Growth media

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, showed an albino phenotype, and secreted one or more brown pigments into growth media . Analysis of growth medium identified tyrosine, phenylalanine, and dihydroxyphenylalanine (L-DOPA) were excreted by the Gna1 strains but not by wild type . The presence of these compounds, and the insensitivity of melanization to tricyclazole suggest that S . nodorum synthesizes melanin via the L-DOPA pathway, the first fungal phytopathogen described to do so . Decreases in protease (and several other depolymerases) activities and sensitivity to osmotic stress were other phenotypes identified in the Gna1 mutants . Gna1 is the first signal transduction gene to be cloned and characterized from S . nodorum and its inactivation has uncovered several previously unknown facets of pathogenicity.

J Appl Microbiol, 2004, 96(6), 1271 - 7
The heat shock response is dependent on the external environment and on rapid ionic balancing by pharmacological agents in Saccharomyces cerevisiae; Vovou I et al.; AIMS: To investigate whether non-preconditioned yeast cells survive under heat shock, when placed in growth medium originated from protected cells and to provide insights into the ionic contribution in the response . METHODS AND RESULTS: The heat shock response was investigated by determining cell viability following exposure of yeast cells to 53 degrees C for 30 min, either in the absence or presence of drugs . Preconditioning was performed by incubating the cultures at 37 degrees C for 2 h . Under heat shock, non-preconditioned cell survival was significantly enhanced by the presence of the cell-free supernatant of resistant cultures . Addition of omeprazole or tetraethylammonium ions during the heat shock resulted in similar increases . Neither amiodarone nor mepivacaine showed any analogous effect . Omeprazole enhanced survival when added before the heat shock, while amiodarone exhibited a cytocidic action . CONCLUSIONS: Rapid balancing of ions may contribute to cell survival during heat shock, while survival under mild stress could probably be co-ordinated by additional events . SIGNIFICANCE AND IMPACT OF THE STUDY: Evidence is provided for the implication of the external environment and ionic homeostasis in the survival of yeast cells under unfavourable environmental conditions . This knowledge may be of importance in controlling both fermentation and therapeutic approaches.

Biopolymers, 2004 May-Jun 5, 74(1-2), 172 - 5
Infrared microspectroscopy of individual human cervical cancer (HeLa) cells suspended in growth medium; Miljkovic M et al.; We report for the first time the infrared spectra of individual human cervical cancer (HeLa) cells suspended in buffer or cell culture medium . Although we did not establish whether these cells were viable at the time of spectral data acquisition, we believe that the methodology used is applicable to the study of live cells . Data were collected either from entire cells, using 25- to 40-microm apertures, or via an imaging approach, where pixels measuring 6.25 x 6.25 microm were assembled to form a map of a cell in suspension . Measurements were carried out both in reflection/absorption and in transmission modes . The results reported here might have far-reaching implications for the use of infrared microspectroscopy to monitor cell proliferation, drug response, and other cell biological parameters in live cells .

Microbiology, 2004 May, 150(Pt 5), 1139 - 45
Production of ammonia by Tritrichomonas foetus and Trichomonas vaginalis; Kleydman Y et al.; Production of ammonia is difficult to find among the various studies of amino acid metabolism in protozoa . Several studies suggest that catabolism of arginine to ammonium is important for the growth of trichomonads . Trichomonads are amitochondriate zooflagellates that thrive under microaerophilic and anaerobic conditions . The authors were able to detect accumulation of ammonium ions and ammonia in cultures of Tritrichomonas foetus and Trichomonas vaginalis, including those resistant to metronidazole . Ammonium ions and ammonia were detected using the indophenol colorimetric method . Cells incubated overnight under an ambient oxygen gas phase had 0.9 mM soluble ammonium (NH(4)(+) and NH(3)) or a 20 % greater concentration of ammonium relative to sterile growth medium that had been incubated similarly . Production of ammonia itself was confirmed by analysis of a wick that was moistened with sulfuric acid (20 mM) and placed above the liquid in sealed cultures of a strain of Trichomonas vaginalis . The wicks from these cultures captured the equivalent of 0.048 mM volatile ammonia (NH(3)) from the liquid as compared to 0.021 mM volatile ammonia from sterile medium after overnight incubation . Intact trichomonads, 0.7 x 10(6) cells ml(-1) equivalent to 0.7 mg protein ml(-1), incubated in Doran's buffer with or without (1 mM) L-arginine produced significant amounts of soluble ammonium (0.07 mM and 0.04 mM, respectively) during 60 min . The results indicate that ammonium ions and the more irritating ammonia are significant metabolites of trichomonads . In addition, based upon end-product amounts, it appears that the rate of arginine metabolism is of the same order of magnitude as that for carbohydrate metabolism by trichomonads.

Spine, 2004 Mar 1, 29(5), 568 - 75
Effect of nicotine on spinal disc cells: a cellular mechanism for disc degeneration; Akmal M et al.; STUDY DESIGN: Experimental investigation to determine the effect of nicotine on intervertebral spinal disc nucleus pulposus (NP) cells cultured in vitro . OBJECTIVES.: To evaluate the effects of nicotine on cell proliferation, extracellular matrix production, and viability of NP cells in three-dimensional alginate constructs cultured in vitro . SUMMARY OF BACKGROUND DATA: Numerous studies confirm that smoking is a strong risk factor for back pain . The most widely accepted explanations for the association between smoking and disc degeneration is malnutrition of spinal disc cells by carboxy-hemoglobin-induced anoxia or vascular disease . Nicotine, a constituent of tobacco smoke, present in most body fluids of smokers is known to have detrimental effects on a variety of tissues . It may also be directly responsible for intervertebral disc (IVD) degeneration by causing cell damage in both the nucleus pulposus and anulus fibrosus . The effect of nicotine on IVD cells has not previously been investigated . METHODS: Bovine chondrocytic intervertebral disc cells were isolated by sequential digestion of nucleus pulposus and seeded in 2% alginate . The constructs were cultured for 21 days either in growth medium containing freebase nicotine (Sigma) at concentrations found in the serum of smokers (25 nmol/L-300 nmol/L) or in standard nicotine free-medium as controls . Samples were collected at time points 3, 7, 14, and 21 days and a quantitative assay was performed for DNA, glycosaminoglycans (GAG), and hydroxyproline . Samples were also processed for qualitative histologic analysis including immunolocalization of collagen types I and II . RESULTS: There was both a dose- and time-dependent response to nicotine, with constructs cultured in low-nicotine concentration media demonstrating an early increase in DNA, GAG, and collagen content, while constructs cultured in high nicotine concentration media demonstrated a late decrease in these parameters . At 25 nmol/L dose of nicotine, there was a significant increase (P < 0.05) in the above parameters at day 7 compared with the controls . At higher doses, there was a significant dose-dependent decrease (P < 0.05) in these parameters compared to controls; however, this was only significant at day 14 for the 300 nmol/L group and at day 21 for the 100 nmol/L, 200 nmol/L, and 300 nmol/L groups . Adverse morphologic changes were observed on histology, which included reduced cell proliferation, disrupted cell architecture, disintegration of cells, and extracellular matrix . Immunohistochemistry revealed the presence of type I collagen in the extracellular matrix rather than the normal type II collagen seen in the controls . CONCLUSIONS: Nicotine has an overall detrimental effect on NP disc cells cultured in vitro . There was significant inhibition of cell proliferation and extracellular matrix synthesis . Nicotine in tobacco smoke may have a role in pathogenesis of disc degeneration.

Cancer Res, 2004 May 1, 64(9), 3002 - 5
A hot spot for RAD51C interactions revealed by a peptide that sensitizes cells to cisplatin; Connell PP et al.; DNA repair via the homologous recombination pathway requires the recombinase RAD51 and, in vertabrates, five RAD51 paralogs . The paralogs form two complexes in solution, a XRCC3/RAD51C heterodimer and a RAD51B/RAD51C/RAD51D/XRCC2 heterotetramer . Mutation of any one of the five paralog genes prevents subnuclear assembly of recombinase at damaged sites and renders cells 30-100 fold sensitive to DNA cross-linking drugs . Phage display was used to isolate peptides that bind the paralog XRCC3 . Sequences of binding peptides showed similarity to residues 14-25 of RAD51C protein . Point mutations in this region of RAD51C altered its interaction with both XRCC3 and RAD51B in a two-hybrid system . A synthetic peptide composed of residues 14-25 of RAD51C fused to a membrane transduction sequence {protein transduction domain 4 (PTD4)}, inhibited subnuclear assembly of RAD51 recombinase, and sensitized Chinese hamster ovary cells to cisplatin when added to growth medium . These results suggest that residues 14-25 of RAD51C contribute to a "hot spot" used in both XRCC3-RAD51C and RAD51B-RAD51C interactions . Peptide-based inhibition of homologous recombination may prove useful for improving the efficacy of existing cancer therapies.

Semin Cell Dev Biol, 2004 Jun, 15(3), 337 - 45
Making new beta cells from stem cells; Colman A; In 2000, Shapiro et al . provided compelling "proof of principle" data showing that the transplantation of human islets, purified from cadaveric material, could restore severely diabetic, Type 1 patients to insulin independence . This demonstration prompted renewed efforts to find an alternative and sustainable source of surrogate islet cells for cell therapy . Experiments involving adult ductal and liver "stem" cells, or embryonic stem cells, are prominent amongst these endeavors and are reviewed in this article . Whilst there are many published claims to success in converting ES cells into insulin secreting, glucose responsive cells, all require careful reinterpretation in the light of findings that cells can adsorb insulin present in growth media . It is likely that work with adult cells is less prone to this potential artifact and significant progress has been made in producing insulin-secreting cells . Assessment of in vivo function in the surrogate cells is most frequently made using cell transplantation into toxin-induced, diabetic mice, but this model is rarely used to maximal advantage . In many cases, it remains unclear whether reductions in the hyperglycemia result from insulin secretion from the transplanted cells or are due to recovery of endogenous islet function . In this latter context, experiments are reviewed where endogenous stimulation of recovery is engendered even by irradiated donor cells.

Yan Ke Xue Bao, 2004 Mar, 20(1), 42 - 7
Muscarinic receptor agonists protect cultured bovine trabecular meshwork cells against apoptosis induced by dexamethasone; Gu Y et al.; PURPOSE: To study whether muscarinic receptor agonists can protect cultured bovine trabecular meshwork cells against apoptosis induced by dexamethasone . METHODS: The third to fifth passages of bovine trabecular meshwork cells were grown to confluence and incubated for 1-14 days in growth media with dexamethasone or pretreatment of pilocarpine or carbachol . The cultures were evaluated for apoptosis by phase-contrast microscopy, fluorescence microscopy, DNA laddering and flow cytometric analysis . RESULTS: Dexamethasone (0.24-0.96 mmol.L-1) induced apoptosis of trabecular meshwork cells in a dose and time-dependent manner . Before 0.48 mmol.L-1 dexamethasone-treatment, 1.84 mmol.L-1 of pilocarpine or 2.74 mmol.L-1 of carbachol added could significantly reduce apoptotic percentage . CONCLUSION: Muscarinic receptor agonists can protect cultured bovine trabecular meshwork cells against apoptosis induced by dexamethasone.

