothelialization and significantly improved endothelium-dependent relaxation when assessed 4 weeks after denudation (n=4 to 5, P<0.05) . Transplantation of EPCs did not affect vasomotor function of arterial smooth muscle cells . Protein array analysis of conditioned media obtained from cultured EPCs demonstrated the ability of these cells to produce and release a number of proangiogenic cytokines . CONCLUSIONS: We conclude that local delivery of cultured circulating EPCs into the lumen of denuded carotid arteries accelerates endothelialization and improves endothelial function . Paracrine effects of EPCs may contribute to regenerative properties of EPCs. J Bacteriol, 2004 Sep, 186(18), 6093 - 100 Biosynthesis of chloro-beta-hydroxytyrosine, a nonproteinogenic amino acid of the peptidic backbone of glycopeptide antibiotics; Puk O et al.; The role of the putative P450 monooxygenase OxyD and the chlorination time point in the biosynthesis of the glycopeptide antibiotic balhimycin produced by Amycolatopsis balhimycina were analyzed . The oxyD gene is located directly downstream of the bhp (perhydrolase) and bpsD (nonribosomal peptide synthetase D) genes, which are involved in the synthesis of the balhimycin building block beta-hydroxytyrosine (beta-HT) . Reverse transcriptase experiments revealed that bhp, bpsD, and oxyD form an operon . oxyD was inactivated by an in-frame deletion, and the resulting mutant was unable to produce an active compound . Balhimycin production could be restored (i) by complementation with an oxyD gene, (ii) in cross-feeding studies using A . balhimycina JR1 (a null mutant with a block in the biosynthesis pathway of the building blocks hydroxy- and dihydroxyphenylglycine) as an excretor of the missing precursor, and (iii) by supplementation of beta-HT in the growth medium . These data demonstrated an essential role of OxyD in the formation pathway of this amino acid . Liquid chromatography-electrospray ionization-mass spectrometry analysis indicated the biosynthesis of completely chlorinated balhimycin by the oxyD mutant when culture filtrates were supplemented with nonchlorinated beta-HT . In contrast, supplementation with 3-chloro-beta-HT did not restore balhimycin production . These results indicated that the chlorination time point was later than the stage of free beta-HT, most likely during heptapeptide synthesis. J Biol Chem, 2004 Nov 12, 279(46), 47520 - 7 Epub 2004 Aug 26. Murine Wnt-1 with an internal c-myc tag recombinantly produced in Escherichia coli can induce intracellular signaling of the canonical Wnt pathway in eukaryotic cells; Fahnert B et al.; Wnt-1 belongs to the Wnt family of secreted glycoproteins inducing an intracellular signaling pathway involved in cell proliferation, differentiation, and pattern formation . The canonical branch is one of three known branches . This is also valid in vitro, and Wnts can be considered beneficial for culturing primary cells from organs, provided Wnts are available and applicable even with cells of different species . It was shown here that internally c-myc-tagged murine Wnt-1 produced in the heterologous host Escherichia coli was appropriate for inducing intracellular signaling of the canonical Wnt pathway in eukaryotic cells via stabilization of cytosolic beta-catenin . The pioneering injection of the protein into the blastocoels of Xenopus laevis embryos led to axis duplication and suppression of head formation . Applying the recombinant murine Wnt-1 to metanephric mesenchyme activated the tubulogenic program . The signal-inducing activity of the recombinant protein was also positively demonstrated in the TOP-flash reporter assay . Although Wnts were purified recently from the growth media of stably transfected eukaryotic cell lines, the production of active Wnt proteins in pro- or eukaryotic microorganisms reportedly has never been successful . Here soluble production in E . coli and translocation into the oxidizing environment of the periplasm were achieved . The protein was purified using the internal c-myc tag . The effect on the eukaryotic cells implies that activity was retained . Thus, this approach could make recombinant murine Wnt-1 available as a good starting point for other Wnts needed, for example, for maintaining and differentiating stem cells, organ restoration therapy, and tissue engineering. J Biol Chem, 2004 Nov 5, 279(45), 46644 - 51 Epub 2004 Aug 27. Bacterial acetone carboxylase is a manganese-dependent metalloenzyme; Boyd JM et al.; Bacterial acetone carboxylase catalyzes the ATP-dependent carboxylation of acetone to acetoacetate with the concomitant production of AMP and two inorganic phosphates . The importance of manganese in Rhodobacter capsulatus acetone carboxylase has been established through a combination of physiological, biochemical, and spectroscopic studies . Depletion of manganese from the R . capsulatus growth medium resulted in inhibition of acetone-dependent but not malate-dependent cell growth . Under normal growth conditions (0.5 microm Mn2+ in medium), growth with acetone as the carbon source resulted in a 4-fold increase in intracellular protein-bound manganese over malate-grown cells and the appearance of a Mn2+ EPR signal centered at g = 2 that was absent in malate-grown cells . Acetone carboxylase purified from cells grown with 50 microm Mn2+ had a 1.6-fold higher specific activity and 1.9-fold higher manganese content than cells grown with 0.5 microm Mn2+, consistently yielding a stoichiometry of 1.9 manganese/alpha2beta2gamma2 multimer, or 0.95 manganese/alphabetagamma protomer . Manganese in acetone carboxylase was tightly bound and not removed upon dialysis against various metal ion chelators . The addition of acetone to malate-grown cells grown in medium depleted of manganese resulted in the high level synthesis of acetone carboxylase (15-20% soluble protein), which, upon purification, exhibited 7% of the activity and 6% of the manganese content of the enzyme purified from acetone-grown cells . EPR analysis of purified acetone carboxylase indicates the presence of a mononuclear Mn2+ center, with possible spin coupling of two mononuclear sites . The addition of Mg.ATP or Mg.AMP resulted in EPR spectral changes, whereas the addition of acetone, CO2, inorganic phosphate, and acetoacetate did not perturb the EPR . These studies demonstrate that manganese is essential for acetone carboxylation and suggest a role for manganese in nucleotide binding and activation. FEMS Microbiol Lett, 2004 Sep 1, 238(1), 241 - 8 Low concentrations of the non-ionic detergent Nonidet P-40 interfere with sterol biogenesis and viability of the yeast Saccharomyces cerevisiae; Hronska L et al.; Mild non-ionic detergents are used for solubilization of hydrophobic substrates in yeast growth media at concentrations 0.1-1% . Our data show that low concentrations of Nonidet P-40 may significantly affect lipid biogenesis in the yeast Saccharomyces cerevisiae . The uptake and esterification of external {4-14C}-cholesterol is strongly reduced in hem1 mutants treated with low concentrations of Nonidet P-40 . Significant inhibitory effect of NP-40 on sterol uptake and esterification was evident both in non-growing and growing cells supplemented with external cholesterol . Increased levels of sterol precursors (squalene, lanosterol) in hem1 cells grown in complex medium with cholesterol indicated general interference of NP-40 with sterol biosynthesis . NP-40 in the growth medium affected also cell viability estimated as the colony forming ability . More attention should be therefore paid to possible effects of mild detergents at low concentrations generally considered to be harmless, especially in cells with disturbed lipid biogenesis. Plant Physiol Biochem, 2004 Jul-Aug, 42(7-8), 639 - 45 Lead accumulation in the aquatic fern Azolla filiculoides; Oren Benaroya R et al.; In this study, we characterized lead (Pb2+) accumulation and storage by the aquatic fern Azolla filiculoides . Lead precipitates were detected in the vacuoles of mesophyll cells of Azolla plants cultured for 6 d in rich growth medium containing 20 mg l(-1) Pb2+ . Energy dispersive spectroscopy (EDS) analysis of the relative element content of leaves collected from these plants revealed a 100% increase in the levels of P, S, Na and Ca and a 40% decrease in Mg and Cl compared to the untreated plants . Both Azolla whole plants and isolated apoplasts were incubated for 6 d in 20 mg l(-1) Pb2+ . Lead content in the whole plant composed 0.37%, 2.3% and 1.8% of the dry weight after 2, 4 and 6 d of growth, respectively, while the isolated Azolla apoplast contained 0.125%, 1.22% and 1.4% Pb2+, respectively . Lead content in Azolla whole plant increase by 200%, 100% and 22% after 2, 4 and 6 d of growth, respectively, when compared to Azolla apoplast . Dark, electron dense deposits of lead were observed in light and transmission electron microscope in leaf cells treated with lead . All the observed lead deposits were localized in vacuoles while larger lead deposits were found in mature leaves than in young leaves . No lead deposits were found in cells of the cyanobiont Anabaena when the plants were exposed to similar conditions . Activity and content of V-H+-ATPase were studied in Azolla plants grown in the presence of 20, 40 and 80 mg l(-1) of lead for a period of 4 d . Activity of V-H+-ATPase was increased by 190%, 210% and 220%, respectively, but the content of V-H+-ATPase was reduced by all lead concentrations . Exp Eye Res, 2004 Aug, 79(2), 157 - 65 Integrin alpha5 expression by the ARPE-19 cell line: comparison with primary RPE cultures and effect of growth medium on the alpha5 gene promoter strength; Proulx S et al.; Primary cultures of human retinal pigment epithelium (RPE) requires young human donors with short post-mortem time and no known retinal diseases . The use of an established human RPE cell line, like ARPE-19, would be a welcomed alternative to primary cultures . This cell line retains many of the characteristics of RPE cells, including cell morphology, functional tight junctions and expression of CRALBP and RPE65 . This study was conducted in order to investigate integrin alpha5 expression at both the gene and protein level in the ARPE-19 cell line and compare the results with those obtained with primary cultures of RPE cells . The potential use of this cell line as a substitute for primary cultures of RPE cells was also considered . Integrin alpha5 protein was detected on RPE and ARPE-19 cultures at different confluencies by immunofluorescence and immunoprecipitation analyses . Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to study alpha5 mRNA levels . Transient transfections were performed in order to compare alpha5 promoter strength in both types of cells . Immunofluorescence studies showed that both primary RPE and ARPE-19 cells yielded similar alpha5 staining patterns at all cell confluencies . Both immunoprecipitation and RT-PCR analyses provided evidence that sub-confluent and confluent RPE and ARPE-19 cells have similar cell surface alpha5 protein and mRNA levels whereas post-confluent cells had a marked decrease in both protein and transcript levels . ARPE-19 cells show a large increase in promoter strength compared to primary cultures . When compared to primary cultures, the cell line exhibited major differences in the way the alpha5 promoter is regulated, even if both cell types are cultured under identical conditions . This study demonstrates that primary cultures of human RPE and ARPE-19 cells show reductions in both the alpha5 protein and the mRNA when cells reach post-confluency . However, major differences have been observed in the strength of the alpha5 promoter between both cell types . We also show that culturing ARPE-19 cells in a different growth medium alters the transcriptional activity directed by the alpha5 promoter. Oecologia, 2005 Jan, 142(1), 11 - 9 Epub 2005 Jan. Lichens show that fungi can acclimate their respiration to seasonal changes in temperature; Lange OL et al.; Five species of lichens, the majority members of a soil-crust community ( Cladonia convoluta, Diploschistes muscorum, Fulgensia fulgens, Lecanora muralis, Squamarina lentigera) showed seasonal changes of temperature sensitivity of their dark respiration (DR) to such an extent that several substantially met the definition of full acclimation, i.e . near identical DR under different nocturnal temperature conditions during the course of the year . C . convoluta, for example, had maximal DR at 5 degrees C of -0.42, -1.11 and -0.09 nmol CO(2) g(-1) s(-1) in autumn, winter, and summer, respectively, a tenfold range . However, at the mean night temperatures for the same three seasons, 9.7 degrees C, 4.2 degrees C and 13.6 degrees C, maximal DR were almost identical at -1.11, -0.93, and -1.45 nmol CO(2) g(-1) s(-1) . The information was extracted from measurements using automatic cuvettes that continuously recorded a sample lichen's gas exchange every 30 min under near-natural conditions . The longest period (for L . muralis) covered 15 months and 22,000 data sets whilst, for the other species studied, data blocks were available throughout the calendar year . The acclimation of DR means that maximal net carbon fixation rates remain substantially similar throughout the year and are not depressed by increased carbon loss by respiration in warmer seasons . This is especially important for lichens because of their normally high rate of DR compared to net photosynthesis . We suggest that lichens, especially soil-crust species, could be a suitable model for fungi generally, a group of organisms for which little is known about temperature acclimation because of the great difficulty in separating the organism from its growth medium . Fungi, whether saprophytic, symbiotic or parasitic, including soil lichens, are important components of soil ecosystems and contribute much of the respired CO(2) from these systems . Temperature acclimation by fungi would mean that expected increases in carbon losses caused by global climate warming from soil ecosystems might not be as extensive as first thought . This would ameliorate this positive feedback loop present in some climate models and might substantially lower the predicted warming. J Med Entomol, 2004 Jul, 41(4), 705 - 11 Pathogenicity of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae to Ixodidae tick species Dermacentor variabilis, Rhipicephalus sanguineus, and Ixodes scapularis; Kirkland BH et al.; Nymphal and adult ticks from three different tick species, Dermacentor variabilis Say, Ixodes scapularis Say, and Rhipicephalus sanguineus Latrielle, were treated with conidia and blastospores of the entomopathogenic fungi Beauveria bassiana (Bals.) Vuill . and Metarhizium anisopliae Metschnikoff . Dose-response experiments indicated that a critical concentration of fungal spores is required for infection and mortality . Over a 28-d time course, fungal suspensions of either B . bassiana or M . anisopliae at 10(8) conidia/ml resulted in 50-70% mortality in adult I . scapularis and R . sanguineus, but <20% mortality in D . variabilis ticks . R . sanguineus nymphs were highly susceptible to both entomopathogenic fungi, displaying >60% mortality within 14 d postinfection and >90% mortality within 21-28 d postinfection . D . variabilis nymphs also were more susceptible than their corresponding adults, displaying mortalities ranging from 20 to 40% 28 d postinfection . I . scapularis nymphs, however, seemed to be slightly less susceptible than adults (45% mortality, 28 d postinfection) . The addition of nutrients to fungal cell suspensions did not have any noticeable effects on mortality toward any of the tick species tested . Significant mortality against D . variabilis adults (approximately 65%) was noted only when B . bassiana fungal cells with growth media carryover were used as the inoculum against the ticks . Entomopathogenic fungi such as B . bassiana and M . anisopliae may have the potential for controlling populations of I . scapularis and R . sanguineus, and under certain conditions D . variabilis . Our results indicate that inoculum conditions can greatly affect successful virulence and subsequent mortality. Biochemistry (Mosc), 2004 Jul, 69(7), 809 - 12 Changes in the Nitrocellulose Molecule Induced by Sulfate-Reducing Bacteria Desulfovibrio desulfuricans 1388 . The Enzymes Participating in This Process; Tarasova NB et al.; The appearance of unsubstituted glucopyranose residues in nitrocellulose (NC) induced by Desulfovibrio desulfuricans was established by (13)C-NMR spectroscopy . After contact with bacterial cells, the degree of substitution by nitro groups in NC decreased from 2.59 to 2.40 . The bacteria possess intra- and extracellular nitroesterase activities, which are responsible for denitration of the polymer . The presence of NC in the growth medium influences the extracellular nitroesterase activity . It is shown that inhibition of enzymatic activity in the presence of NC is caused by appearance of nitrates in the culture medium . Nitrate and nitrite reductases of dissimilatory type reduce nitrates . The data suggest consideration of bacteria belonging to the Desulfovibrio genus as the initial agent in utilization of an unnatural polymer--nitrocellulose--in a microbial consortium. J Plant Physiol, 2004 Jul, 161(7), 867 - 72 Carbohydrate metabolism in growing rice seedlings under arsenic toxicity; Jha AB et al.; We studied in the seedlings of two rice cultivars (Malviya-36 and Pant-12) the effect of increasing levels of arsenic in situ on the content of sugars and the activity of several enzymes of starch and sucrose metabolism: alpha-amylase (EC 3.2.1.1), beta-amylase (EC 3.2.1.2), starch phosphorylase (EC 2.4.1.1), acid invertase (EC 3.2.1.26), sucrose synthase (EC 2.4.1.13) and sucrose phosphate synthase (EC 2.4.1.14) . During a growth period of 10-20 d As2O3 at 25 and 50 microM in the growth medium caused an increase in reducing, non-reducing and total soluble sugars . An increased conversion of non-reducing to reducing sugars was observed concomitant with As toxicity . The activities of alpha-amylase, beta-amylase and sucrose phosphate synthase declined, whereas starch phosphorylase, acid invertase and sucrose synthase were found to be elevated . Results indicate that in rice seedlings arsenic toxicity causes perturbations in carbohydrate metabolism leading to the accumulation of soluble sugars by altering enzyme activity . Sucrose synthase possibly plays a positive role in synthesis of sucrose under As-toxicity. Appl Biochem Biotechnol, 1999 Spring, 77-79, 681 - 8 Lignin Peroxidase Production by Streptomyces viridosporus T7A: Nitrogen Nutrition Optimization Using Glucose as Carbon Source; Zerbini JE et al.; The production of lignin peroxidase by Streptomyces viridosporus T7A was studied in submerged batch fermentations using growth media containing 6.5 g/L yeast extract and 2.5-10.0 g/L glucose, corresponding to carbon to nitrogen (C/N) ratios from 7.1-12.4 . The kinetics for biomass and enzyme accumulation and glucose consumption were followed allowing definition of optimized conditions for enzyme production . Considering the physiological response of the microorganism in relation to enzyme production, a sharp increase on enzyme activity was consistently observed upon glucose depletion, indicating glucose regulation . In accordance to that the plot of maximal enzyme vs maximal enzyme per gram of glucose consumption showed a linear inversely proportional relationship, indicating that the characteristics of the metabolic pool at the studied C/N ratios affected enzyme biosynthesis even after glucose depletion. Acta Biomed Ateneo Parmense, 2004, 75 Suppl 1, 18 - 22 Psychobiology of the amniotic environment; Benassi L et al.; Water, basic element of amniotic fluid (A.F.), is closely related to Life, Fertility and Motherhood in several cultures and religions . Through material evidences of an essential growth medium and useful diagnostic source, a new concept grow up: the fluid as a first real environment in which fetus lives and acts . Many studies confirm that in A.F . fetus starts his character-building, his memory and his intelligence . The fluid seems to be the first means of learning and acknowledgement . Sounds, smells and tastes are perceived as well as emotions and fears . Urinoterapy and staminal cells sampling shows how A.F . can be considered as an additional terapeutic resource. Acta Crystallogr D Biol Crystallogr, 1996 Jul, 52(Pt 4), 806 - 12 Contaminant effects on protein crystal morphology in different growth environments; Hirschler J; Contaminant effects on the morphology of turkey egg-white lysozyme (TEWL) crystals grown in ungelled and in gelled growth media have been investigated . The latter may serve as a model system for future microgravity experiments . Hen egg-white lysozyme (HEWL) was added as the contaminant at levels ranging from 4 up to 70%(w/w) (total protein) . Morphology measurements indicate a contaminant effect leading to a shortening along the c axis of the crystal . This shortening effect depends on the contaminant concentration . It is attenuated and varies more regularly in gelled than in ungelled growth media . The specificity of the HEWL contaminant effect was verified by addition of ribonuclease A, which did not influence crystal morphology . Contaminant inclusion into the growing TEWL crystals could be calculated directly from equilibrium protein concentration measurements . The level of HEWL inclusion is closely related to the concentration of HEWL in the growth solutions . The specificity of the observed effect as well as the differences between the two growth media are discussed. Environ Sci Technol, 2004 Jul 15, 38(14), 4002 - 11 In situ metal precipitation in a zinc-contaminated, aerobic sandy aquifer by means of biological sulfate reduction; Janssen GM et al.; The applicability of in situ metal precipitation (ISMP) based on bacterial sulfate reduction (BSR) with molasses as carbon source was tested for the immobilization of a zinc plume in an aquifer with highly unsuitable initial conditions (high Eh, low pH, low organic matter content, and low sulfate concentrations), using deep wells for substrate injection . Batch experiments revealed an optimal molasses concentration range of 1-5 g/L and demonstrated the necessity of adding a specific growth medium to the groundwater . Without this growth medium, even sulfate, nitrogen, phosphorus, and potassium addition combined with pH optimization could not trigger biological sulfate reduction . In column experiments, precipitation of ZnS(s) was induced biologically as well as chemically (by adding Na2S) . In both systems, zinc concentrations dropped from about 30 mg/L to below 0.02 mg/L . After termination of substrate addition the biological system showed continuation of BSR for at least 2 months, suggesting the insensitivity of the sulfate reducing system for short stagnations of nutrient supply, whereas in the chemical system an immediate increase of Zn concentrations was observed . A pilot experiment conducted in situ at the zinc-contaminated site showed a reduction of zinc concentrations from around 40 mg/L to below 0.01 mg/L . Termination of substrate supply did not result in an immediate stagnation of the BSR process, but continuation of BSR was observed for at least 5 weeks. J Clin Microbiol, 2004 Aug, 42(8), 3475 - 82 Interlaboratory comparison of results of susceptibility testing with caspofungin against Candida and Aspergillus species; Odds FC et al.; Seventeen laboratories participated in a study of interlaboratory reproducibility with caspofungin microdilution susceptibility testing against panels comprising 30 isolates of Candida spp . and 20 isolates of Aspergillus spp . The laboratories used materials supplied from a single source to determine the influence of growth medium (RPMI 1640 with or without glucose additions and antibiotic medium 3 {AM3}), the same incubation times (24 h and 48 h), and the same end point definition (partial or complete inhibition of growth) for the MIC of caspofungin . All tests were run in duplicate, and end points were determined both spectrophotometrically and visually . The results from almost all of the laboratories for quality control and reference Candida and Aspergillus isolates tested with fluconazole and itraconazole matched the NCCLS published values . However, considerable interlaboratory variability was seen in the results of the caspofungin tests . For Candida spp . the most consistent MIC data were generated with visual "prominent growth reduction" (MIC(2)) end points measured at 24 h in RPMI 1640, where 73.3% of results for the 30 isolates tested fell within a mode +/- one dilution range across all 17 laboratories . MIC(2) at 24 h in RPMI 1640 or AM3 also gave the best interlaboratory separation of Candida isolates of known high and low susceptibility to caspofungin . Reproducibility of MIC data was problematic for caspofungin tests with Aspergillus spp . under all conditions, but the minimal effective concentration end point, defined as the lowest caspofungin concentration yielding conspicuously aberrant hyphal growth, gave excellent reproducibility for data from 14 of the 17 participating laboratories. Protein Sci, 2004 Sep, 13(9), 2388 - 97 Epub 2004 Aug 04. Characterization of SLAC: a small laccase from Streptomyces coelicolor with unprecedented activity; Machczynski MC et al.; Laccases and other four-copper oxidases are usually constructed of three domains: Domains one and three house the copper sites, and the second domain often helps form a substrate-binding cleft . In contrast to this arrangement, the genome of Streptomyces coelicolor was found to encode a small, four-copper oxidase that lacks the second domain . This protein is representative of a new family of enzymes--the two-domain laccases . Disruption of the corresponding gene abrogates laccase activity in the growth media . We have recombinantly expressed this enzyme, called SLAC, in Escherichia coli and characterized it . The enzyme binds four copper ions/monomer, and UV-visible absorption and EPR measurements confirm that the conserved type 1 copper site and trinuclear cluster are intact . We also report the first known paramagnetic NMR spectrum for the trinuclear copper cluster of a protein from the laccase family . The enzyme is highly stable, retaining activity as a dimer in denaturing gels after boiling and SDS treatment . The activity of the enzyme against 2,6-dimethoxyphenol (DMP) peaks at an unprecedentedly high pH (9.4), whereas the activity against ferrocyanide decreases with pH . SLAC binds negatively charged substrates more tightly than positively charged or uncharged molecules. Appl Environ Microbiol, 2004 Aug, 70(8), 4748 - 55 New strategies for cultivation and detection of previously uncultured microbes; Stevenson BS et al.; An integrative approach was used to obtain pure cultures of previously uncultivated members of the divisions Acidobacteria and Verrucomicrobia from agricultural soil and from the guts of wood-feeding termites . Some elements of the cultivation procedure included the following: the use of agar media with little or no added nutrients; relatively long periods of incubation (more than 30 days); protection of cells from exogenous peroxides; and inclusion of humic acids or a humic acid analogue (anthraquinone disulfonate) and quorum-signaling compounds (acyl homoserine lactones) in growth media . The bacteria were incubated in the presence of air and in hypoxic (1 to 2% O(2) {vol/vol}) and anoxic atmospheres . Some bacteria were incubated with elevated concentrations of CO(2) (5% {vol/vol}) . Significantly more Acidobacteria were found on isolation plates that had been incubated with 5% CO(2) . A simple, high-throughput, PCR-based surveillance method (plate wash PCR) was developed . This method greatly facilitated detection and ultimate isolation of target bacteria from as many as 1,000 colonies of nontarget microbes growing on the same agar plates . Results illustrate the power of integrating culture methods with molecular techniques to isolate bacteria from phylogenetic groups underrepresented in culture. J Biomed Biotechnol, 2004, 2004(3), 150 - 155 A Simple Chemical Method for Rendering Wild-Type Yeast Permeable to Brefeldin A That Does Not Require the Presence of an erg6 Mutation; Pannunzio VG et al.; The present work aims to develop a growth medium to render a wild-type strain of Saccharomyces cerevisiae permeable to the antifungal drug Brefeldin A . In the current study, a synthetic medium containing 0.1% L-proline and supplemented with $3.0\times 10;{-3}$ % SDS is employed . When Brefeldin A is added to this medium, a wild-type strain shows increased growth sensitivity and a diminished transport of the amino acid L-leucine . Since Brefeldin A exerts its effect on the endoplasmic reticulum and the Golgi apparatus, the medium permits the study of the drug effect on the intracellular traffic of L-leucine permeases. Appl Microbiol Biotechnol . 2004 Jul 28; {Epub ahead of print} Biotechnological aspects of the production of the anticancer drug podophyllotoxin; Farkya S et al.; The natural lignan podophyllotoxin, a dimerized product of two phenylpropanoid moieties which occurs in a few plant species, is a pharmacologically important compound for its anticancer activities . It is used as a precursor for the chemical synthesis of the anticancer drugs etoposide, teniposide and etopophose . The availability of this lignan is becoming increasingly limited because of the scarce occurrence of its natural sources and also because synthetic approaches for its production are still commercially unacceptable . Biotechnological production using cell culture may be considered as an alternative source . Selection of the best performing cell line, its maintenance and stabilization are necessary prerequisites for its production in bioreactors and subsequent scale-up of the cultivation process to the industrial level . Scale-up of growth and product yield depends on a multitude of factors, such as growth medium, physicochemical conditions, seed inoculum, type of reactor and processing conditions . The composition of the growth medium, elicitors and precursors, etc . can markedly influence the production . Optimum levels of parameters that facilitate high growth and product response in cell suspensions of Podophyllum hexandrum have already been determined by statistical design . P . hexandrum cells have successfully been cultivated in a 3-l stirred-tank bioreactor under low shear conditions in batch and fed-batch modes of operation . The batch kinetic data were used to identify the mathematical model which was then used to develop nutrient-feeding strategies for fed-batch cultivation to prolong the productive log phase of cultivation . An improvement in the production of podophyllotoxin to 48.8 mg l(-1) in a cell culture of P . hexandrum was achieved, with a corresponding volumetric productivity of 0.80 mg l(-1) day(-1), when the reactor was operated in continuous cell-retention mode . Efforts are being made to further enhance its production levels by the development of hairy root culture or by varying the channeling of precursors towards the desired biosynthetic pathway by molecular approaches. Oncol Rep, 2004 Sep, 12(3), 615 - 9 Cell growth after withdrawal of gefitinib ("Iressa", ZD1839) in human lung cancer cells; Satoh H et al.; Gefitinib ("Iressa", ZD1839) is a selective epidermal growth factor receptor-tyrosine kinase inhibitor, which blocks signal transduction pathways implicated in proliferation and survival of cancer cells . However, in vitro and in vivo studies concerning cell growth after withdrawal of gefitinib are limited . To determine whether cancer cells would resume proliferation upon removal of gefitinib, an in vitro study was undertaken using 4 human non-small cell lung cancer cell lines . With immunocytochemical staining and Western blot analysis, we confirmed positive expression of EGFR in these cell lines . We next evaluated growth inhibition before and after withdrawal of gefitinib . After incubation with 0-100 microM gefitinib for 24 h, medium containing gefitinib was removed, followed by addition of fresh growth medium to cell cultures . MTT assays were performed daily over a 6-day time course . Even at very low levels of gefitinib (<1 microM), these 4 TKB cells had 1-10% growth inhibition, respectively . With these levels of gefitinib, continued inhibitory effects after withdrawal of getitinib were observed in 3 of the 4 cell lines . Furthermore, none of the 4 cell lines, including the cell line which had abolished growth inhibition, showed accelerated re-growth rate after the withdrawal of gefitinib even in these exposure conditions . Based on our in vitro experiments, additional in vivo studies, which can compare the pre- and post-treatment growth rate, will be necessary to help better understand the mechanisms behind cell growth recovery after temporary gefitinib treatment. Prostaglandins Other Lipid Mediat, 2004 Apr, 73(3-4), 237 - 47 Exogenous prostaglandin E2 inhibits TPA induced matrix metalloproteinase-9 production in MCF-7 cells; Reno F et al.; Elevated levels of prostaglandin E2 (PGE2) have been reported in many high metastatic human breast cancers, but no relationship between exogenous PGE2 activity, expression of matrix metalloproteinases (MMPs) and metastasis in human tumor cells has been reported . The poorly invasive human breast cancer cell line MCF-7 was cultured for 24h in the presence of both phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 nM) and PGE2 (1 microM) and the activity of MMP-9, one of the MMPs involved in metastasis, was measured, in growth medium by gelatin substrate zymography . TPA induced a strong production of MMP-9 while exogenous PGE2 had no effect on the basal MMP-9 level, but inhibited the TPA induced enzyme expression and matrigel invasiveness . We showed that MCF-7 cells expressed EP2, EP3 and EP4 receptors for PGE2 and that its action was probably mediated by EP4 receptor and adenylyl cyclase activation while cAMP dependent PKA was not involved in the process of inhibition of MMP-9 production . These findings suggest a possible inhibitory role for exogenous PGE2 in the metastatic process development. Mol Cell Proteomics, 2004 Nov, 3(11), 1065 - 82 Epub 2004 Nov. Comparative proteomes of the proliferating C(2)C(12) myoblasts and fully differentiated myotubes reveal the complexity of the skeletal muscle differentiation program; Tannu NS et al.; When cultured in low serum-containing growth medium, the mouse C(2)C(12) cells exit cell cycle and undergo a well-defined program of differentiation that culminates in the formation of myosin heavy chain-positive bona fide multinucleated muscle cells . To gain an understanding into this process, we compared total, membrane- and nuclear-enriched proteins, and phospho-proteins from the proliferating C(2)C(12) cells and the fully differentiated myotubes by the combined methods of two-dimensional PAGE, quantitative PDQuest image analysis, and MS . Quantification of more than 2,000 proteins from C(2)C(12) myoblasts and myotubes revealed that a vast majority of the abundant proteins appear to be relegated to the essential, housekeeping and structural functions, and their steady state levels remain relatively constant . In contrast, 75 proteins were highly regulated during the phenotypic conversion of rapidly dividing C(2)C(12) myoblasts into fully differentiated, multi-nucleated, post-mitotic myotubes . We found that differential accumulation of 26 phospho-proteins also occurred during conversion of C(2)C(12) myoblasts into myotubes . We identified the differentially expressed proteins by MALDI-TOF-MS and LC-ESI-quadrupole ion trap MS/MS . We demonstrate that more than 100 proteins, some shown to be associated with muscle differentiation for the first time, that regulate inter- and intracellular signaling, cell shape, proliferation, apoptosis, and gene expression impinge on the mechanism of skeletal muscle differentiation. Plant Physiol Biochem, 2004 Feb, 42(2), 97 - 102 L-Myo-inositol 1-phosphate synthase in the aquatic fern Azolla filiculoides; Benaroya RO et al.; L-Myo-inositol 