J Glaucoma, 2004 Jun, 13(3), 200 - 9
Antiglaucoma eye drop pulses--increased interleukin-6 secretion by Tenon's capsule fibroblast cultures; Koh SW et al.; PURPOSE: Long-term antiglaucoma eye drop therapy prior to trabeculectomy is a risk factor for surgical failure resulting from Tenon's capsule fibrosis at the fistula site . The study tested the hypothesis that secretion of the proinflammatory cytokine interleukin (IL)-6 by wounded Tenon's capsule-fibroblasts is elevated by prior long-term antiglaucoma eye drop treatment . METHODS: Fibroblast cultures were established from Tenon's capsule biopsies during trabeculectomy . Twice daily and for four and a half days, confluent secondary (2-4 passages) cultures were treated (30 minutes at 37 degrees C) with the following drugs (diluted at 1:400-1:100): 0.2% brimonidine-tartrate (Alphagan), 2.0% dorzolamide-HCl (Trusopt), 0.5% timolol-maleate (Timoptic), 2.0% dorzolamide-HCl/0.5% timolol-maleate (Cosopt), 2 and 4% pilocarpine-HCl (Akarpine and Pilocar), 0.005% latanoprost (Xalatan), placebos for Trusopt and Timoptic, and 0.01% benzalkonium chloride . Subsequently, cultures were wounded by removing cells grown on half of each culture dishes along with the medium and conditioned for 20 hours in serum-free growth medium, which was then collected for ELISA for IL-6 (and TNF-alpha and IL-1 beta) . Cultures were grown for four additional days to show the maintenance of culture sterility . RESULTS: Latanoprost, pilocarpine-HCl, and timolol-maleate increased IL-6 levels in the conditioned medium in a dilution factor-dependent manner (P < 0.05, ANOVA) . IL-6 concentrations were increased most significantly by latanoprost and were (pg/ml; mean +/- SEM; N = 3 cultures) 186 +/- 37, 187 +/- 33, 295 +/- 46 and 336 +/- 76 in cultures treated at 1:400, 1:250, 1:150, and 1:100 dilutions, respectively, whereas those of six control cultures averaged 80 +/- 9 . Benzalkonium chloride, brimonidine-tartrate, dorzolamide-HCl, Cosopt placebo, Timoptic placebo, and dorzolamide-HCl/timolol-maleate did not significantly elevate IL-6 concentrations . IL-1 beta and TNF-alpha were not detected in the medium of control cultures and those treated with pilocarpine (1:200) . CONCLUSIONS: The present study demonstrated for the first time that the level of IL-6 secretion by wounded Tenon's capsule fibroblast cell cultures was increased by repeat pulsing of these cultures with some, but not all, antiglaucoma eye drops prior to wounding.

Arterioscler Thromb Vasc Biol, 2004 Jul, 24(7), 1217 - 22 Epub 2004 Apr 29.
AIF-1 expression modulates proliferation of human vascular smooth muscle cells by autocrine expression of G-CSF; Chen X et al.; OBJECTIVE: Allograft inflammatory factor-1 (AIF-1) is associated with vascular smooth muscle cell (VSMC) activation and vascular injury . The purpose of this study was to characterize the molecular mechanism of AIF-1 growth-enhancing effects in human VSMC . METHODS AND RESULTS: Primary human VSMCs were stably transduced with AIF-1 retrovirus (RV) . Impact on cell growth was evaluated by the increase in cell number, and the effects on gene expression were determined by cDNA microarray analysis . AIF-RV overexpressing cells grew significantly more rapidly than empty-RV control cells in growth medium and serum-reduced medium (P<0.01 and 0.02, respectively) . cDNA microarray analysis and Western blotting on serum-starved AIF-1-transduced VSMCs identified increased mRNA expression of several cell cycle proteins and, surprisingly, the cytokine G-CSF . Addition of G-CSF caused a 75% increase in proliferation of VSMCs in the absence of serum growth factors . The proliferative effects of AIF-1 were abrogated by neutralizing antibodies to G-CSF (P<0.05), and AIF-1-transduced VSMCs are chemotactic for human monocytes . Increased expression of G-CSF and colocalization with AIF-1 positive cells were seen in diseased, not normal human coronary arteries . CONCLUSIONS: This study indicates that AIF-1 enhances VSMC growth by autocrine production of G-CSF, and AIF-1 expression may influence VSMC-inflammatory cell communication.

Yeast, 2004 Apr 30, 21(6), 519 - 30
Differential regulation by glucose and fructose of a gene encoding a specific fructose/H+ symporter in Saccharomyces sensu stricto yeasts; Rodrigues de Sousa H et al.; Saccharomyces cerevisiae transports fructose through a facilitated diffusion system common to other hexoses and mediated by the Hxt proteins . The related species S . pastorianus (carlsbergensis) and S . bayanus produce, in addition, a specific fructose/H(+) symporter . We have previously cloned a gene (FSY1) encoding the active fructose symporter from S . pastorianus PYCC 4457 . Expression of Fsy1p in a S . cerevisiae mutant (hxt-null) devoid of the facilitated diffusion system allows growth on fructose but not on glucose . Here we present results concerning the regulation of Fsy1p expression, both in S . pastorianus and S . bayanus, where it occurs naturally, and in suitably engineered S . cerevisiae transformants . To that purpose, we made use of both Northern blot analysis and a Fsy1p-GFP fusion protein . The expression of Fsy1p is strongly regulated by both the carbon source and its concentration in the growth medium . In S . pastorianus, as well as in S . bayanus, very low concentrations of either fructose or glucose induced expression but higher sugar concentrations prevented transcription of the gene . Glucose was considerably more effective than fructose in repressing FSY1 expression . Proper regulation of the gene in S . cerevisiae seems to be exquisitely dependent on sugar transport . Analysis of Fsy1 expression in S . cerevisiae mutants shows that repression is mainly dependent on Mig1p, the final effector of the main glucose repression pathway . Interestingly, Mig1p also seems to mediate repression of FSY1 expression by high maltose concentrations .

Yeast, 2004 Apr 30, 21(6), 463 - 71
Screening for novel essential genes of Saccharomyces cerevisiae involved in protein secretion; Davydenko SG et al.; We describe here a screening procedure devised for searching new genes involved in protein secretion in Saccharomyces cerevisiae . The screening procedure takes advantage of yeast strains constructed within the EUROFAN project, in which the promoters of the novel essential genes were replaced by the doxycycline-regulated tetO(7)-CYC1 promoter . This promoter is active in normal growth medium but results in downregulation of the gene in the presence of doxycycline . The yeast cells were grown in the presence or absence of doxycycline, and both the growth and secretion of the heat shock protein, Hsp150p, into the culture medium were determined . In seven strains there was a specific effect on protein secretion . In a strain in which the RPN5 gene was downregulated, the level of secreted Hsp150p was increased compared to the control culture . When RER2 was downregulated, cells secreted Hsp150p that was not of the mature size . In five strains, secretion was more severely reduced than cell growth . One of these downregulated genes, YGL098w, was recently reported to encode an ER-located t-SNARE, USE1 . Four of the genes detected, NOG2, NOP15, RRP40 and SDA1, encode proteins involved in ribosome assembly, suggesting a possible new signalling pathway between ribosome biogenesis and production of secreted proteins . The results obtained here indicate that the present screen could be successfully used in larger scale to identify novel secretion-related genes .

J Agric Food Chem, 2004 May 5, 52(9), 2615 - 22
Application of high-Cu compost to dill and peppermint; Zheljazkov VD et al.; A controlled environment experiment was conducted to determine the effect of amending soil with various rates of high-Cu compost (0, 20, 40, and 60% compost/soil by volume) on dill (Anethum graveolens L.) and peppermint (Mentha X piperita L.) yields, on fractionation of Cu and Zn in soils, on elemental composition of soil and tissue, and on the essential oils . The compost contained about 2000 mg kg(-)(1) of Cu . Dill yields were greatest in the 20 or 40% treatments, but peppermint yields were greatest in the 20% treatment . Compost additions increased soil pH and electrical conductivity (EC), HNO(3) extractable soil B, Ca, K, Mg, Mn, P, S, Na, and Pb . Additions of high-Cu compost to soil increased tissue P, S, and Na in both crops and Mn, Mo, and Zn in dill but decreased tissue Ca, Cd, and Fe in both crops and Mn, Mo, and Zn in peppermint, increased Cu in all soil fractions including exchangeable, and increased tissue Cu of dill and peppermint as compared to unamended soil . Addition of 60% of high-Cu compost to soil resulted in 760-780 mg kg(-)(1) Cu in the growth medium . Nevertheless, Cu content in both crops reached only 12 mg kg(-)(1) DW in the 60% compost treatment, which is below the toxicity levels for plants and below the upper chronic dietary exposure for animals . The application of high-Cu compost altered chemical composition of dill and peppermint essential oils, but oils were free of Cu, Zn, Cd, Ni, Cr, and Pb . Results from this study suggest that mature composts with concentrations of Cu and Zn of 2008 and 321 mg/kg, respectively, can be used as a soil conditioner without risk for phytotoxicity or risk of increasing the normal range of Cu and Zn in crop tissue . However, the long-term effect of the accumulation of heavy metals in soils following repeated compost applications needs to be carefully considered.

Neuroreport, 2004 Jan 19, 15(1), 5 - 8
Hepatocyte growth factor promotes neuronal differentiation of neural stem cells derived from embryonic stem cells; Kato M et al.; We previously reported that hepatocyte growth factor (HGF) promoted proliferation of neurospheres and neuronal differentiation of neural stem cells (NSCs) derived from mouse embryonic brain . In this study, spheres from mouse embryonic stem (ES) cells were generated by floating culture following co-culture on PA6 stromal cells . In contrast to the behavior of the neurospheres derived from embryonic brain, addition of HGF to the growth medium of the floating cultures decreased the number of spheres derived from ES cells . When spheres were stained using a MAP-2 antibody, more MAP-2-positive cells were observed in spheres cultured with HGF . When HGF was added to the growth and/or differentiation medium, more MAP-2-positive cells were also obtained . These results suggest that HGF promotes neuronal differentiation of NSCs derived from ES cells.

Mol Cell Proteomics, 2004 Jul, 3(7), 729 - 35 Epub 2004 Apr 21.
Quantitative cancer proteomics: stable isotope labeling with amino acids in cell culture (SILAC) as a tool for prostate cancer research; Everley PA et al.; Microarrays have been the primary means for large-scale analyses of genes implicated in cancer progression . However, more recently a need has been recognized for investigating cancer development directly at the protein level . In this report, we have applied a comparative proteomic technique to the study of metastatic prostate cancer . This technology, termed stable isotope labeling with amino acids in cell culture (SILAC), has recently gained popularity for its ability to compare the expression levels of hundreds of proteins in a single experiment . SILAC makes use of (12)C- and (13)C-labeled amino acids added to the growth media of separately cultured cell lines, giving rise to cells containing either "light" or "heavy" proteins, respectively . Upon mixing lysates collected from these cells, proteins can be identified by tandem mass spectrometry . The incorporation of stable isotopes also allows for a quantitative comparison between the two samples . Using this method, we compared the expression levels for more than 440 proteins in the microsomal fractions of prostate cancer cells with varying metastatic potential . Of these, 60 were found elevated greater than 3-fold in the highly metastatic cells, whereas 22 were reduced by equivalent amounts . Western blotting provided further confirmation of the mass spectrometry-based quantification . Our results demonstrate the applicability of this novel approach toward the study of cancer progression using defined cell lines.

J Gen Microbiol, 1991 Dec, 137 ( Pt 12), 2787 - 96
Age-dependent changes in extracellular proteins, aminopeptidase and proteinase activities in Frankia isolate BR; Muller A et al.; To investigate protein secretion by the nitrogen-fixing actinomycete Frankia isolate BR, we designed a rapid DEAE adsorption, salt elution and Biogel P6DG desalination method to concentrate protein from the growth medium . Secreted proteins reached a maximum concentration (5.6 gm l-1) in the medium at growth arrest . Analysis by SDS-PAGE detected up to 63 extracellular polypeptides when Frankia cells were grown under stirred conditions in BAP medium supplemented with phosphatidylcholine and MES buffer and 65 proteins in stirred BAP media alone . The pattern of extracellular polypeptides changed during growth . Several extracellular proteolytic activities were detected and compared with intracellular ones . The substrate specificity of the extracellular and intracellular aminopeptidase activities were the same . Also, the electrophoretic migration patterns of secreted and intracellular aminopeptidases could not be distinguished . Secretion of the proline-specific aminopeptidase FAP proteinase (PF) were secreted: 10 had the same electrophoretic mobility as their intracellular counterparts after SDS-gelatine-PAGE while five (PF - 39.5, PF - 38.5, PF - 36.5, PF - 25.5 and PF - 20.5 kDa) had a different electrophoretic mobility and, therefore, appeared to be exclusively extracellular . At least seven extracellular proteinases appeared to increase coordinately in activity shortly before growth arrest.

Arch Oral Biol, 2004 Jun, 49(6), 485 - 92
Effect of strain on bone nodule formation by rat osteogenic cells in vitro; Visconti LA et al.; The purpose of this study was to assess in vitro bone nodule formation by cells exposed to a range of microstrain, at a sub-optimal oscillation frequency for bone formation . Fetal rat calvarial cells experienced a Flexercell regimen within either FLEX I (deformable) or FLEX II (non-deformable) substrates . Cells in FLEX I plates were exposed to growth medium only; those in FLEX II plates were exposed to either growth medium only, or growth medium + 10(-7) M IGF-1 . Cell numbers were assessed from 1 to 6 days . Other cells were exposed to the Flexercell regimen (-2 kPa, 0.05 Hz) for 1-3 (Group 1), 3-6 (Group 2), 1-9 (Group 3) or 10-15 (Group 4) days and were maintained, at other times, under standard conditions . After 21 days, nodules were counted within each well and within the compression, <999, 1000-4900, 5000-9999, 10,000-14,999 and 15,000-25,000 microstrain regions of the FLEX I membrane . Cyclic deformation inhibited cell numbers from 1 to 6 days, compared to control or IGF-1 groups (P<0.001) . The number of nodules in Groups 2 and 4 were greater than Groups 1 or 3 (P<0.001), but not different from control or IGF-1 groups . Compression or tensile microstrain significantly affected nodule formation in all groups, with Group 4 producing more nodules than other groups in most microstrain regions . Thus, the number of bone nodules produced by osteogenic cell cultures exposed to cyclic deformation was significantly affected by the timing of initiation and the characteristics and magnitude of the deformation regimen.

J Cell Physiol, 2004 Jun, 199(3), 364 - 70
Senescence occurs with hTERT repression and limited telomere shortening in human oral keratinocytes cultured with feeder cells; Kang MK et al.; We investigated the phenotypic and molecular alterations in normal human oral keratinocytes (NHOK) during in vitro replication in two different culture conditions . The cells were cultured either in chemically defined Keratinocyte Growth Medium (KGM) without feeder layers or in serum-containing flavin-adenine dinucleotide (FAD) medium with feeder layers . Primary NHOK underwent 22 +/- 3 population doublings (PDs) in KGM and 42 +/- 4 PDs in FAD medium, reflecting 52% increase in replication capacity with feeder layers . In both culture conditions, exponentially replicating NHOK demonstrated telomerase activity and expression of human telomerase reverse transcriptase (hTERT) gene . Telomerase activity and hTERT expression were rapidly diminished in senescing NHOK, which exhibited small decrease of telomere length for the remaining limited cellular replications until the complete arrest of cell division . However, telomere length in senescent NHOK was 6.7 +/- 0.5 kilobase pairs (kbps), significantly longer than that (5.12 kbps) of senescent human fibroblasts . The onset of senescence was accompanied with marked induction of p16(INK4A), and this occurred in both culture systems using either KGM or FAD medium . These results indicate that replicative senescence of NHOK is associated with loss of telomerase activity followed by limited telomere shortening .

Ann Vasc Surg . 2004 Apr 21; {Epub ahead of print}
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.

Environ Pollut, 1996, 94(2), 217 - 25
An assessment of sulphide oxidation in abandoned base-metal tailings, Te Aroha, New Zealand; John Morrell W et al.; Tailings from the Tui base-metal mine were characterized using a variety of techniques including scanning electron microscopy (SEM) and flame atomic absorption spectroscopy (FAAS), to assess their potential for use as a plant growth medium . With the notable exception of Pb (10 568 mg kg(-1)), 'total' metal concentrations in the surface tailings (0-200mm) were relatively low (Cu, 113; Fe, 3660; Zn, 486 mg kg(-1)) . The theoretical acid generating potential (TAGP) and 'total' concentrations of Cu, Fe and Zn of the tailings, were found to increase greatly with depth, reflecting an increase in the abundance of chalcopyrite (CuFeS(2)), pyrite (FeS(2)) and sphalerite (ZnS), as detected by X-ray diffraction (XRD) analysis . SEM micrographs indicate that the distribution of sulphide minerals in the tailings was originally uniform with depth . The depletion of Cu, Fe and Zn in the surface tailings is considered to be a result of sulphide oxidation, as evidenced by the craggy and highly irregular morphology of the sulphide particles and the high hydrogen ion activity (pH 2.3-4.0) in this zone . The persistence of high concentrations of acid-generating sulphide minerals between 200 and 600 mm has important implications in determining strategies for revegetating the tailings.

Environ Pollut, 2000 Mar, 107(3), 465 - 72
The effect of ozone on below-ground carbon allocation in wheat; McCrady JK et al.; Short-term (14)CO(2) pulse and chase experiments were conducted in order to investigate the effect of ozone on below-ground carbon allocation in spring wheat seedlings (Triticum aestivum L . 'ANZA') . Wheat seedlings were grown in a sand-hydroponic system and exposed to either high ozone (38-40 ppm-h) or low ozone (23-31 ppm-h) for 21 days in a series of replicated experiments . Following the ozone exposures, the plants were pulsed with (14)CO(2) and allocation of (14)C-labeled photosynthate was measured in the plant and growth media . Soluble root exudates were measured, without disturbing the plant roots, 24 h after the (14)CO(2) pulse . Shoot biomass was reduced by 17% for the high ozone and 9% for the low ozone exposures, relative to control treatments . Root biomass was reduced by 9% for the high ozone exposures, but was not significantly different than the controls for the low ozone . The amount of (14)C activity in the shoot and root tissue 24 h after the (14)CO(2) pulse, normalized to tissue weight, total (14)CO(2) uptake, or the total (14)C retention in each plant, was not affected by either high or low ozone exposures . The amount of (14)C activity measured in the growth media solution surrounding the roots increased 9% for the high ozone exposures, and after normalizing to root size or root (14)C activity, the growth media solution (14)C activity increased 29 and 40%, respectively . Total respiration of (14)CO(2) from the ozone-treated plants decreased, but the decrease was not statistically significant . Our results suggest that soluble root exudation of (14)C activity to the surrounding rhizosphere increases in response to ozone . Increased root exudation to the rhizosphere in response to ozone is contrary to reports of decreased carbon allocation below ground and suggests that rhizosphere microbial activity may be initially stimulated by plant exposure to ozone.

Environ Pollut, 1988, 52(3), 203 - 19
The effect of thallium on the growth of Lemna minor and plant tissue concentrations in relation to both exposure and toxicity; Kwan KH et al.; The effect of thallium on frond production and the rates of increase in surface area and fresh weight in Lemna minor were examined in terms of concentrations of the element in the growth medium and in the plant . The toxicity indices, EC(50) and threshold concentration, indicate that curtailment of frond expansion occurs at a lower thallium exposure than inhibition of frond multiplication . Production of smaller fronds, rather than chlorosis, was the most notable initial response to thallium stress and, below the threshold of toxicity, enhanced multiplication rates were recorded . Subsequent recovery in thallium-free medium was significant in the region of EC(50) exposures and below, but very little recovery occurred at higher exposures . Lemna minor has a remarkable capacity to accumulate thallium, and accumulation factors of 88 x 10(3) at low exposures, and 6 x 10(3) at high exposures, were recorded, which would suggest some form of active uptake.

Environ Pollut, 1990, 67(1), 61 - 77
The impact of aluminium on green algae isolated from two hydrochemically different headwater streams, Bavaria, Germany; Lindemann J et al.; Two strains of green algae (Scenedesmus sp . and Chlorella sp.) have been isolated from two headwater streams differing in stream chemistry (Eger river: acidic, low alkalinity; Puttlach river: slightly alkaline, high alkalinity) . In this study the growth response of these indigenous algae to increased concentrations of aluminium (Al) is investigated . A semi-continuous culture technique was used for Al-toxicity studies . Algal response was determined by calculating growth rates from turbidity and cell counts . Those Al-species which are well known to be toxic were estimated by equilibrium calculations using the WATEQ computer program (Truesdall & Jones, 1973) . The pH-value of the culture media was usually pH=5, except for one of the test series . Tested concentrations ranged from c=4 to 220 micromol litre(-1) . The isolated strains of green algae were highly sensitive to increased Al-concentrations . The strain isolated from the Puttlach river was more sensitive than the Eger river algae . A total growth inhibition occurs at Al-concentrations of c=4 micromol litre(-1) if the whole Al was added at once . If Al was added gradually into the growth media the response of the algae was delayed . This is due to Al-enrichment in cells . In our long term toxicity studies, growth inhibition occurs even at nearly neutral pH-conditions (pH=6.5) although Al toxicity is expected at pH-values less than pH=5.5 . This new result confirms the need of long-term studies in continuous cultures under simulated natural conditions . This might be the only way to achieve valid conclusions about the fate and the toxicity of environmental pollutants.

Environ Pollut, 1990, 67(3), 279 - 86
The in-vitro response of pollen germination and tube length to different types of acidity; Paoletti E et al.; Pollen germination and tube growth are among the most sensitive responses to atmospheric pollution . Both these are inhibited by the acidity of the growth medium . Pollen grains from two species (Pinus cembra L . and Sambucus nigra L.) were germinated in media over a range of pHs (5.0, 4.5, 4.0, 3.5, 3.0 and 2.5) and six types of acidity (H(2)SO(4), HNO(3), H(2)SO(4): HNO(3) in 1:1, 2:1, 3:1 and 5:1 ratio) . Pollen of the Gymnosperm is shown to be more resistant to acidity in the medium . Sulphuric acid alone and the ratio 2:1 with nitric acid are demonstrated to be the more harmful for P . cembra and S . nigra, respectively . The latter species was sensitive to all mixtures, particularly in respect to germination percentage.

Environ Pollut, 1994, 86(2), 201 - 6
90Sr uptake by Pinus ponderosa and Pinus radiata seedlings inoculated with ectomycorrhizal fungi; Entry JA et al.; Strontium-90 ((90)Sr) is a radionuclide characteristic of fallout from nuclear reactor accidents and nuclear weapons testing . Prior studies have shown that Pinus ponderosa and P . radiata seedlings can remove appreciable quantities of (90)Sr from soil and store it in plant tissue . In this study, we inoculated P . ponderosa and P . radiata seedlings with one of five isolates of ectomycorrhizal fungi . Inoculated and noninoculated (control) seedlings were compared for their ability to remove (90)Sr from an organic growth medium . Seedlings were grown in a growth chamber in glass tubes containing 165 cm(3) of sphagnum peat moss and perlite (1 : 1 (v/v)) and, except in the controls, the fungal inoculum . After 3 months, 5978 Bq of (90)Sr in 1 ml of sterile, distilled, deionized water was added . Seedlings were grown for an additional month and then harvested . P . ponderosa seedlings with ectomycorrhizae accumulated 3.0-6.0% of the (90)Sr; bioconcentration ratios (Bq (90)Sr cm(-3) plant tissue/Bq (90)Sr cm(-3) growth medium) ranged from 98-162 . Ectomycorrhizal P . radiata seedlings accumulated 6.0-6.9% of the (90)Sr; bioconcentration ratios ranged from 88-133 . Nonmycorrhizal P . ponderosa and P . radiata seedlings accumulated only 0.6 and 0.7% of the (90)Sr and had bioconcentration ratios of 28 and 27, respectively . Ectomycorrhizal P . ponderosa and P . radiata seedlings are able to remove 3-5 times more (90)Sr from contaminated soil than seedlings without ectomycorrhizae.