1-phosphate synthase (INPS EC 5.5.1.4) catalyzes the conversion of D-glucose 6-phosphate to L-myo-inositol 1-phosphate . INPS is a key enzyme involved in the biosynthesis of phytate which is a common form of stored phosphates in higher plants . The present study monitored the increase of INPS expression in Azolla filiculoides resulting from exposure to inorganic phosphates, metals and salt stress . The expression of INPS was significantly higher in Azolla plants that were grown in rich mineral growth medium than those maintained on nutritional growth medium . The expression of INPS protein and corresponding mRNA increased in plants cultured in minimal nutritional growth medium when phosphate or Zn2+, Cd2+ and NaCl were added to the growth medium . When employing rich mineral growth medium, INPS protein content increased with the addition of Zn2+, but decreased in the presence of Cd2+ and NaCl . These results indicated that accumulation of phytate in Azolla is a result of the intensified expression of INPS protein and mRNA, and its regulation may be primarily derived by the uptake of inorganic phosphate, and Zn2+, Cd2+ or NaCl. Genetics, 2004 Jul, 167(3), 1109 - 21 Defects arising from whole-genome duplications in Saccharomyces cerevisiae; Andalis AA et al.; Comparisons among closely related species have led to the proposal that the duplications found in many extant genomes are the remnants of an ancient polyploidization event, rather than a result of successive duplications of individual chromosomal segments . If this interpretation is correct, it would support Ohno's proposal that polyploidization drives evolution by generating the genetic material necessary for the creation of new genes . Paradoxically, analysis of contemporary polyploids suggests that increased ploidy is an inherently unstable state . To shed light on this apparent contradiction and to determine the effects of nascent duplications of the entire genome, we generated isogenic polyploid strains of the budding yeast Saccharomyces cerevisiae . Our data show that an increase in ploidy results in a marked decrease in a cell's ability to survive during stationary phase in growth medium . Tetraploid cells die rapidly, whereas isogenic haploids remain viable for weeks . Unlike haploid cells, which arrest growth as unbudded cells, tetraploid cells continue to bud and form mitotic spindles in stationary phase . The stationary-phase death of tetraploids can be prevented by mutations or conditions that result in growth arrest . These data show that whole-genome duplications are accompanied by defects that affect viability and subsequent survival of the new organism. Indian J Exp Biol, 2004 Jan, 42(1), 26 - 35 Goat serum: an alternative to fetal bovine serum in biomedical research; Paranjape S; Serum is frequently added to the defined basal medium as a source of certain nutritional and macromolecular growth factors essential for cell growth . Although a number of synthetic media have been prepared serum continues to be used in cell culture by many investigators . The best supplementation to a basal medium is fetal bovine serum (FBS) that is most frequently used for all types of cell cultures . During last four decades National Institute of Virology, Pune, has been working on isolation and identification of viruses from clinical specimens, employing tissue culture . Initially FBS was used for this purpose . However, due to its prohibitive cost and uncertain supply an alternative was sought . Commercially available sera from newborn calf, sheep, horse, human and serum obtained from goat blood (available from local abattoir) were tried . Goat serum (GS) was found to be suitable for most of the cell lines and primary cultures . Primary cultures from guinea pig embryo, monkey kidney, chick embryo, mouse peritoneal macrophages, and established cell lines were prepared and grown in growth media supplemented with GS . These cultures were studied for their morphology and growth in comparison with cultures grown in FBS containing media, and were used for mass cultivation of cells, quantitation and susceptibility of various virus strains, studies on effects of different nutrients and natural substances on cellular metabolism and virus replication, epitope analysis of various strains of Japanese encephalitis (JE) virus, strain differentiation studies, studies on antibody dependent plaque enhancement, assay of murine migration inhibition factor . Monoclonal antibodies against JE virus adapted to GS were characterised for their retention of functionalities . The results were comparable to those of cell cultures grown in FBS containing media . Similar results on chromosome studies were obtained from patient's whole blood cultures prepared in GS and FBS containing growth media . Organ cultures from mammalian, reptile and avian hosts; successfully grown in GS supplemented growth media, were used for different virological studies . Growth media supplemented with GS were used for in vitro cultivation of malarial parasites . Thus since the last three decades many scientists are using GS in place of FBS, in various fields of biomedical research . The present article reviews an account of the same. J Vet Med A Physiol Pathol Clin Med, 2004 May, 51(4), 167 - 70 Growth of differentiated ovine tracheal epithelial cells in vitro; Radi ZA et al.; Culture of ovine tracheal epithelial cells is a useful tool for conducting various in vitro studies . We describe herein an in vitro technique and the conditions for culturing primary epithelial cells derived from tracheas of adult sheep . Ovine tracheas were surgically removed from 2- to 3-month-old healthy sheep and tracheal epithelial cells were isolated by 0.15% pronase digestion . After epithelial cells isolation, a Millicell insert with porous membrane was coated with 0.05% human placental collagen and the epithelial cells were added to the membrane . To create an air-liquid interface environment for the cells, the apical compartment of the membrane containing the tracheal epithelial cells was left exposed to 5% CO(2) at 37 degrees C for 2 days then increased to 9% CO(2) while cells in the basolateral compartment underneath the membrane contained the growth medium necessary for cells nourishment . Pepsin digestion was more effective in reducing the number of fibroblasts than other procedures . Cells were allowed to grow for 6-7 days to form a confluent monolayer and nearly 21 days for cilia formation on the apical surface as determined by light microscopy of haematoxylin and eosin-stained sections of membranes . In order to further confirm the epithelial origin of cells, cells were stained for cytokeratin antigen by immunohistochemistry . Most ciliated epithelial cells were immunoreactive for cytokeratin . This is the first report of differentiated ovine tracheal epithelial cells growth and isolation . This technique can be used in numerous in vitro investigative studies in ovine species as an animal model for human disease. J Neurosci Res, 2004 Aug 15, 77(4), 475 - 86 Human skin-derived stem cells migrate throughout forebrain and differentiate into astrocytes after injection into adult mouse brain; Belicchi M et al.; Recent evidence indicates that neural stem cell properties can be found among a mammalian skin-derived multipotent population . A major barrier in the further characterization of the human skin-derived neural progenitors is the inability to isolate this population based on expression of cell surface markers . Our work has been devoted to purified human skin-derived stem cells that are capable of neural differentiation, based on the presence or absence of the AC133 cell surface marker . The enriched skin-derived AC133(+) cells express the CD34 and Thy-1 antigens . These cells cultured in a growth medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) proliferate, forming spheres, and differentiate in vitro into neurons, astrocytes, and rarely into oligodendrocytes . Single cells from sphere cultures initiated from human purified AC133(+) cells were replated as single cells and were able to generate new spheres, demonstrating the self-renewing ability of these stem cell populations . Brain engraftment of cells obtained from human purified AC133(+)-derived spheres generated different neural phenotypes: immature neurons and a most abundant population of well differentiated astrocytes . The AC133-derived astrocytes assumed perivascular locations in the frontal cortex . No donor-derived oligodendrocytes were found in the transplanted mouse brains . Several donor small, rounded cells that expressed endothelial markers were found close to the host vessel and near the subventricular zone . Thus, mammalian skin AC133-derived cells behave as a multipotent population with the capacity to differentiate into neural lineages in vitro and, prevalently, endothelium and astrocytes in vivo, demonstrating the great plasticity of these cells and suggesting potential clinical application . J Struct Funct Genomics, 2004, 5(1-2), 87 - 93 Production in two-liter beverage bottles of proteins for NMR structure determination labeled with either 15N- or 13C-15N; Zhao Q et al.; The use of 2-L polyethylene terephthalate beverage bottles as a bacterial culture vessel has been recently introduced as an enabling technology for high-throughput structural biology {Sanville Millard, C . et al., 2003 . Protein Express . Purif . 29, 311-320} . In the article following this one {Stols et al., this issue, pp . 95-102}, this approach was elaborated for selenomethionine labeling used for multiwavelength anomalous dispersion phasing in the X-ray crystallographic determinations of protein structure . Herein, we report an effective and reproducible schedule for uniform 15N- and 13C-labeling of recombinant proteins in 2-L beverage bottles for structural determination by NMR spectroscopy . As an example, three target proteins selected from Arabidopsis thaliana were expressed in Escherichia coli Rosetta (DE3)/pLysS from a T7-based expression vector, purified, and characterized by electrospray ionization mass spectrometry and NMR analysis by 1H-15N heteronuclear single quantum correlation spectroscopy . The results show that expressions in the unlabeled medium provide a suitable control for estimation of the level of production of the labeled protein . Mass spectral characterizations show that the purified proteins contained a level of isotopic incorporation equivalent to the isotopically labeled materials initially present in the growth medium, while NMR analysis of the {U-15N}-labeled proteins provided a convenient method to assess the solution state properties of the target protein prior to production of a more costly double-labeled sample. Biometals, 2004 Aug, 17(4), 379 - 87 Copper-induced oxidative stress and antioxidant defence in Arabidopsis thaliana; Drazkiewicz M et al.; Content of reactive oxygen species (ROS): O2*-, H2O2 and OH* as well as activities of antioxidant enzymes: superoxide dismutase (SOD), guaiacol peroxidase (POX) and catalase (CAT) were studied in leaves of Arabidopsis thaliana ecotype Columbia, treated with Cu excess (0, 5, 25, 30, 50, 75, 100, 150 and 300 microM) . After 7 days of Cu action ROS content and the activity of SOD and POX increased, while CAT activity decreased in comparison with control . Activities of SOD, POX and CAT were correlated both with Cu concentration (0-75 microM) in the growth medium and with OH* content in leaves . Close correlation was also found between OH* content and Cu concentration . Oxidative stress in A . thaliana under Cu treatment expressed in elevated content of O2*-, H2O2 and OH* in leaves . To overcome it very active the dismutase- and peroxidase-related (and not catalase-related, as in other plants) ROS scavenging system operated in A . thaliana . Visual symptoms of phytotoxicity: chlorosis, necrosis and violet colouring of leaves as well as a reduction of shoot biomass occurred in plants. Plant J, 2004 Aug, 39(3), 403 - 14 Arabidopsis Yellow Stripe-Like2 (YSL2): a metal-regulated gene encoding a plasma membrane transporter of nicotianamine-metal