Assay Drug Dev Technol, 2004 Feb, 2(1), 31 - 8
A simple quantitative method for evaluation of angiogenesis activity; Wang HS et al.; Angiogenesis plays a major role in many physiological and pathological processes . Pathological development of diseased conditions like growth and metastasis of solid tumors and psoriasis is associated with angiogenesis . Assays developed, thus far, for evaluation of angiogenesis activity are qualitative or semiquantitative . In vivo angiogenesis assays are more physiologically relevant than in vitro models and, however, time-consuming, labor-intensive, and expensive . The ex vivo rat aorta tube formation model has been demonstrated to correlate well to the physiological conditions . The present study established a reproducible and quantitative assay for evaluating angiogenesis with rat aorta ring cultures . Rat thoracic aortas were harvested, cross-sectioned into rings of 1-mm thickness using a set of aligned blades, and cultured in a three-dimensional extracellular matrix . Endothelial cells outgrow consistently from the aorta rings cultured in endothelial cell growth medium . Angiogenic activity was quantified by a colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazolium/phenazine methosulfate method . The colorimetric assay was reproducible, and its results were compared in parallel with that of the imaging analysis method . IC(50) values of several known antiangiogenics, SU5416, suramin, paclitaxel, and 2-methoxyestradiol, were determined and were comparable to those obtained using the imaging analysis method . We have established a simple, reproducible, and quantitative assay for evaluation of angiogenesis activity with the cultured rat aorta ring, which can be used to screen for angiogenics and angiostatics.

Kidney Int, 2004 Apr, 65(4), 1252 - 61
Testosterone promotes apoptotic damage in human renal tubular cells; Verzola D et al.; BACKGROUND: Apoptosis is a mode of cell death that participates in the kidney physiologic remodeling processes and is thought to contribute to cell loss and kidney structural damage in chronic renal diseases . Gender is one factor which contributes to accelerated nephron loss, with progression more rapid in men than in women in diabetic and nondiabetic chronic renal diseases . Mechanisms by which androgens may cause higher rate of progression of chronic renal diseases in men are poorly explored . METHODS: In this study, to investigate the role of androgens on apoptotic damage and its associated mechanisms, we examined the effects of testosterone (T) (0.1 nmol/L to 1 micromol/L) on apoptosis, and apoptosis-related proteins in a proximal human tubule cell line (HK-2 cells) . Additional experiments were performed in primary cultures of proximal tubular epithelial cells (PTECs) . Cells were grown to subconfluence in normal growth medium, and apoptotic damage was induced by serum deprivation for 24 to 48 hours . Cycloheximide, flutamide (a T-receptor antagonist), 17-beta estradiol, or caspase inhibitors were added to cultures that were successively processed for terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end-labeling (TUNEL) analysis, annexin V/propidium iodide staining, immunofluorescence, or immunoblots to identify effects and apoptotic pathways that could be modulating cell survival . RESULTS: Both morphologic analysis by annexin V/propidium iodide staining and TUNEL showed that physiologic T levels (1 to 10 nmol/L) induced a significant increase in apoptosis both in HK-2 cells and PTECs . In both types of cell lines pretreatment with the androgen receptor antagonist flutamide prevented the T-induced apoptosis . T-induced apoptosis was enhanced by treatment with cycloheximide and prevented by 17beta-estradiol . Fas, Fas ligand (FasL), and Fas-associating death domain containing protein (FADD) were clearly up-regulated within 48 hours of T treatment in HK-2 cells . Also, T significantly increased the expression of Bax protein (P < 0.01 vs . control) (an effect which was blocked by flutamide), and decreased the expression of Bcl-2 . Western blot analysis showed that caspase-3 was activated . Moreover, cleavage into an 85-kD poly(ADP-ribose) polymerase-1 (PARP-1) terminal breakdown product was detectable . The changes in cellular morphology induced by T at 48 hours were no longer observed after the addition of caspase-8, caspase-9, and caspase-3 inhibitors to the culture medium . CONCLUSION: These results indicate that T increases the permissiveness of proximal tubule kidney cells to apoptotic effects by triggering an apoptotic pathway involving caspase activation, Fas up-regulation, and FasL expression, thus potentially interacting with mechanisms of cell loss which have been already shown to be activated in chronic renal diseases . This is consistent with a role for T in promoting renal injury in men.

Virus Res, 2004 Jun 15, 102(2), 221 - 4
Inactivation of selected picornaviruses by high hydrostatic pressure; Kingsley DH et al.; The potential of high hydrostatic pressure processing (HPP) to inactivate Aichi virus (AiV), human parechovirus-1 (HPeV-1), and the coxsackievirus strains A9 and B5 was investigated . For coxsackievirus A9 (CAV9), a 5-min HPP treatment in minimum essential growth medium (MEM) supplemented with 10% fetal bovine sera (FBS) resulted in 3.4-, 6.5-, and 7.6-log(10) tissue culture infectious dose (50%) (TCID(50)) reductions at 400, 500, and 600megaPascals (MPa), respectively . For HPeV-1, a 5-min treatment in MEM with 10% FBS resulted in reductions of 1.3-, 4.3-, and 4.6-log(10) TCID(50) at 400, 500, and 600MPa, respectively; however, AiV and coxsackievirus B5 (CBV5) in MEM supplemented with 2 and 10% FBS, respectively, remained fully infectious after a 5-min treatment at 600MPa . These data establish that different picornaviruses have widely variable pressure inactivation thresholds in response to HPP.

Biotechnol Bioeng, 2004 May 5, 86(3), 251 - 60
Metabolic pathway analysis of yeast strengthens the bridge between transcriptomics and metabolic networks; Cakir T et al.; Central carbon metabolism of the yeast Saccharomyces cerevisiae was analyzed using metabolic pathway analysis tools . Elementary flux modes for three substrates (glucose, galactose, and ethanol) were determined using the catabolic reactions occurring in yeast . Resultant elementary modes were used for gene deletion phenotype analysis and for the analysis of robustness of the central metabolism and network functionality . Control-effective fluxes, determined by calculating the efficiency of each mode, were used for the prediction of transcript ratios of metabolic genes in different growth media (glucose-ethanol and galactose-ethanol) . A high correlation was obtained between the theoretical and experimental expression levels of 38 genes when ethanol and glucose media were considered . Such analysis was shown to be a bridge between transcriptomics and fluxomics . Control-effective flux distribution was found to be promising in in silico predictions by incorporating functionality and regulation into the metabolic network structure .

Chemosphere, 2004 Jun, 55(9), 1159 - 68
Cadmium uptake and translocation in tumbleweed (Salsola kali), a potential Cd-hyperaccumulator desert plant species: ICP/OES and XAS studies; de la Rosa G et al.; Cadmium is a heavy metal, which, even at low concentrations, can be highly toxic to the growth and development of both plants and animals . Plant species vary extensively in their tolerance to excess cadmium in a growth medium and very few cadmium-tolerant species have been identified . In this study, tumbleweed plants (Salsola kali) grown in an agar-based medium with 20 mgl(-1) of Cd(II) did not show phytotoxicity, and their roots had the most biomass (4.5 mg) (P < 0.05) compared to the control plants (2.7 mg) as well as other treated plants . These plants accumulated 2696, 2075, and 2016 mg Cd kg(-1) of dry roots, stems, and leaves, respectively . The results suggest that there is no restricted cadmium movement in tumbleweed plants . In addition, the amount of Cd found in the dry leaf tissue suggests that tumbleweed could be considered as potential cadmium hyperaccumulating species . X-ray absorption spectroscopy studies demonstrated that in roots, cadmium was bound to oxygen while in stems and leaves, the metal was attached to oxygen and sulfur groups . This might imply that some small organic acids are responsible for Cd transport from roots to stems and leaves . In addition, it might be possible that the plant synthesizes phytochelatins in the stems, later coordinating the absorbed cadmium for transport and storage in cell structures . Thus, it is possible that in the leaves, Cd either exists as a Cd-phytochelatin complex or bound to cell wall structures . Current studies are being performed in order to elucidate the proposed hypothesis .

J Appl Microbiol, 2004, 96(5), 1117 - 23
Carbohydrate carbon sources induce loss of flocculation of an ale-brewing yeast strain; Soares EV et al.; AIMS: To identify the nutrients that can trigger the loss of flocculation under growth conditions in an ale-brewing strain, Saccharomyces cerevisiae NCYC 1195 . METHODS AND RESULTS: Flocculation was evaluated using the method of Soares, E.V . and Vroman, A . {Journal of Applied Microbiology (2003) 95, 325} . Yeast growth with metabolizable carbon sources (glucose, fructose, galactose, maltose or sucrose) at 2% (w/v), induced the loss of flocculation in yeast that had previously been allowed to flocculate . The yeast remained flocculent when transferred to a medium containing the required nutrients for yeast growth and a sole nonmetabolizable carbon source (lactose) . Transfer of flocculent yeast into a growth medium with ethanol (4% v/v), as the sole carbon source did not induce the loss of flocculation . Even the addition of glucose (2% w/v) or glucose and antimycin A (0.1 mg l(-1)) to this culture did not bring about loss of flocculation . Cycloheximide addition (15 mg l(-1)) to glucose-growing cells stopped flocculation loss . CONCLUSIONS: Carbohydrates were the nutrients responsible for stimulating the loss of flocculation in flocculent yeast cells transferred to growing conditions . The glucose-induced loss of flocculation required de novo protein synthesis . Ethanol prevented glucose-induced loss of flocculation . This protective effect of ethanol was independent of the respiratory function of the yeast . SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the elucidation of the role of nutrients in the control of the flocculation cycle in NewFlo phenotype yeast strains.

J Environ Qual, 2004 Mar-Apr, 33(2), 740 - 8
Bermudagrass sod growth and metal uptake in coal combustion by-product-amended media; Schlossberg MJ et al.; Coal combustion by-products (CCB) include fly ash and bottom ash and are generated nationally at rates of 10(8) Mg yr(-1) . Land applications of CCB have improved physicochemical properties of soil, yet inherent bulkiness and trace metal content of CCB often limit their use . Likewise, utilization of biosolids and manure as fertilizer can be problematic due to unfavorable nutrient ratios . A 2-yr field study evaluated environmental and technical parameters associated with CCB-organic waste utilization as growth media in turfgrass sod production . Experimental growth media formulated with CCB and organic waste and a sand-compost control mixture were uniformly spread at rates from 200 to 400 m3 ha(-1) and sprigged with hybrid bermudagrass {Cynodon dactylon (L.) Pers . x C . transvaalensis Burtt-Davy} . Leaf clippings were collected and analyzed for total elemental content each year . In Year 2, growth media samples were collected during establishment 47 and 84 days after planting (DAP) and viable Escherichia coli organisms were quantified . At harvest (99 or 114 DAP), sod biomass and physicochemical properties of the growth media were measured . During sod propagation, micronutrient and metal content in leaf clippings varied by growth media and time . After 47 d of typical sod field management, viable E . coli pathogens were detected in only one biosolids-amended plot . No viable E . coli were measured at 84 DAP . In both years, sod biomass was greatest in media containing biosolids and fly ash . Following installation of sod, evaluations did not reveal differences by media type or application volume . Using CCB-organic waste mixes at the rates described herein is a rapid and environmentally safe method of bermudagrass sod production.

J Environ Qual, 2004 Mar-Apr, 33(2), 458 - 64
Dredged Illinois River sediments: plant growth and metal uptake; Darmody RG et al.; Sedimentation of the Illinois River in central Illinois has greatly diminished the utility and ecological value of the Peoria Lakes reach of the river . Consequently, a large dredging project has been proposed to improve its wildlife habitat and recreation potential, but disposal of the dredged sediment presents a challenge . Land placement is an attractive option . Previous work in Illinois has demonstrated that sediments are potentially capable of supporting agronomic crops due to their high natural fertility and water holding capacity . However, Illinois River sediments have elevated levels of heavy metals, which may be important if they are used as garden or agricultural soil . A greenhouse experiment was conducted to determine if these sediments could serve as a plant growth medium . A secondary objective was to determine if plants grown on sediments accumulated significant heavy metal concentrations . Our results indicated that lettuce (Lactuca sativa L.), barley (Hordeum vulgare L.), radish (Raphanus sativus L.), tomato (Lycopersicon lycopersicum L.), and snap bean (Phaseolus vulagaris L . var . humillis) grown in sediment and a reference topsoil did not show significant or consistent differences in germination or yields . In addition, there was not a consistent statistically significant difference in metal content among tomatoes grown in sediments, topsoil, or grown locally in gardens . In the other plants grown on sediments, while Cd and Cu in all cases and As in lettuce and snap bean were elevated, levels were below those considered excessive . Results indicate that properly managed, these relatively uncontaminated calcareous sediments can make productive soils and that metal uptake of plants grown in these sediments is generally not a concern.

Appl Opt, 2004 Apr 1, 43(10), 2079 - 88
Design, construction, characterization, and application of a hyperspectral microarray scanner; Sinclair MB et al.; We describe the design, construction, and operation of a hyperspectral microarray scanner for functional genomic research . The hyperspectral instrument operates with spatial resolutions ranging from 3 to 30 microm and records the emission spectrum between 490 and 900 nm with a spectral resolution of 3 nm for each pixel of the microarray . This spectral information, when coupled with multivariate data analysis techniques, allows for identification and elimination of unwanted artifacts and greatly improves the accuracy of microarray experiments . Microarray results presented in this study clearly demonstrate the separation of fluorescent label emission from the spectrally overlapping emission due to the underlying glass substrate . We also demonstrate separation of the emission due to green fluorescent protein expressed by yeast cells from the spectrally overlapping autofluorescence of the yeast cells and the growth media.

Am J Physiol Heart Circ Physiol, 2004 Aug, 287(2), H512 - 7 Epub 2004 Apr 08.
Modulation of the vascular response to injury by autologous blood-derived outgrowth endothelial cells; Gulati R et al.; Delivery of a heterogeneous population of cells with endothelial phenotype derived from peripheral blood has been shown to improve vascular responses after balloon arterial injury in an endothelium-dependent manner . Refinement of culture techniques has enabled the generation of outgrowth endothelial cells (OECs), a homogeneous population of distinctly endothelial cells expanded from circulating progenitor cells . The present study tested the hypothesis that OEC delivery would confer vascular protection after balloon arterial injury in a rabbit model . Rabbit peripheral blood mononuclear cells (PBMCs) were cultured in endothelial growth medium for 4-5 wk, yielding proliferative OECs with distinct endothelial phenotype (morphology, incorporation of acetylated LDL, and expression of endothelial nitric oxide synthase and caveolin-1 but not CD14) . Animals underwent balloon carotid injury immediately followed by local delivery of autologous OECs for 20 min . Fluorescent-labeled OECs were detected in all layers at 4 wk, with immunostaining revealing maintenance of endothelial phenotype (von Willebrand factor-positive and RAM-11-negative) by luminal and nonluminal cells . To evaluate functional effects, additional animals received autologous OECs, saline, or freshly harvested PBMCs as noncultured cell controls by local dwell after balloon injury . Local OEC delivery improved endothelium-dependent vasoreactivity (P < 0.05 vs . saline and PBMC) and similarly reduced neointimal formation (P < 0.05 vs . saline and PBMC) . These data suggest that OECs can be detected in injured arterial segments at 4 wk . Moreover, delivery of OECs confers greater vascular protection than PBMCs or saline controls and may thus offer a novel, autologous strategy to limit the response to mechanical injury.

Hum Exp Toxicol, 2004 Feb, 23(2), 61 - 5
The radiation-induced bystander effect: evidence and significance; Azzam EI et al.; A multitude of biological effects observed over the past two decades in various in vivo and in vitro cell culture experiments have indicated that low dose/low fluence ionizing radiation has significantly different biological responses than high dose radiation . Exposure of cell populations to very low fluences of alpha-particles or incorporated radionuclides results in significant biological effects occurring in both the irradiated and nonirradiated cells in the population . Cells recipient of growth medium from irradiated cultures can also respond to the radiation exposure . This phenomenon, termed the 'bystander response', has been postulated to impact both the estimation of risks of exposure to ionizing radiation and radiotherapy . Amplification of radiation-induced cytotoxic and genotoxic effects by the bystander effect is in contrast to the observations of adaptive responses, which are generally induced following exposure to low dose, low linear energy transfer radiation and which tend to attenuate radiation-induced damage . In this article, the evidence for existence of radiation-induced bystander effects and our current knowledge of the biochemical and molecular events involved in mediating these effects are described . Potential similarities between factors that mediate the radiation-induced bystander and adaptive responses are highlighted.

Appl Biochem Biotechnol, 2004 Spring, 113-116, 161 - 71
Effects of carbon source on expression of alcohol oxidase activity and on morphologic pattern of YR-1 Strain, a filamentous fungus isolated from petroleum-contaminated soils; Robelo CR et al.; Soluble alcohol oxidase (AO) activity was detected in the supernatant fraction of a high-speed centrifugation procedure after ballistic cellular homo-genization to break the mycelium from a filamentous fungus strain named YR-1, isolated from petroleum-contaminated soils . AO activity from aerobically grown mycelium was detected in growth media containing different carbon sources, including alcohols and hydrocarbons but not in glucose . In previous work, zymogram analysis conducted with crude extracts from aerobic mycelium of YR-1 strain indicated the existence of two AO enzymes originally named AO-1 and AO-2 . In the present study, we were able to separate the AO-1 band into two bands depending on culture conditions, carbon source, and polyacrylamide gel electrophoresis (PAGE) separation conditions; the enzyme activity pattern in zymograms from cell-free extracts exhibited three different bands after native PAGE . New nomenclature was used for upper bands AO-1 and AO-2 and lower band AO-3, respectively . The expression of AO activity was studied in the absence of glucose in the culture media and in the presence of hydrocarbons or petroleum as sole carbon source, suggesting that AO expression could be subjected to two regulatory possibilities: carbon catabolite regulation by glucose and induction by hydrocarbons . The possibility of catabolic inhibition of AO by glucose in the active enzyme was also tested, and the results confirm that this kind of regulatory mechanism is not present in AO activity.

J Biol Chem, 2004 Jun 25, 279(26), 27428 - 39 Epub 2004 Mar 30.
A novel cAMP-response element, CRE1, modulates expression of nor-1 in Aspergillus parasiticus; Roze LV et al.; The level of aflatoxin accumulation in the filamentous fungus Aspergillus parasiticus is modulated by a variety of environmental cues . The presence of glucose (a preferred carbon source) in liquid and solid glucose minimal salts (GMS) growth media strongly stimulated aflatoxin accumulation . Peptone (a non-preferred carbon source) in peptone minimal salts (PMS) media stimulated only low levels of aflatoxin accumulation . Glucose stimulated transcription of the aflatoxin structural genes ver-1 and nor-1 to similar intermediate levels in liquid GMS, while on solid media, ver-1 transcription was stimulated to 20-fold higher levels than nor-1 . PMS liquid and solid media stimulated very low or non-detectable levels of transcription of both genes . Electrophoretic mobility shift analysis using a nor-1 promoter fragment (norR) and A . parasiticus cell protein extracts revealed specific DNA-protein complexes of different mobility on GMS and PMS solid and liquid media . An imperfect cAMP-response element, CRE1, was identified in norR that mediated formation of the specific DNA-protein complexes . Mutation in CRE1 or AflR1 (AflR cis-acting site) caused up to a 3-fold decrease in cAMP-mediated stimulation of nor-1 promoter activity on GMS agar . South-Western blot analysis identified a 32-kDa protein that specifically bound to norR . p32 could be co-immunoprecipitated by anti-AflR antibody and co-purified with an AflR-maltose-binding protein fusion demonstrating a physical interaction between AflR and p32 in vitro . We hypothesize that p32 assists AflR in binding to the nor-1 promoter, thereby modulating nor-1 gene expression in response to environmental cues.

Can J Microbiol, 2004 Feb, 50(2), 121 - 6
Biotransformation of the Streptomyces scabies phytotoxin thaxtomin A by the fungus Aspergillus niger; Lazarovits G et al.; Of several hundred microorganisms randomly selected from the environment, only a fungal isolate identified as Aspergillus niger van Tiegham var . niger was found to transform the phytotoxin thaxtomin A to much less toxic metabolites . The rate and extent of transformation of thaxtomin A was tested under a variety of conditions, including different growth media, biomass concentrations, incubation periods, and shaker speeds . Under optimum conditions the fungus converted thaxtomin A into two major and five minor metabolites . The two major metabolites and three of the five minor metabolites were fully characterized by a combination of mass spectral and nuclear magnetic resonance techniques . When assayed on aseptically produced mini-tubers, the major metabolites proved to be much less phytotoxic than thaxtomin A.

Biotechnol Lett, 2004 Feb, 26(3), 257 - 62
Isolation of a beta-carotene over-producing soil bacterium, Sphingomonas sp; Silva C et al.; A carotenoid-accumulating bacterium isolated from soil, identified as a Sphingomonas sp., grew at 0.18 h(-1) and produced 1.7 mg carotenoids g(-1) dry cell, among which beta-carotene (29% of total carotenoids) and nostoxanthin (36%) . A mutant strain, obtained by treatment with ethyl methanesulfonate, accumulated up to 3.5 mg carotenoids g(-1) dry cell . Accumulation of beta-carotene by this strain depended on the oxygenation of the growth medium, with maximal accumulation (89%) occurring under limiting conditions . Beta-carotene accumulation could be further enhanced by incubating the cells in the presence of glycerol (either not or only slowly assimilated) and yeast extract resulting in an accumulation of 5.7 mg beta-carotene g(-1) dry cell wt . The strain used lactose as carbon source with similar biomass and carotenoid production, providing a viable alternative use for cheese whey ultra-filtrate.

Biotechnol Lett, 2004 Feb, 26(3), 213 - 6
Improved technique for rapid and non-destructive in vitro differentiation between resistant and susceptible banana clones of Fusarium oxysporum f . sp . cubense; Companioni B et al.; Culture filtrates of Fusarium oxysporum f . sp . cubense were applied to field-grown banana leaves of susceptible and resistant clones . The difference in leaf lesions, measured after 48 h, varied from 13 to 51 mm2 depending on the composition of the growth medium.