complexes; DiDonato RJ Jr et al.; The Yellow Stripe-Like (YSL) family of proteins has been identified based on sequence similarity to maize Yellow Stripe1 (YS1), the transporter responsible for the primary uptake of iron from the soil . YS1 transports iron that is complexed by specific plant-derived Fe(III) chelators called phytosiderophores (PS) . Non-grass species of plants neither make nor use PS, yet YSL family members are found in non-grass species (monocot, dicot, gymnosperm, and moss species) including Arabidopsis thaliana . YSLs in non-grasses have been hypothesized to transport metals complexed by nicotianamine (NA), an iron chelator that is structurally similar to PS and which is found in all higher plants . Here we show that Arabidopsis YSL2 (At5g24380) transports iron and copper when these metals are chelated by NA . YSL2 is expressed in many cell types in both roots and shoots, suggesting that diverse cell types obtain metals as metal-NA complexes . YSL2 transcription is regulated by the levels of iron and copper in the growth medium . Based on its expression pattern, a major function of the YSL2 appears to be in the lateral movement of metals in the vasculature. J Neurosci Res, 2004 Aug 1, 77(3), 453 - 61 Comparative study of cell culture and purification methods to obtain highly enriched cultures of proliferating adult rat Schwann cells; Mauritz C et al.; We present here a fast protocol that could be used to obtain highly purified cultures of maximal proliferating adult rat Schwann cells . These adult rat Schwann cells can be transfected in a nonbiological way using the physical transfection method of electroporation . Schwann cells are decisive in recovery of peripheral nerves after injury . In a clinical context, the use of enriched adult Schwann cells is necessary for autologous cell transplantation within nerve transplants for peripheral nerve repair . Different parameters such as tissue preparation, culture conditions, and protocols for enrichment, elevation of proliferation rates, and transfection were evaluated in cell cultures harvested from adult rat peripheral nerves . Cell preparation from in vivo predegenerated adult rat sciatic nerves combined with the use of melanocyte growth medium supplemented with forskolin, fibroblast growth factor (FGF)-2, and pituitary extract as a selective, serum-free culture medium, with a secondary cell-enrichment step using specific detachment, resulted in highly enriched cultures of adult rat Schwann cells (>90%) with enhanced proliferation rates (>or=40%) . About 20% of these adult Schwann cells could be modified genetically using an optimized electroporation protocol . Appl Environ Microbiol, 2004 Jul, 70(7), 3831 - 8 Purification and characterization of two distinct metalloproteases secreted by the entomopathogenic bacterium Photorhabdus sp . strain Az29; Cabral CM et al.; Photorhabdus sp . strain Az29 is symbiotic with an Azorean nematode of the genus Heterorhabditis in a complex that is highly virulent to insects even at low temperatures . The virulence of the bacteria is mainly attributed to toxins and bacterial enzymes secreted during parasitism . The bacteria secrete proteases during growth, with a peak at the end of the exponential growth phase . Protease secretion was higher in cultures growing at lower temperatures . At 10 degrees C the activity was highest and remained constant for over 7 days, whereas at 23 and 28 degrees C it showed a steady decrease . Two proteases, PrtA and PrtS, that are produced in the growth medium were purified by liquid chromatography . PrtA was inhibited by 1,10-phenantroline and by EDTA and had a molecular mass of 56 kDa and an optimal activity at pH 9 and 50 degrees C . Sequences of three peptides of PrtA showed strong homologies with alkaline metalloproteases from Photorhabdus temperata K122 and Photorhabdus luminescens W14 . Peptide PrtA-36 contained the residues characteristic of metzincins, known to be involved in bacterial virulence . In vitro, PrtA inhibited antibacterial factors of inoculated Lepidoptera and of cecropins A and B . PrtS had a molecular mass of 38 kDa and was inhibited by 1,10-phenanthroline but not by EDTA . Its activity ranged between 10 and 80 degrees C and was optimal at pH 7 and 50 degrees C . PrtS also destroyed insect antibacterial factors . Three fragments of PrtS showed homology with a putative metalloprotease of P . luminescens TTO1 . Polyclonal antibody raised against PrtA did not recognize PrtS, showing they are distinct molecules. J Cell Sci, 2004 Jul 1, 117(Pt 15), 3141 - 52 The necessity of mitochondrial genome DNA for normal development of Dictyostelium cells; Chida J et al.; Most unexpectedly, there is now increasing evidence that mitochondria have novel and crucial functions in the regulatory machinery of the growth/differentiation transition, cell-type determination, cellular movement and pattern formation . Here we created rho delta cells with a reduced amount (about 1/4) of mitochondrial DNA (mtDNA) from Dictyostelium discoideum Ax-2 cells, by exposing Ax-2 cells to ca . 30 microg/ml of ethidium bromide (EtBr) in axenic growth medium . Importantly, the rho delta cells exhibited a series of fascinating behaviors: when they were starved, they showed a marked delay of differentiation and stopped their development at the slug stage, thus failing to construct fruiting bodies . Moreover, cell patterning and cell-type proportioning were found to be greatly modified in slugs (referred to as rho delta slugs) derived from rho delta cells . That is, prestalk differentiation was significantly enhanced in rho delta slugs, while prespore differentiation was markedly inhibited . In addition, the clear anterior prestalk/posterior prespore pattern was considerably disturbed in rho delta slugs, presumably because of incomplete sorting between the two types of differentiated cells . After the assay of phototaxis, rho delta slugs also exhibited highly disordered movement towards the light source . Taken together, these results suggest that mtDNA might have important multiple functions in a variety of cellular processes during Dictyostelium development. Am J Surg, 2004 Jul, 188(1A Suppl), 36 - 41 Wet wound healing: from laboratory to patients to gene therapy; Eriksson E et al.; Wet treatment of wounds has been used as an "irrigation" method since the seventh century . We have developed the concept of an in vivo tissue culture that facilitates wound healing and allows tissue engineering . A transparent, flexible, round chamber provides the wet environment . This system heals clean wounds as fast or faster than any other method, with less scarring . It allows delivery of analgesics, antibiotics, growth factors, growth media, and cells into the chamber, becoming a platform for tissue engineering . Gene therapy of the wound can be done in the chamber with growth factor and other genes . A tetracycline switch allows precise timing and amounts of expression and provides the opportunity for sequential expression of genes delivered at the same time. Proteomics, 2004 Jul, 4(7), 2005 - 13 Comparative proteome analysis of Hansenula polymorpha DL1 and A16; Kim YH et al.; Proteomic responses of methylotrophic yeasts (Hansenula polymorpha DL1 and A16) to growth medium tuning by carbon source shift (glycerol-->methanol) were monitored and analyzed by two-dimensional gel electrophoresis . Through comparative analyses of two-dimensional gels, intracellular yeast proteins with complex expression patterns were systematically sorted into: (1) proteins that are commonly expressed with comparable high abundance in both strains; (2) strain-specific proteins that are expressed at high level only in a particular strain; (3) strain-specific and methanol-induced proteins that are expressed only in the presence of methanol; and (4) strain-specific and constitutively-expressed proteins that are expressed consistently irrespective of carbon source shift without extreme change in expression level . Among the DL1-specific proteins belonging to group four, the four proteins showing the highest expression levels in the course of the fermentation process were identified as: glucose-6-phosphate dehydrogenase, isocitrate lyase, succinyl-CoA synthetase, and glycerol-3-phosphate dehydrogenase . From these results, it is suggested that DL1 has distinct metabolic characteristics including enhanced metabolic activities both in glycerol uptake and the glyoxylate bypass cycle, as compared to A16 . This is likely to explain why the DL1 strain shows a significantly higher rate of glycerol and methanol consumption during the fermentation process . Our systematic approach to the analysis of proteomic responses and the detailed analysis results reported here will be useful to better understand the global physiology of H . polymorpha, as proteome databases for various methylotrophic yeasts are established. J Nat Prod, 2004 Jun, 67(6), 1014 - 7 Biotransformation of imperatorin by Aspergillus flavus; Teng WY et al.; Imperatorin (1) was metabolized by Aspergillus flavus, in growth media, to give five metabolites . On the basis of their physical data, the structures of the five metabolites were elucidated as xanthotoxol (2), E-trichoclin (3), Z-trichoclin (4), E-imperatorin acid (5), and Z-imperatorin acid (6); among these, 4, 5, and 6 were characterized as new coumarins . The five metabolites 2-6 were tested for anti-hepatitis B virus activity in vitro and found to be less active than the parent compound 1. Plant Cell Physiol, 2004 Jun, 45(6), 703 - 11 A novel ethanol-hypersensitive mutant of Arabidopsis; Hirayama T et al.; A novel ethanol-hypersensitive mutant, geko1 (gek1), was isolated from Arabidopsis thaliana . The gek1 mutant displays an enhanced sensitivity (10-100 times greater than the wild type) to ethanol in growth medium, while it grows normally in the absence of ethanol, and responds normally to other alcohols and to environmental stresses such as heat shock and high salinity . The ethanol-hypersensitive phenotype of gek1 requires alcohol dehydrogenase activity, indicating that gek1 is sensitive not to ethanol itself but to the metabolites of ethanol . Consistent with this, gek1 shows enhanced sensitivity to acetaldehyde in the medium . The endogenous acetaldehyde levels were not different between gek1-2 and wild-type seedlings treated with ethanol . These results indicate that the ethanol hypersensitivity of gek1 is due to an enhanced sensitivity to acetaldehyde toxicity, instead of abnormally elevated accumulation of toxic acetaldehyde, which has been thought to be the major cause of ethanol toxicity in mammal cells. Antimicrob Agents Chemother, 2004 Jul, 48(7), 2531 - 7 Saccharomyces cerevisiae multidrug transporter Qdr2p (Yil121wp): localization and function as a quinidine resistance determinant; Vargas RC et al.; This work reports the functional analysis of Saccharomyces cerevisiae open reading frame YIL121w, encoding a member of a family of drug:H(+) antiporters with 12 predicted membrane-spanning segments (DHA12 family) . Like its close homologue Qdr1p, Yil121wp was localized at the plasma membrane, and its increased expression also led to increased tolerance to the antiarrhythmia and antimalarial drug quinidine . The quinidine resistance phenotype was confirmed for different yeast strains and growth media, including a prototrophic strain, and YIL121w was named the QDR2 gene . Both QDR1 and QDR2 were also implicated in yeast resistance to the herbicide barban (4-chloro-2-butynyl {3-chlorophenyl} carbamate), and the genes are functionally interchangeable with respect to both resistance phenotypes . The average intracellular pH of a yeast population challenged with quinidine added to the acidic growth medium was significantly below the intracellular pH of the unstressed population, suggesting plasma membrane permeabilization by quinidine with consequent increase of the H(+) influx rate . For the same extracellular quinidine concentration, internal acidification was more intense for the Deltaqdr2 deletant compared with the parental strain . Although QDR2 transcription was not enhanced in response to quinidine, the results confirmed that Qdr2p is involved in the active export of quinidine out of the cell, thus conferring resistance to the drug. Anal Bioanal Chem, 2004 Mar, 378(6), 1601 - 7 Fourier-transform infrared spectroscopic study of the interactions of selenium species with living bacterial cells; Feo JC et al.; A study of the interactions of several selenium species with living bacterial cells was carried out by Fourier-transform infrared (FT-IR) spectroscopy . Bacterial cells consisted of an Escherichia coli strain (K-12) cultivated in a growth medium based on glucose contaminated with selenium species . Equilibrium between the analyte in the solution and the extraction medium was established, and then the effects of selenium species upon the external membrane of the living bacterial cells were characterized by performing FT-IR spectroscopy of whole cells . The presence of the toxicants at various concentrations in the culture medium had an effect on the FT-IR spectra, and the concentration of the selenium species was determined directly in the biomass by FT-IR spectroscopy . The intensity ratios between several absorption lines, which varied as a function of the concentration of the selenium species, were used as the analytical signal . Electronic Supplementary Material: Supplementary material is available for this article if you access the article at A link in the frame on the left on that page takes you directly to the supplementary material. Can J Microbiol, 2004 Apr, 50(4), 261 - 7 Isolation of antibiotic-resistant and antimetabolite-resistant mutants of Frankia strains EuI1c and Cc1.17; Myers AK et al.; Antibiotic-resistant and antimetabolite-resistant mutants of the nitrogen-fixing symbiotic bacterium Frankia were isolated to provide strains with genetic backgrounds amenable to genetic analysis . The lethal and mutagenic effects of ethyl methanesulfonate (EMS) and UV light on four Frankia strains were investigated . UV irradiation or EMS treatment of strain EuI1c cells resulted in the formation of two different colony types: rough and smooth . The smooth colonies were conditional sporulation mutants . In the case of EMS-induced cells of strain Cc1.17, resistance to lincomycin, ampicillin, and 5-fluorouracil occurred at a frequency of 1 x 10(-5), 1 x 10(-5), and 4 x 10(-5), respectively . The lincomycin-resistant mutants produced a yellow-tan pigment that was released into the growth medium . Resistance to tetracycline and lincomycin with EMS-induced cells of strain EuI1c occurred at a frequency of 3.2 x 10(-3) and 4.7 x 10(-4), respectively . These strains will be useful for the development of genetic methods for Frankia. J Exp Bot, 2004 Aug, 55(404), 1861 - 70 Epub 2004 Jun 18. Towards dissecting nutrient metabolism in plants: a systems biology case study on sulphur metabolism; Nikiforova VJ et al.; A genomics analysis on sulphur metabolism has been conducted at the level of transcriptomics and metabolomics . The analysis of these data after applying bioinformatic tools is to reveal novel findings . The findings are discussed and the knowledge obtained from comparable analyses on sulphur metabolism and other plant nutrient genomic studies is reviewed . The analysis of the response of the transcriptome and metabolome to sulphur deprivation in the growth medium provides a tool set for the analysis of comparable genomics studies of other nutrients . The goal of this 'sulphobolomics' (i.e . sulphur genomics and metabolome analysis) approach, and of other investigations, is to describe in a holistic way the biochemical, molecular, and physiological response of a plant to nutrient starvation, here sulphate, or, more generally, to alterations and imbalances in nutrient availability . Eventually, this analysis will provide a case study for a systems biology approach. J Bacteriol, 2004 Jul, 186(13), 4254 - 61 Energetics of gliding motility in Mycoplasma mobile; Jaffe JD et al.; Mycoplasma mobile glides on surfaces at up to 7 microm/s by an unknown mechanism . We studied the energetics that power gliding by using a novel, growth medium-free system . We found that cells could glide in defined media if the glass substrate is preconditioned by exposure to horse serum . The active component that potentiates gliding is sensitive to proteinase K treatment . We used the defined medium system to test the effect of various inhibitors, ionophores, and poisons on motility of M . mobile . Valinomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), N,N'-dicyclohexylcarbodiimide, phenamil, amiloride, rifampin, and puromycin had no short-term effects on gliding . We also confirmed that we were able to modulate the membrane potential with valinomycin and FCCP by using a potential-sensitive dye . Shifting the pH likewise had no effect on motility . These results rule out the use of conventional ion motive forces to power gliding . Arsenate had a dramatic inhibitory effect on gliding, and both the speed and the fraction of cells moving tracked ATP levels . Sodium orthovanadate had a slight but significant inhibitory effect on gliding . Taken together, these results suggest that the motor system of M . mobile is likely an ATPase or is directly coupled to an ATPase. J Drug Target, 2004 Jan, 12(1), 11 - 8 Efficient gene delivery with serum into human cancer cells using targeted anionic liposomes; Mady MM et al.; Success of human gene therapy depends upon the development of delivery vehicles or vectors, which can selectively deliver therapeutic genes to target cells with efficiency and safety . Previous studies have shown an efficient, systemic trans-gene expression in many cell lines (in vitro) by using an anionic liposomal vector, based on the composition of retroviral envelopes (artificial viral envelopes, AVEs) . The AVE-liposomes and their complexes with plasmid (DNA) were characterized according to zeta potential measurements and transmission electron microscopy (TEM) . We successfully demonstrated that AVE liposomes, dispersed in 10% serum-containing growth medium, efficiently delivered plasmid DNA to HuH-7 (human hepatoma cell line) cells . We assessed the utility of liver-targeted vesicles as a drug/gene delivery system for the treatment of liver diseases . We found that small unilamellar AVE vesicles containing 15 mol% digalactosyl diglyceride (DGDG) are efficiently targeted to the liver via the hepatic asialoglycoprotein receptor. J Biol Chem, 2004 Aug 20, 279(34), 35353 - 9 Epub 2004 Jun 16. Regulation of the yeast EKI1-encoded ethanolamine kinase by inositol and choline; Kersting MC et al.; Regulation of the EKI1-encoded ethanolamine kinase by inositol and choline was examined in Saccharomyces cerevisiae . Transcription of the EKI1 gene was monitored by following the expression of beta-galactosidase activity driven by a P(EKI1)-lacZ reporter gene . The addition of inositol to the growth medium resulted in a dose-dependent decrease in EKI1 expression . Supplementation of choline to inositol-containing growth medium brought about a further decrease in expression, whereas choline supplementation alone had no effect . Analysis of EKI1 expression in ino2Delta, ino4Delta, and opi1Delta mutants indicated that the transcription factors Ino2p, Ino4p, and Opi1p played a role in this regulation . Moreover, mutational analysis showed that the UAS(INO) element in the EKI1 promoter was required for the inositol-mediated regulation . The regulation of EKI1 expression by inositol and choline was confirmed by corresponding changes in ethanolamine kinase mRNA, protein, and activity levels . The repression of ethanolamine kinase by inositol supplementation correlated with a decrease in the incorporation of ethanolamine into CDP-ethanolamine pathway intermediates and into phosphatidylethanolamine and phosphatidylcholine. Biotechnol Lett, 2004 May, 26(9), 747 - 51 Improved use of organic phosphate by Skeletonema costatum through regulation of Zn2+ concentrations; Shi Y et al.; The maximum growth rate (1.4-2 x 10(5) cells ml(-1) d(-1)), cell final yields (2.6-5.2 x 10(5) cells ml(-1)) and extracellular alkaline phosphatase activity (2.4-10.6 microg phosphate released ml(-1) h(-1)) of the red tide alga, Skeletonema costatum, increased when Zn2+ was increased from 0 to 24 pM, but decreased with 66 pM Zn2+ in growth medium with glycerophosphate as the sole phosphorus source . Extracellular carbonic anhydrase activity and the affinity for HCO3- and CO2 uptake increased when Zn2+ was increased from 0 to 12 pM, but then decreased at higher concentrations . The results suggested that utilization of organic phosphate required more Zn2+ than the uptake of inorganic carbon did, while utilization of dissolved inorganic carbon by Skeletonema costatum was very sensitive to Zn2+ concentration variations. J Biol Chem, 2004 Aug 20, 279(34), 35273 - 80 Epub 2004 Jun 12. Low affinity orthophosphate carriers regulate PHO gene expression independently of internal orthophosphate concentration in Saccharomyces cerevisiae; Pinson B et al.; Phosphate is an essential nutrient that must be taken up from the growth medium through specific transporters . In Saccharomyces cerevisiae, both high and low affinity orthophosphate carriers allow this micro-organism to cope with environmental variations . Intriguingly, in this study we found a tight correlation between selenite resistance and expression of the high affinity orthophosphate carrier Pho84p . Our work further revealed that mutations in the low affinity orthophosphate carrier genes (PHO87, PHO90, and PHO91) cause deregulation of phosphate-repressed genes . Strikingly, the deregulation due to pho87Delta, pho90Delta, or pho91Delta mutations was neither correlated to impaired orthophosphate uptake capacity nor to a decrease of the intracellular orthophosphate or polyphosphate pools, as shown by (31)P NMR spectroscopy . Thus, our data clearly establish that the low affinity orthophosphate carriers affect phosphate regulation independently of intracellular orthophosphate concentration through a new signaling pathway that was found to strictly require the cyclin-dependent kinase inhibitor Pho81p . We propose that phosphate-regulated gene expression is under the control of two different regulatory signals as follows: the sensing of internal orthophosphate by a yet unidentified protein and the sensing of external orthophosphate by low affinity orthophosphate transporters; the former would be required to maintain phosphate homeostasis, and the latter would keep the cell informed on the medium phosphate richness. J Neurobiol, 2004 Jul, 60(1), 51 - 60 Nicotine decreases agrin signaling and acetylcholine receptor clustering in C2C12 myotube culture; Ferayorni AJ et al.; The clustering of acetylcholine receptors (AChRs) in skeletal muscle fibers is a critical event in neuromuscular synaptogenesis . AChRs in concert with other molecules form postsynaptic scaffolds in response to agrin released from motor neurons as motor neurons near skeletal muscle fibers in development . Agrin drives an intracellular signaling pathway that precedes AChR clustering and includes the tyrosine phosphorylation of AChRs . In C2C12 myotube culture, agrin application stimulates the agrin signaling pathway and AChR clustering . Previous studies have determined that the frequency of spontaneous AChR clustering is decreased and AChRs are partially inactivated when bound by the acetylcholine agonist nicotine . We hypothesized that nicotine interferes with AChR clustering and consequent postsynaptic scaffold formation . In the present study, C2C12 myoblasts were cultured with growth medium to stimulate proliferation and then differentiation medium to stimulate fusion into myotubes . They were bathed in a physiologically relevant concentration of nicotine and then subject to agrin treatment after myotube formation . Our results demonstrate that nicotine decreases agrin-induced tyrosine phosphorylation of AChRs and decreases the frequency of spontaneous as well as agrin-induced AChR clustering . We conclude that nicotine interferes with