J Food Prot, 2004 Mar, 67(3), 438 - 47
Ethylene modulates development and toxin biosynthesis in aspergillus possibly via an ethylene sensor-mediated signaling pathway; Roze LV et al.; Ethylene, a biologically active natural compound, inhibited aflatoxin accumulation by Aspergillus parasiticus on a solid growth medium in a dose-dependent manner at concentrations of 0.1 to 150 ppm . The activity of the nor-1 promoter (an early aflatoxin gene) was reduced to nondetectable levels by similar quantities of ethylene, suggesting that the inhibitory effect on toxin synthesis occurred, at least in part, at the level of transcription . The inhibitory effect of ethylene on aflatoxin accumulation was also observed when A . parasiticus was grown on raw peanuts . Under similar growth conditions and doses, ethylene strongly inhibited development of asci and ascospores in Aspergillus nidulans, with no detectable effect on Hulle cell formation, conidiation, or sterigmatocystin accumulation . During early growth, A . parasiticus and A . nidulans produced ethylene with approximately twofold higher quantities measured in continuous light than in the dark . 1-Methylcyclopropene (an inhibitor of ethylene receptors in plants), light, CO2, temperature, and growth medium composition altered the effect of ethylene on A . nidulans and A . parasiticus . These observations are consistent with the existence of an ethylene sensor molecule that mediates the function of an ethylene-responsive signaling pathway(s) in Aspergillus.

J Bacteriol, 2004 Apr, 186(7), 2156 - 63
Selenocysteine-containing proteins in anaerobic benzoate metabolism of Desulfococcus multivorans; Peters F et al.; The sulfate-reducing bacterium Desulfococcus multivorans uses various aromatic compounds as sources of cell carbon and energy . In this work, we studied the initial steps in the aromatic metabolism of this strictly anaerobic model organism . An ATP-dependent benzoate coenzyme A (CoA) ligase (AMP plus PPi forming) composed of a single 59-kDa subunit was purified from extracts of cells grown on benzoate . Specific activity was highest with benzoate and some benzoate derivatives, whereas aliphatic carboxylic acids were virtually unconverted . The N-terminal amino acid sequence showed high similarities with benzoate CoA ligases from Thauera aromatica and Azoarcus evansii . When cultivated on benzoate, cells strictly required selenium and molybdenum, whereas growth on nonaromatic compounds, such as cyclohexanecarboxylate or lactate, did not depend on the presence of the two trace elements . The growth rate on benzoate was half maximal with 1 nM selenite present in the growth medium . In molybdenum- and/or selenium-depleted cultures, growth on benzoate could be induced by addition of the missing trace elements . In extracts of cells grown on benzoate in the presence of {75Se}selenite, three radioactively labeled proteins with molecular masses of approximately 100, 30, and 27 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis . The 100- and 30-kDa selenoproteins were 5- to 10-fold induced in cells grown on benzoate compared to cells grown on lactate . These results suggest that the dearomatization process in D . multivorans is not catalyzed by the ATP-dependent Fe-S enzyme benzoyl-CoA reductase as in facultative anaerobes but rather involves unknown molybdenum- and selenocysteine-containing proteins.

Environ Technol, 2004 Jan, 25(1), 89 - 100
Detoxification of olive mill wastewater using superabsorbent polymers; Davies LC et al.; The detoxification of agro-industrial effluents using superabsorbent polymers is a new and innovative process . Olive mill wastewater constitutes a major environmental problem in Mediterranean countries due to the large volumes generated, the seasonality of the industry, and the high content of polyphenols and organic matter . The application of superabsorbent polymers allows olive mill wastewater to be used as a fertilizer, as it is immobilized, increasing the biological activity that decreases its phytotoxicity, thus making its water, organic matter and mineral content usable for plant nutrition . Various parameters that characterise olive mill wastewater were evaluated after absorption in 2 different superabsorbent polymers (SAP1 and SAP2) . The organic matter was equally distributed in both phases, while there was a concentration of protein and sodium in solution . The K:Na ratio decreased from 70:1 to 2:1 . The polyphenol desorption from the gel into solution was found to follow Fick's law . The mass transfer coefficients were 0.147 min(-1) and 0.0085 min(-1) for SAP1 and SAP2, respectively . Phytotoxicity tests were carried out with SAP2 . Olive mill wastewater in SAP2 with polyphenol concentrations up to 200 mg l(-1) revealed no phytotoxicity, and even stimulated Lepidium sativum growth, while olive mill wastewater without superabsorbent polymer revealed growth inhibition for all concentrations tested . Caffeic acid degradation by the immobilised biomass followed zero order kinetics . Degradation constants of 0.087 mg l(-1) min(-1) gSAP2(-1) and 1.156 mg l(-1) min(-1) gSAP2(-1) were found . Fungi that developed in the plant growth medium were identified as Aspergillus sp . and Penicillium sp.

Pharmazie, 2004 Feb, 59(2), 93 - 8
Synthesis and antifungal properties of compounds which target the alpha-aminoadipate pathway; Palmer DR et al.; Fungi synthesize lysine via the alpha-aminoadipate pathway, which is not found in plants or animals . This pathway has been proposed as a target for antifungal agents, but until now no reports have appeared to test this proposal . Hampering studies on the susceptibility of filamentous fungi such as those of the clinically important genus Aspergillus is the fact that growth quantitation is notoriously difficult . We have used the recently-reported XTT-based method of biomass quantitation to measure the susceptibility of Aspergillus nidulans strain A28 to growth suppression by novel compounds designed to target early steps in the alpha-aminoadipate lysine biosynthesis pathway, specifically those steps involving (R)-homocitrate and (2R,3S)-homoisocitrate . Three compounds show moderate inhibition of fungal growth, which can be partially restored by the presence of lysine in the growth medium.

Biochim Biophys Acta, 2004 Mar 11, 1697(1-2), 123 - 35
HPr kinase/phosphorylase, a Walker motif A-containing bifunctional sensor enzyme controlling catabolite repression in Gram-positive bacteria; Poncet S et al.; Carbon catabolite repression (CCR) in Gram-positive bacteria is regulated by the bifunctional enzyme HPr kinase/phosphorylase (HprK/P) . This enzyme catalyses the ATP- as well as the pyrophosphate-dependent phosphorylation of Ser-46 in HPr, a phosphocarrier protein of a sugar transport and phosphorylation system . HprK/P also catalyses the pyrophosphate-producing, inorganic phosphate-dependent dephosphorylation (phosphorolysis) of seryl-phosphorylated HPr (P-Ser-HPr) . P-Ser-HPr functions as catabolite co-repressor by interacting with the LacI/GalR-type repressor, catabolite control protein A (CcpA), and allowing it to bind to operator sites preceding catabolite-regulated transcription units . HprK/P thus indirectly controls the expression of about 10% of the genes of Gram-positive bacteria . The two antagonistic activities of HprK/P are regulated by intracellular metabolites, which change their concentration in response to the absence or presence of rapidly metabolisable carbon sources (glucose, fructose, etc.) in the growth medium . Biochemical and structural studies revealed that HprK/P exhibits no similarity to eukaryotic protein kinases and that it contains a Walker motif A (or P-loop) as nucleotide binding site . Interestingly, HprK/P has a structural fold resembling that in kinases phosphorylating certain low molecular weight substrates such as nucleosides, nucleotides or oxaloacetate . The structures of the complexes of HprK/P with HPr and P-Ser-HPr have also been determined, which allowed proposing a detailed mechanism for the kinase and phosphorylase functions of HprK/P.

Peptides, 2003 Nov, 24(11), 1815 - 21
Activity of dermaseptin K4-S4 against foodborne pathogens; Yaron S et al.; Dermaseptin S4 and its substituted derivative K(4)-S4 were investigated against various food-related pathogenic bacteria in culture media . K(4)-S4, but not the native peptide displayed significant growth inhibitory activity against all bacteria tested . Next, activity of K(4)-S4 against Escherichia coli O157:H7 was defined in terms of milieu dependencies . Salt-dependent kinetic studies in growth medium indicated that the peptide's antibacterial activity is maintained at fairly high (up to 600mM) NaCl concentrations but inhibited at higher concentrations . Similarly, antibacterial activity was reduced at high but not low pH conditions . Importantly, antibacterial activity was significantly maintained at temperatures lower than 37 degrees C and significantly enhanced at 42 degrees C . With respect to bactericidal kinetics, negative cultures were obtained in LB as well as in commercial apple juice, respectively, within 1 and 2h treatment, at twice the minimal inhibitory concentration (MIC) value . Overall, the data collected is indicative of a certain interest for dermaseptin derivatives as potential food preservatives.

Mech Ageing Dev, 2004 Mar, 125(3), 237 - 49
Enhancement of Fas-mediated apoptosis in ageing human keratinocytes; Wang X et al.; Cellular senescence and apoptosis are two metabolically related and seemingly synergistic processes that are involved in tissue maintenance and homeostasis, anti-tumor protection, and age-related diseases . Despite this apparent co-operativity, senescence can inhibit apoptosis in certain conditions . Here, we describe senescence-apoptosis relationships in human epidermal cells by comparing apoptosis-related effector concentrations in keratinocyte cultures and epidermal skin cells at various stages of ageing . Using western blots, flow cytometry, enzyme-linked immuno-sorbent assay (ELISA) and immunofluorescence, we determined the amounts of apoptotic effectors in aged cells compared to young ones, in parallel with beta-galactosidase activity at neutral pH (senescence-associated beta-galactosidase, SA beta-gal), found to be a good indicator of cellular ageing . We observed increased levels of several Fas-mediated apoptosis effectors (Fas, Fas ligand, FADD, FLICE), both in cell cultures at advanced passages and in skin cells of aged donors (above 45 years) . Furthermore, we found that while the pro-apoptotic p53 increased, the anti-apoptotic Bcl-2 declined . In spite of this, the extent of spontaneous apoptosis did not change in senescent keratinocyte cultures . The cells, however, became notably more susceptible to apoptosis when kept in exhausted growth medium, or upon Fas receptor activation by anti-Fas antibody binding . Our results are consistent with recent findings in senescent fibroblasts, showing that the death-signaling pathway is enhanced at senescence.

J Appl Microbiol, 2004, 96(4), 894 - 902
Calibration of the impedance method for rapid quantitative estimation of Escherichia coli in live marine bivalve molluscs; Dupont J et al.; AIM: Calibration of impedance measurement was performed vs the Association Francoise de Normalisation (AFNOR) MPN method with a view to rapid enumeration of Escherichia coli in live marine bivalve molluscs . METHODS AND RESULTS: Linear regression models between log10 MPN and detection time (DT) were adjusted for several shellfish types, growth media, and impedance instruments (BacTrac and Malthus systems) . Escherichia coli concentrations could be estimated from DT using a single regression line for BacTrac 4100 with M1 medium (R2 = 87.8%) and Malthus with M2 medium (R2 = 86.7%), and two regression lines for BacTrac 4110 with M2 medium (R2 = 86.4 and 88.2%) . The uncertainty of the predicted bacterial concentration was around +/-0.43 log unit for duplicate sample analysis . The impedance signal was attributable to E . coli in 99% of cases . All cultures containing E . coli produced an impedance signal with BacTrac 4100 and BacTrac 4110, whereas 5.6% did not exhibit a signal with Malthus . CONCLUSIONS: Impedance measurement is a possible alternative to the MPN method for rapid quantitative estimation of E . coli in live bivalve shellfish . SIGNIFICANCE AND IMPACT OF THE STUDY: The impedance method reduces analysis handling time considerably and is much easier to use than the MPN method . Moreover, results can be obtained within 5-10 h, allowing rapid intervention to ensure public health protection in case of shellfish contamination.