postsynaptic scaffold formation by preventing the tyrosine phosphorylation of AChRs, an agrin signaling event that precedes AChR clustering . J Appl Microbiol, 2004, 97(1), 124 - 33 Source of tryptone in growth medium affects oxidative stress resistance in Escherichia coli; De Spiegeleer P et al.; AIMS: To investigate the influence of the source of tryptone in the growth medium on the resistance of Escherichia coli to various types of oxidative stress . METHODS AND RESULTS: Cultures of Escherichia coli MG1655 were grown in Luria-Bertani (LB) medium at 37 degrees C to stationary phase, harvested, and subsequently subjected to various types of oxidative stress . A marked difference in oxidative stress sensitivity was observed depending on the origin of the tryptone in the LB medium used to grow the cultures . Cells harvested from LB containing tryptone from source x (LBx) were more sensitive to inactivation by the superoxide generating compound plumbagin and by t-butyl peroxide, and to growth inhibition by the lactoperoxidase enzyme system, than cells harvested from LB containing tryptone from source y (LBy) . By monitoring expression of a panel of stress gene promotors linked to the gfp (green fluorescent protein) gene, and using Delta2-22 alkaline phosphatase as a probe for disulphide bridge formation from protein sulphydryl groups, it was demonstrated that a greater cytoplasmic oxidative stress existed in cells during growth in LBy than in LBx . CONCLUSIONS: Depending on the source of tryptone, bacteria may experience different levels of oxidative stress in tryptone-containing nonselective growth media . Although these levels of oxidative stress are subinhibitory, they may trigger a stress response that makes the bacteria more resistant to a subsequent exposure to a lethal or inhibitory level of oxidative stress . SIGNIFICANCE AND IMPACT OF THE STUDY: This work highlights the importance of controlling very subtle differences in composition of nonselective growth media in studies on bacterial physiology. J Agric Food Chem, 2004 Jun 16, 52(12), 3894 - 9 Bioavailability of cadmium-organic complexes to soil alga--an exception to the free ion model; Krishnamurti GS et al.; It is generally considered that cadmium bioavailability shows a considerable dependence on chemical speciation of Cd in solution, correlates best with the activity of free metal ion (Cd2+) in solution, and is largely indifferent to soluble metal complexes . The role of soluble organic matter (DOM) and soluble metal-organic complexes in metal bioavailability and toxicity, however, is not clear . Growth studies with a soil alga (Chlorococcum sp.) were conducted on a growth medium and pore water of Cookes Plain soil (Paleuxeralf), spiked with Cd as Cd(NO3)2 . Speciation of the Cd in pore water, and in growth medium with and without citrate, was performed using the MINTEQA2 computer model incorporating updated values of the stability constants of Cd-DOM complexes, as well as using anode stripping voltammetry . Analysis of the toxicity data showed that Cd-citrate, as well as the Cd-DOM complexes, is bioavailable and contributes toward the toxicity to alga . These data contradict the long-held notion that Cd-DOM complexes are not bioavailable to soil biota although they may increase the mobility of Cd. Chembiochem, 2004 Apr 2, 5(4), 500 - 7 Exploring recombinant flavonoid biosynthesis in metabolically engineered Escherichia coli; Watts KT et al.; Flavonoids are important plant-specific secondary metabolites synthesized from 4-coumaroyl coenzyme A (CoA), derived from the general phenylpropanoid pathway, and three malonyl-CoAs . The synthesis involves a plant type III polyketide synthase, chalcone synthase . We report the cloning and coexpression in Escherichia coli of phenylalanine ammonia lyase, cinnamate-4-hydroxylase, 4-coumarate:CoA ligase, and chalcone synthase from the model plant Arabidopsis thaliana . Simultaneous expression of all four genes resulted in a blockage after the first enzymatic step caused by the presence of nonfunctional cinnamate-4-hydroxylase . To overcome this problem we fed exogenous 4-coumaric acid to induced cultures . We observed high-level production of the flavanone naringenin as a result . We were also able to produce phloretin by feeding cultures with 3-(4-hydroxyphenyl)propionic acid . Feeding with ferulic or caffeic acid did not yield the corresponding flavanones . We have also cloned and partially characterized a new tyrosine ammonia lyase from Rhodobacter sphaeroides . Tyrosine ammonia lyase was substituted for phenylalanine ammonia lyase and cinnamate-4-hydroxylase in our E . coli clones and three different growth media were tested . After 48 h induction, high-level production (20.8 mg L(-1)) of naringenin in metabolically engineered E . coli was observed for the first time. Plant Physiol, 2004 Jun, 135(2), 1050 - 8 Epub 2004 Jun 04. Cell cycle modulation in the response of the primary root of Arabidopsis to salt stress; West G et al.; Salt stress inhibits plant growth and development . We investigated the importance of cell cycle regulation in mediating the primary root growth response of Arabidopsis to salt stress . When seedlings were transferred to media with increasing concentrations of NaCl, root growth rate was progressively reduced . At day 3 after transfer of seedlings to growth medium containing 0.5% NaCl the primary roots grew at a constant rate well below that prior to the transfer, whereas those transferred to control medium kept accelerating . Kinematic analysis revealed that the growth reduction of the stressed roots was due to a decrease in cell production and a smaller mature cell length . Surprisingly, average cell cycle duration was not affected . Hence, the reduced cell production was due to a smaller number of dividing cells, i.e . a meristem size reduction . To analyze the mechanism of meristem size adaptation prior to day 3, we investigated the short-term cell cycle events following transfer to saline medium . Directly after transfer cyclin-dependent kinase (CDK) activity and CYCB1;2 promoter activity were transiently reduced . Because protein levels of both CDKA;1 and CDKB1;1 were not affected, the temporary inhibition of mitotic activity that allows adaptation to the stress condition is most likely mediated by posttranslational control of CDK activity . Thus, the adaptation to salt stress involves two phases: first, a rapid transient inhibition of the cell cycle that results in fewer cells remaining in the meristem . When the meristem reaches the appropriate size for the given conditions, cell cycle duration returns to its default. Acta Pharmacol Sin, 2004 Jun, 25(6), 827 - 32 Stimulation of mucin secretion from human bronchial epithelial cells by mast cell chymase; He SH et al.; AIM: To investigate the effect of chymase on the mucin secretion from human bronchial epithelial cells . METHODS: Primarily-cultured human bronchial epithelial (PCHBE) cells and normal human bronchial epithelial (NHBE) cells were cultured with chymase or other stimulus in a mixture of bronchial epithelial growth medium (BEGM) and Dulbecco's modified Eagle's medium (DMEM), and the quantities of stimulatory mucin release were recorded . MUC5AC mucin was measured with an ELISA and dolichos biflorus agglutinin (DBA) mucin was determined with an enzyme linked DBA assay . RESULTS: A dose-dependent secretion of DBA mucin from PCHBE cells was observed with chymase with a maximum secretion of 98 % above baseline being achieved following 3 h incubation . The action of chymase started from 1 h, peaked at 3 h and dramatically decreased at 20 h following incubation . Chymase was able to also stimulate approximately 38 % increase in MUC5AC mucin release from PCHBE cells, and about 121 % increase in DBA mucin release from NHBE cells . A chymase inhibitor soybean trypsin inhibitor (SBTI) was able to inhibit up to 85 % chymase induced mucin release, indicating that the enzymatic activity was essential for the actions of chymase on bronchial epithelial cells . CONCLUSION: Chymase is a potent stimulus of mucin secretion from human bronchial epithelial cells . It can contribute to mucus hypersecretion process in the patients with chronic obstructive pulmonary disease or asthma. Histol Histopathol, 2004 Jul, 19(3), 735 - 42 Immuno-histochemical expression of alpha1, alpha2 and alpha3 integrin subunits during angiogenesis in vitro; Suda H et al.; Aortic explants were obtained from mouse fetuses and cultured in collagen gels . Immuno-fluorescence microscopy, antibodies (anti alpha1, alpha2 and alpha3 integrin subunits) were used . Fibroblastic cells migrated from the aortic explant after one day of cultivation . The migrating cells located in the peripheral part of the aortic explant were positive for alpha1 and alpha2 integrin subunit antibodies . Immuno-fluorescence-positive staining for the alpha3 integrin subunit antibody was clearly seen in the migrating cells located near the aortic explant and surrounding tube-like structures . In an immuno-electron microscope study performed by pre-embedding immuno labeling, gold particles associated with the alpha3 integrin subunit were found to reside on the membranes of the cells surrounding the capillary-like tubes . Two synthetic peptides, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) and KDGEA (Lys-Asp-Gly-Glu-Ala), were added to the growth medium to study their effects on cell migration . KDGEA, a compound containing the recognition sequence for alpha2beta1 integrin, decreased cell migration, while GRGDSP exhibited no effect . The migration of fibroblastic cells is an important phenomenon for tube formation . The present study suggested that the alpha1 and alpha2 integrin subunits are both involved in the cell migration, and more specifically, that the alpha2 integrin subunit participates in cell migration through the KDGEA sequence . The alpha3 integrin subunit played a role in tube formation. Integr Cancer Ther, 2004 Jun, 3(2), 173 - 88 Cancer and complementary and alternative medicine in Italy: personal observations and historical considerations; Moss RW; This article contains observations and historical considerations on cancer and complementary and alternative medicine (CAM) in Italy, a country that has a great tradition in medical research, going back to the Renaissance . However, Italy does not have a strong tradition of using CAM approaches in the treatment of cancer . While surveys show that the Italian population is eager to learn more about CAM, the medical profession there is largely dismissive of these methods . In 1997-1998, the notorious Luigi Di Bella affair occurred in Italy, when a professor of physiology at Modena proposed a nonconventional approach to cancer treatment, based on the off-label use of somatostatin . This treatment found champions in the media and general public but was opposed by most of the medical profession . Although clinical trials later demonstrated that it had no efficacy, the affair divided Italian public opinion and nearly brought down the national government . Italy no longer has prominent proponents of nonconventional treatments in cancer . However, it continues to have innovative scientists who do important work that is consonant with a CAM approach . This article considers the work of 3 such scientists: Paolo Lissoni, MD, of Monza (Milan), who has carried out numerous clinical trials with the pineal hormone melatonin; Giancarlo Pizza, MD, of Bologna, who has done extensive work on the use of transfer factor and other immunomodulators in the treatment of renal cell and other kinds of cancer; and Aldo Mancini, MD, of Naples, who has isolated a mutated form of Mn-SOD-2 from the growth medium of a unique liposarcoma cell line . These scientists have introduced some flexibility into a rigid state-run hospital system by offering patients innovative treatment options in the context of approved clinical trials. Int J Food Microbiol, 2004 Jun 15, 93(3), 325 - 33 New method for rapid and sensitive quantification of sulphide-producing bacteria in fish from arctic and temperate