Tissue Eng, 2004 Jan-Feb, 10(1-2), 145 - 55
Serum-free growth medium sustains commitment of human articular chondrocyte through maintenance of Sox9 expression; Malpeli M et al.; Human articular cartilage heals poorly in adults and current surgical procedures do not provide long-term repair . Cell therapy and tissue engineering could become the treatment of choice, but suffer a major limitation as chondrocytes in vitro lose the differentiated phenotype . In vivo, the chondrogenic lineage is specified by transcription factor Sox9 . Thus, cell-based therapy could be successful if Sox9 expression and chondrogenic commitment of the expanded cells were preserved . To achieve this goal, we developed a serum-free medium that supports cell proliferation and preserves the differentiation potential . Indeed, expression of Sox9 is maintained when the conventionally used serum is substituted for by this defined supplement . Spontaneous cartilage formation after expansion in serum-free medium is obtained in vitro in a high-density pellet culture and confirmed in vivo in a functional assay in immunodeficient mice . By contrast, cells grown in serum lose the expression of Sox9 and fail to reform cartilage both in vitro and in vivo unless they are rescued by chondrogenic inducers such as transforming growth factor beta(1) and dexamethasone . Our data emphasize the importance of the microenvironment in modulating commitment, plasticity, and phenotype of chondrocytes, and provide an experimental system to study their physiological or pathological metabolism in a controlled context.

Tree Physiol, 2004 May, 24(5), 551 - 60
Physiological responses of wild type and putrescine-overproducing transgenic cells of poplar to variations in the form and concentration of nitrogen in the medium; Minocha R et al.; We determined: (a) the physiological consequences of overproduction of putrescine in transgenic poplar (Populus nigra x maximoviczii) cells expressing an ornithine decarboxylase transgene; and (b) effects of variation in nitrogen (N) concentration of the medium on cellular polyamine concentration in transgenic and non-transgenic cells . Cells grown in the presence of supplemental (to the normal concentrations of N sources in the growth medium) and reduced amounts of NH(4)NO(3) and KNO(3) were used to study effects on membrane permeability, mitochondrial respiratory activity, protein accumulation, growth rates and changes in cellular polyamine concentration . The N concentration of the MS medium was not a limiting factor for continued overproduction of putrescine in transgenic cells . However, continued supplies of NH(4) (+) and NO(3) (-) were required to maintain homeostatic amounts of putrescine in both cell lines . The presence of high amounts of putrescine in transgenic cells had significant effects on the physiological parameters measured . Compared with non-transgenic cells, transgenic cells had greater plasma membrane permeability, less tolerance to NH(4)NO(3), more tolerance to KNO(3), and accumulated higher amounts of soluble protein.

Genome Res, 2004 Mar, 14(3), 380 - 90
Control of yeast filamentous-form growth by modules in an integrated molecular network; Prinz S et al.; On solid growth media with limiting nitrogen source, diploid budding-yeast cells differentiate from the yeast form to a filamentous, adhesive, and invasive form . Genomic profiles of mRNA levels in Saccharomyces cerevisiae yeast-form and filamentous-form cells were compared . Disparate data types, including genes implicated by expression change, filamentation genes known previously through a phenotype, protein-protein interaction data, and protein-metabolite interaction data were integrated as the nodes and edges of a filamentation-network graph . Application of a network-clustering method revealed 47 clusters in the data . The correspondence of the clusters to modules is supported by significant coordinated expression change among cluster co-member genes, and the quantitative identification of collective functions controlling cell properties . The modular abstraction of the filamentation network enables the association of filamentous-form cell properties with the activation or repression of specific biological processes, and suggests hypotheses . A module-derived hypothesis was tested . It was found that the 26S proteasome regulates filamentous-form growth.

Leukemia, 2004 Jun, 18(6), 1064 - 71
Modulation of cell cycle by graded expression of MLL-AF4 fusion oncoprotein; Caslini C et al.; Acute lymphoblastic leukemia (ALLs) expressing MLL-AF4, the fusion product of t(4;11)(q21;q23), show marked leucocytosis and extramedullary disease in multiple organs, respond poorly to chemotherapy and have poor prognosis . In vitro, leukemic cells with the t(4;11) show resistance to serum deprivation-induced or interferon gamma-regulated CD95-mediated apoptosis . In addition, t(4;11) cells have prolonged doubling time and lower percentage of cells in cycle compared to non-t(4;11) B lineage cell lines . In this study, we examine the time- and level-dependent effects of MLL-AF4 conditional expression on cell cycle and differentiation of myelomonocytic leukemia cell line U937 . By varying the concentration of tetracycline in growth media, we found that increasing levels of MLL-AF4 expression result in a progressive decrease in growth rate and fraction of S phase cells, paralleled by an increase in percentage of cells expressing CD11b . Our results demonstrate a dosage-dependent effect of MLL-AF4 fusion oncoprotein on cell cycle progression, with increasing expression levels resulting in the accumulation in G1, prolonged doubling time, both findings that might be responsible for the increased resistance to etoposide-mediated cytotoxicity . We propose the cell cycle control exerted by MLL-AF4 may be responsible of resistance to cell-death promoting stimuli in leukemia carrying the t(4;11) translocation.

J Virol, 2004 Mar, 78(6), 2682 - 92
Requirements for CEACAMs and cholesterol during murine coronavirus cell entry; Thorp EB et al.; Previous reports have documented that cholesterol supplementations increase cytopathic effects in tissue culture and also intensify in vivo pathogenicities during infection by the enveloped coronavirus murine hepatitis virus (MHV) . To move toward a mechanistic understanding of these phenomena, we used growth media enriched with methyl-beta-cyclodextrin or cholesterol to reduce or elevate cellular membrane sterols, respectively . Cholesterol depletions reduced plaque development 2- to 20-fold, depending on the infecting MHV strain, while supplementations increased susceptibility 2- to 10-fold . These various cholesterol levels had no effect on the binding of viral spike (S) proteins to cellular carcinoembryonic antigen-related cell adhesion molecule (CEACAM) receptors, rather they correlated directly with S-protein-mediated membrane fusion activities . We considered whether cholesterol was indirectly involved in membrane fusion by condensing CEACAMs into "lipid raft" membrane microdomains, thereby creating opportunities for simultaneous binding of multiple S proteins that subsequently cooperate in the receptor-triggered membrane fusion process . However, the vast majority of CEACAMs were solubilized by cold Triton X-100 (TX-100), indicating their absence from lipid rafts . Furthermore, engineered CEACAMs appended to glycosylphosphatidylinositol anchors partitioned with TX-100-resistant lipid rafts, but cells bearing these raft-associated CEACAMs were not hypersensitive to MHV infection . These findings argued against the importance of cholesterol-dependent CEACAM localizations into membrane microdomains for MHV entry, instead suggesting that cholesterol had a more direct role . Indeed, we found that cholesterol was required even for those rare S-mediated fusions taking place in the absence of CEACAMs . We conclude that cholesterol is an essential membrane fusion cofactor that can act with or without CEACAMs to promote MHV entry.

Environ Toxicol Chem, 2004 Feb, 23(2), 371 - 82
Effects of 25 pharmaceutical compounds to Lemna gibba using a seven-day static-renewal test; Brain RA et al.; Antibiotics are known to have antichloroplastic properties, but their effects on aquatic higher plants are virtually unknown . In order to address this issue, 25 pharmaceuticals, including 22 antibiotics, were assessed for phytotoxicity to the aquatic higher plant Lemna gibba . A 7-d static-renewal test was used, and plants were treated with 0, 10, 30, 100, 300, and 1,000 microg/L of pharmaceutical-containing growth media . Phytotoxicity was assessed using multiple growth and biochemical endpoints . Effective concentration (EC)50, EC25, and EC10 values as well as tests for significant differences between treatments and controls lowest-observed-effect concentration (LOECs) were calculated for each endpoint . Twelve different classes of antibiotics were assessed; however, only members of the fluoroquinolone, sulfonamide, and tetracycline classes of antibiotics displayed significant phytotoxicity . The most toxic members of each of these classes tested were lomefloxacin, sulfamethoxazole, and chlortetracycline, with wet weight EC25 values of 38, 37, and 114 microg/L, respectively . Injury symptoms were comparatively uniform and consistent among chemical classes while degree of phytotoxicity varied considerably . Both of these criteria varied markedly between classes . Wet mass was consistently the most sensitive endpoint above 100 microg/L; conversely, frond number was the most sensitive below 100 microg/L . Pigment endpoints were significantly less sensitive than growth endpoints.

J Chromatogr A, 2004 Feb 20, 1027(1-2), 147 - 54
Use of headspace solid-phase microextraction and headspace sorptive extraction for the detection of the volatile metabolites produced by toxigenic Fusarium species; Demyttenaere JC et al.; An efficient methodology was developed to determine the growth of toxigenic Fusarium spp., based on headspace solid-phase microextraction (SPME) and stir bar sorptive extraction of the fungal volatile metabolites produced . SPME and headspace sorptive extraction (HSSE) were used to monitor the de novo production of sesquiterpene hydrocarbons, such as trichodiene, a volatile marker and intermediate in the biosynthesis of trichothecenes . On growth media such as malt extract agar and potato dextrose agar, it was found that trichodiene was produced by toxigenic strains of Fusarium sambucinum and Fusarium sporotrichioides . It was the main volatile metabolite in the headspace extract of the cultures . On the other hand, deoxynivalenol producing Fusarium graminearum showed a completely different pattern of volatile sesquiterpenes and could easily be distinguished from a zearalenone producing strain of F . graminearum based on the headspace profile . Hence, it can be concluded that headspace analysis of volatile fungal metabolites by SPME and HSSE in combination with gas chromatography/mass spectrometry is a suitable monitoring technique to differentiate toxigenic strains of Fusarium.

Tree Physiol, 1992 Jun, 10(4), 357 - 66
Effects of acidity and detergent on in vitro pollen germination and tube growth in forest tree species; Paoletti E; The presence of 1 to 3 mg 1(-1) sodium dodecylbenzensulfonate detergent, or a growth medium pH of 4.0-5.0, inhibited pollen germination and pollen tube elongation more in broad-leaved trees than in conifers . In the broad-leaved species, pollen germination and pollen tube elongation showed similar sensitivities to detergent and acidity, whereas in the conifers, pollen tube elongation was more sensitive than pollen germination to detergent and acidity . Although the germination percentage was low, conifer pollen germinated in the presence of detergent concentrations up to 15 mg l(-1) and at acidities as low as pH 2.5 or 3.0 . Pollen germination of most broad-leaved species was completely inhibited in the presence of detergent concentrations of more than 3-5 mg l(-1); the only exceptions were some entomophilous species (e.g., Salix caprea L.) in which the ability of the pollen to germinate in high pollutant concentrations could be related to the presence of tryphyne.

Biotechnol Bioeng, 2004 Mar 20, 85(6), 666 - 75
Application of Plackett-Burman design and response surface methodology to achieve exponential growth for aggregated shipworm bacterium; Ahuja SK et al.; Here we report the successful implementation of the Plackett-Burman multifactorial design to screen the limiting components for growth and subsequent use of the response surface methodology (RSM) to design a medium that supported exponential growth of the aggregated morphology of the shipworm bacterium, Teredinobacter turnirae . The results obtained with the help of Plackett-Burman design indicated limitations of three components in the growth medium, MnCl2.4H2O, Na2CO3, and K2HPO4 . The concentrations of these three components were further optimized using RSM . By increasing the concentrations of the above-mentioned components by 4-fold, 12-fold, and 12-fold, respectively, it became possible to achieve exponential growth of the culture .