waters; Skjerdal OT et al.; The offensive, fishy, rotten H2S-off-odours in spoiled, aerobically and cold stored fish from arctic and temperate waters are generally caused by sulphide-producing bacteria (SPB), mainly Shewanella putrefaciens . In the present work, a new, rapid, simple and accurate method for estimation of the SPB content in fish from these areas is described . The quantification is based on the formation rate of iron sulphide during growth of SPBs incubated at 30 degrees C in a liquid growth medium containing cysteine, sodium thiosulphate and iron(III)citrate as specific substrates for iron sulphide formation . The iron sulphide turns the medium grey and masks the background fluorescence in the medium when the SPB content in the assay is approximately 10(9) cfu/ml . The fluorescence change could be detected instrumentally and the colour change visually . The method was developed and evaluated in tests with S . putrefaciens CCUG 13452 DT as well as naturally occurring SPBs in cod, salmon, wolf fish and coal fish . A linear correlation between the SPB count and detection time was obtained over the entire range from 1 to 10(9) cfu SPB/g, corresponding to detection times 17 and 1 h, respectively . The correlation is described by the equation: log cfu/g fish= -0.59(+/- 0.17) x DT+ 9.65(+/- 0.09), where DT is the detection time in hours . The model was valid for all the tested fish species and all tested naturally occurring SPBs in these species . The regression coefficients (R2) for cod, coal fish, wolf fish and salmon were 0.99, 0.92, 0.97 and 0.97, respectively . The detection level of the method is 1 SPB per sample tube, corresponding to 16 cfu/g fish . The method could be used to predict the remaining shelf life of the fish for different markets, even when the time-temperature history during storage of the fish is unknown. FEMS Microbiol Lett, 2004 Jun 1, 235(1), 101 - 8 Characterisation and expression of a gene encoding a mutarotase from the fungus Rhizopus nigricans; Vilfan T et al.; A gene coding for a mutarotase was isolated and characterised from the filamentous fungus Rhizopus nigricans . In order to determine the encoded enzyme's activity a recombinant protein was prepared in the baculovirus expression system and the mutarotase activity was determined . Expression studies showed that the gene is repressed by high as well as low concentrations of glucose and derepressed during deficiency of glucose . Besides the regulation at the level of transcription, an accelerative effect of glucose in growth medium on the mutarotase mRNA decay was also demonstrated . Moreover, a Southern hybridisation performed at lower temperatures suggested that the R . nigricans genome harbours a nucleotide sequence, that is homologous to the isolated gene. Methods, 2004 Jul, 33(3), 213 - 9 In vivo labeling of fission yeast DNA with thymidine and thymidine analogs; Sivakumar S et al.; In vivo labeling of DNA with thymidine and thymidine analogs has long been a cornerstone of replication studies . Unfortunately, yeast lack a thymidine salvage pathway and thus do not incorporate exogenous thymidine . Specifically, yeast neither efficiently take up exogenous thymidine from their growth media nor phosphorylate it to thymidylate, the precursor of dTTP . We have overcome these problems in fission yeast by expressing the human equilibrative nucleoside transporter 1 (hENT1) along with herpes simplex virus thymidine kinase (tk) . hENT1 tk cells are healthy and efficiently incorporate exogenous thymidine and thymidine analogs . We present protocols for labeling DNA with tritiated thymidine, for in situ detection of incorporated BrdU by immunofluorescence, for double labeling with CldU and IdU, for CsCl gradient separation of IdU-labeled DNA, and for using hENT1 and tk as both positive and negative selection markers. Antimicrob Agents Chemother, 2004 Jun, 48(6), 2190 - 8 Structure and association of human lactoferrin peptides with Escherichia coli lipopolysaccharide; Chapple DS et al.; An 11-amino-acid amphipathic synthetic peptide homologous to a helical region on helix 1 of human lactoferrin HLP-2 exhibited bactericidal activity against Escherichia coli serotype O111, whereas an analogue synthesized with Pro substituted for Met, HLP-6, had greatly reduced antimicrobial activity . The bactericidal activity of HLP-2 was 10-fold greater than that of HLP-6 in both buffer and growth medium by time-kill assays . These assays also showed a pronounced lag phase that was both concentration and time dependent and that was far greater for HLP-2 than for HLP-6 . Both peptides, however, were shown to be equally efficient in destabilizing the outer membrane when the hydrophobic probe 1-N-phenylnaphthylamine was used and to have the same lipopolysaccharide (LPS) binding affinity, as shown by polymyxin B displacement . Circular dichroism (CD) spectroscopy was used to study the structure and the organization of the peptides in solution and upon interaction with E . coli LPS . In the presence of LPS, HLP-2 and HLP-6 were found to bind and adopt a beta-strand conformation rather than an alpha-helix, as shown by nonimmobilized ligand interaction assay-CD spectroscopy . Furthermore, this assay was used to show that there is a time-dependent association of peptide that results in an ordered formation of peptide aggregates . The rate of interpeptide association was far greater in HLP-2 LPS than in HLP-6 LPS, which was consistent with the lag phase observed on the killing curves . These results allow us to propose a mechanism by which HLP-2 folds and self-assembles at the outer membrane surface before exerting its activity. Org Lett, 2004 May 27, 6(11), 1753 - 6 Synthesis of 4-fluorinated UDP-MurNAc pentapeptide as an inhibitor of bacterial growth; Ueda T et al.; 4-Fluorinated UDP-MurNAc pentapeptide, 2, has been synthesized . In our previous study, UDP-MurNAc pentapeptide analogue 1 was found to be incorporated into the bacterial cell wall through biosynthesis . Compound 2 showed growth-inhibition activity against Gram-positive bacteria when it was added to growth media at 0.01 mg/mL . {structure--see text} Arch Toxicol, 2004 Oct, 78(10), 565 - 74 Epub 2004 May 19. Mitochondrial viability and apoptosis induced by aluminum, mercuric mercury and methylmercury in cell lines of neural origin; Toimela T et al.; Mercury and aluminum are considered to be neurotoxic metals, and they are often connected with the onset of neurodegenerative diseases . In this study, mercuric mercury, methylmercury and aluminum were studied in three different cell lines of neural origin . To evaluate the effects, mitochondrial cytotoxicity and apoptosis induced by the metals were measured after various incubation times . SH-SY5Y neuroblastoma, U 373MG glioblastoma, and RPE D407 retinal pigment epithelial cells were subcultured to appropriate cell culture plates and 0.01-1,000 microM concentrations of methylmercury, mercuric and aluminum chloride were added into the growth medium . In the assay measuring the mitochondrial dehydrogenase activity, WST-1, the cultures were exposed for 15 min, 24 or 48 h before measurement . Cells were allowed to recover from the exposure in part of the study . Apoptosis induced by the metals was measured after 6-, 24- and 48-h exposure times with the determination of activated caspase 3 enzyme . Mitochondrial assays showed a clear dose-response and exposure time-response to the metals . The most toxic was methylmercury (EC50 ~0.8 microM, 48 h), and the most sensitive cell line was the neuroblastoma cell line SH-SY5Y . Furthermore, there was marked mitochondrial activation, especially in connection with aluminum and methylmercury at low concentrations . This activation may be important during the initiation of cellular processes . All the metals tested induced apoptosis, but with a different time-course and cell-line specificity . In microscopic photographs, glioblastoma cells formed fibrillary tangles, and neuroblastoma cells settled along the fibrilles in cocultures of glial and neuronal cell lines during aluminum exposure . The study emphasized the toxicity of methylmercury to neural cells and showed that aluminum alters various cellular activities. J Bacteriol, 2004 Jun, 186(11), 3640 - 8 Dimethylselenide demethylation is an adaptive response to selenium deprivation in the archaeon Methanococcus voltae; Niess UM et al.; The archaeon Methanococcus voltae needs selenium for optimal growth . A gene group most likely involved in the demethylation of dimethylselenide was discovered, the expression of which is induced upon selenium deprivation . The operon comprises open reading frames for a corrinoid protein and two putative methyltransferases . It is shown that the addition of dimethylselenide to selenium-depleted growth medium relieves the lack of selenium, as indicated by the repression of a promoter of a transcription unit encoding selenium-free hydrogenases which is normally active only upon selenium deprivation . Knockout mutants of the corrinoid protein or one of the two methyltransferase genes did not show repression of the hydrogenase promoter in the presence of dimethylselenide . The mutation of the other methyltransferase gene had no effect . Growth rates of the two effective mutants were reduced compared to wild-type cells in selenium-limited medium in the presence of dimethylselenide. Aquat Toxicol, 2004 Jun 10, 68(2), 121 - 8 Relationship between uptake capacity and differential toxicity of the herbicide atrazine in selected microalgal species; Weiner JA et al.; Microalgal species vary in their sensitivity to the triazine herbicide, atrazine . This study examined both atrazine uptake and cellular characteristics of microalgae to determine if either can be used to predict algal sensitivity . Standard toxicity tests were performed on five microalgal species, each representing a different algal division or habitat . Test species listed in order of increasing sensitivity were: Isochrysis galbana, Dunaliella tertiolecta, Phaeodactylum tricornutum, Pseudokirchneriella subcapitata, and Synechococcus sp . Each species was exposed to 14C-atrazine at its growth rate EC50 concentration (44-91 microg/L) . At five time-points over 96 h, samples were filtered to collect algae and washed with unlabeled atrazine to displace labeled atrazine loosely absorbed to the cell surface . Radioactivity present on filters and in the growth medium was measured by liquid scintillation counting . Relationships between algal species-sensitivity to atrazine and compound uptake, cell dry weight, cell volume, and cell surface area were determined by linear regression analysis . Cell size measurements (based on dry weight, biovolume, and surface area) were significantly correlated with atrazine uptake (R2 > 0.45, P-value < 0.05) . There was a significant correlation between atrazine uptake and species-sensitivity to atrazine (R2 = 0.5413 , P-value = 0.0012) . These results indicate that smaller cells with greater surface area to volume ratios will incorporate more atrazine, and in general, will be more sensitive to atrazine exposure . However, I . galbana, with small cell size and relatively high atrazine uptake was the least sensitive species tested . This species and others may have mechanisms to compensate for atrazine stress that make predicting responses of microalgal communities difficult. Mol Plant Microbe Interact, 2004 May, 17(5), 456 - 66 The disruption of a Galpha subunit sheds new light on the pathogenicity of Stagonospora nodorum on wheat; Solomon PS et al.; Gna1, a gene encoding a Galpha subunit, a key component of signal transduction pathways, has been cloned and characterized in the wheat pathogen Stagonospora nodorum . Analysis of Gna1 expression during infection revealed a slight decrease in transcript levels shortly after germination, after which levels steadily increased until sporulation . Inactivation of Gna1 had a pleiotropic effect on phenotype . The Gna1 mutants were less pathogenic, attributed to coinciding with a defect in direct penetration . Also, Gna1 mutants were unable to sporulate,