Tree Physiol, 1995 Feb, 15(2), 95 - 103
Influence of controlled water supply on shoot and root development of young peach trees; Hipps NA et al.; Three controlled water supply treatments were applied to 1-year-old peach trees grown in root observation boxes . The treatments were: I(0), growth medium maintained at 50% field capacity; I(1), water supplied when daily net tree stem diameter change was negative or zero for 1 day; I(3) as for I(1) except that water was applied after net daily stem diameter change was negative or zero for 3 consecutive days . Trees in treatment I(0) had the greatest mean daily first-order shoot growth rates, and trees in treatment I(3) had the lowest shoot growth rates . Because leaf production rate (apparent plastochron) of first-order shoots was unaffected by treatment, differences in shoot length were due to differences in internode extension and not to the number of internodes . Trees in treatment I(0) had a greater number of second-order shoot axes than trees in treatment I(1) or I(3) . Furthermore, an increase in the rate of growth of the first-order shoot axis was associated with an increased tendency for branching (i.e., the development of sylleptic second-order shoots) . Increased leaf length was also associated with more frequent watering . Trees in treatment I(0) had the greatest root lengths and dry weights, and this was attributed to a greater number of first-and second-order (lateral) root axes compared with trees in the I(1) and I(3) treatments . The extension rate and apical diameter of first-order roots were reduced by the I(3) treatment . The density of second-order roots along primary root axes was not affected by any of the treatments.

Curr Cancer Drug Targets, 2004 Feb, 4(1), 53 - 64
Stress signaling from irradiated to non-irradiated cells; Azzam EI et al.; Evidence accumulated over the past two decades has indicated that exposure of cell populations to ionizing radiation results in significant biological effects occurring in both the irradiated and non-irradiated cells in the population . This phenomenon, termed the "bystander response", has been shown to occur both in vitro and in vivo . Experiments have indicated that genetic alterations, changes in gene expression and lethality occur in bystander cells that neighbor directly irradiated cells . Furthermore, cells recipient of growth medium harvested from irradiated cultures exhibit responses similar to those of the irradiated cells . Several mechanisms involving secreted soluble factors, gap-junction intercellular communication and oxidative metabolism have been proposed to regulate the radiation-induced bystander effect . In this review, our current knowledge of this phenomenon and its potential impact both on the estimation of risks of exposure to low doses/low fluences of ionizing radiation and on radiotherapy is discussed.

J Trauma, 2004 Feb, 56(2), 363 - 7
Bone debris: dead matter or vital osteoblasts; Hoegel F et al.; BACKGROUND: Mechanical manipulation, pressure, and temperature increase can induce bone necrosis during intramedullary reaming . METHODS: In this study, the bone debris obtained after reaming 18 sheep tibiae was analyzed to investigate its vitality by measuring alkaline phosphatase activity . Two different reamer designs were used for the project . Bone cells were first cultivated in a specific growth medium, counted 3 weeks after the reaming procedure, and then cultivated for another 5 weeks . RESULTS: At the end of the project, qualitative evaluation showed positive alkaline phosphatase activity in most of the cases, and quantitative evaluation also showed enzyme activity . The positive alkaline phosphatase results were independent of the reamer sizes and reamer design . No significant results were obtained from a comparison of different reamer sizes and designs . This indicates that osteoblasts survive after correctly performed reaming . CONCLUSION: The results prove the vitality of the bone debris and confirm clinical observations.

J Biomater Appl, 2004 Jan, 18(3), 209 - 22
Adhesion and proliferation of human dermal fibroblasts on collagen matrix; Croce MA et al.; The purpose of this study was to evaluate adhesion and growth of human dermal fibroblasts on a 0.150 mm-thick matrix of reconstituted collagen isolated from horse tendon . Collagen was extracted and polymerized according to the standard procedures (Opocrin, Corlo, Modena, Italy) . By light microscopy, the bottom surface of the matrix appeared linear and compact, whereas the superficial one was indented and less homogeneous . By scanning electron microscopy, the collagen fibrils had different diameters and the great majority of them was oriented parallel to the surface of the gel . By transmission electron microscopy, collagen fibrils showed the typical banding . Human dermal fibroblasts were seeded on the collagen matrix, previously equilibrated in growth medium . Fibroblast proliferation stopped in the second week and was always significantly lower than that of the same cell strain seeded on plastic and cultured in parallel . By light microscopy, after six days culture, cells formed a confluent multilayer on the surface of the gel . By scanning and transmission electron microscopy, fibroblasts appeared flat and adherent to the matrix . Contacts of cells among themselves and with the collagen fibrils were observed . Fibroblasts never moved into the collagen gel . In conclusion, human dermal fibroblasts can be grown in a three-dimensional matrix made by horse tendon that, on the other hand, seems to condition their proliferation rate.

Appl Environ Microbiol, 2004 Feb, 70(2), 656 - 63
Growth characteristics of Bartonella henselae in a novel liquid medium: primary isolation, growth-phase-dependent phage induction, and metabolic studies; Chenoweth MR et al.; Bartonella henselae is a zoonotic pathogen that usually causes a self-limiting infection in immunocompetent individuals but often causes potentially life-threatening infections, such as bacillary angiomatosis, in immunocompromised patients . Both diagnosis of infection and research into the molecular mechanisms of pathogenesis have been hindered by the absence of a suitable liquid growth medium . It has been difficult to isolate B . henselae directly from the blood of infected humans or animals or to grow the bacteria in liquid culture media under laboratory conditions . Therefore, we have developed a liquid growth medium that supports reproducible in vitro growth (3-h doubling time and a growth yield of approximately 5 x 10(8) CFU/ml) and permits the isolation of B . henselae from the blood of infected cats . During the development of this medium, we observed that B . henselae did not derive carbon and energy from the catabolism of glucose, which is consistent with genome nucleotide sequence data suggesting an incomplete glycolytic pathway . Of interest, B . henselae depleted amino acids from the culture medium and accumulated ammonia in the medium, an indicator of amino acid catabolism . Analysis of the culture medium throughout the growth cycle revealed that oxygen was consumed and carbon dioxide was generated, suggesting that amino acids were catabolized in a tricarboxylic acid (TCA) cycle-dependent mechanism . Additionally, phage particles were detected in the culture supernatants of stationary-phase B . henselae, but not in mid-logarithmic-phase culture supernatants . Enzymatic assays of whole-cell lysates revealed that B . henselae has a complete TCA cycle . Taken together, these data suggest B . henselae may catabolize amino acids but not glucose to derive carbon and energy from its host . Furthermore, the newly developed culture medium should improve isolation of B . henselae and basic research into the pathogenesis of the bacterium.

PDA J Pharm Sci Technol, 2003 Nov-Dec, 57(6), 399 - 403
The influence of incubation conditions in sterility tests; Bugno A et al.; Studies were conducted to evaluate the effects of media, incubation temperature and duration on the detection of bacteria and fungal growth using British, United States and Brazilian compendial sterility test methods . 5 to 50 CFU of nine different microorganisms (including both anaerobic and aerobic bacteria and molds) were used to contaminate test units containing various growth media (sobean-casein digest, thioglycollate, Sabourand and Causen broths) . Test units were incubated at temperatures ranging from 12 to 42 degrees C for 1 to 28 days . Inoculations were conducted according to compendial procedures . Optimal detection conditions were obtained at 22 to 32 degrees C over 14 days using soybean casein digest and thioglycollate broths.

Mol Biotechnol, 2004 Feb, 26(2), 101 - 10
Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli: isolation and some properties of the enzyme; Grzybowska B et al.; Pyrococcus woesei (DSM 3773) alpha-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)alpha-amyl and pYTB2alpha-amyl vectors obtained were used for expression of thermostable alpha-amylase or fusion of alpha-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively . As compared with other expression systems, the synthesis of alpha-amylase in fusion with intein in E . coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation-they exhibit only 35% of total cell activity-and high productivity of the soluble enzyme form (195,000 U/L of the growth medium) . The thermostable alpha-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75 degrees C in the presence of thiol compounds . The recombinant enzyme has maximal activity at pH 5.6 and 95 degrees C . The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C . Maltose was the main end product of starch hydrolysis catalyzed by this alpha-amylase . However, small amounts of glucose and some residual unconverted oligosaccharides were also detected . Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 151 - 5
Application of a MTT assay for screening nutritional factors in growth media of primary sponge cell culture; Zhang X et al.; Marine sponges (Porifera) are producers of the largest variety of bioactive compounds among benthic marine organisms . In vitro culture of marine sponge cells has been proposed for the sustainable production of these pharmacologically interesting compounds from marine sponges but with limited success . The development of a suitable growth medium is an essential prerequisite for sponge cells grown in vitro . The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was adapted to screen for potential nutritional factors in formulating a growth medium for primary cell culture of Suberites domuncula . In 96-well plates, the effects of nutritional factors including glutamine, pyruvate, iron citrate, silicon, RPMI 1640, and Marine Broth 2216 on the viable cell density were examined in primary cell culture of S . domuncula 36 h after inoculation . Ferric iron (Fe(3+)) and pyruvate were found to significantly improve cell viability in a dose-dependent manner . Silicon and glutamine showed limited improvements at certain concentrations . The supplement of RPMI 1640 and Marine Broth 2216 did not increase cell viability . As a result, several improved media able to maintain higher cell viability in a short-term culture of primary sponge cells could be formulated.

Plast Reconstr Surg, 2004 Feb, 113(2), 595 - 602; discussion 603-4
Contractile skeletal muscle tissue-engineered on an acellular scaffold; Borschel GH et al.; For the reconstructive surgeon, tissue-engineered skeletal muscle may offer reduced donor-site morbidity and an unlimited supply of tissue . Using an acellularized mouse extensor digitorum longus muscle as a scaffold, the authors produced engineered skeletal muscle capable of generating longitudinal force . Eight extensor digitorum longus muscles from adult mice were made acellular using a protocol developed in the authors' laboratory . The acellular muscles were then placed in a bath of 20% fetal bovine serum in Dulbecco's modified Eagle's medium and 100 U/ml penicillin for 1 week at room temperature . C2C12 myoblasts were injected into the acellular muscle matrix using a 26-gauge needle and a 100-microl syringe . The resulting constructs were placed in growth medium for 1 week at 37 degrees C under 5% carbon dioxide, with media changes every 48 hours . The constructs were then placed in differentiation medium for 1 week, with media changes every 48 hours . Isometric contractile force testing of the constructs demonstrated production of longitudinal contractile force on electrical stimulation . A length-tension, or Starling, relationship was observed . Light and electron microscopy studies demonstrated recapitulation of some of the normal histologic features of developing skeletal muscle.

J Agric Food Chem, 2004 Feb 11, 52(3), 462 - 6
Growth inhibition of prostate cancer cells by epigallocatechin gallate in the presence of Cu2+; Yu HN et al.; Green tea is an effective chemopreventive agent to human prostate cancer adenoma (PCA) . Epigallocatechin gallate (EGCG) inhibited the growth of PCA cells and induced apoptosis . Cu(2+) is a trace element necessary to our health . Many studies proved that bioactivity of EGCG is altered in the presence of Cu(2+) . We investigated the effects of EGCG on PCA cells in the presence of Cu(2+) . Also, we explored potential mechanisms via measurement of the relative chemiluminescence of growth medium for PCA cells . Chemiluminscence can be an indication of free radicals . Our test results showed that the addition of EGCG and Cu(2+) to the growth medium decreased the relative viability of androgen-sensitive and androgen-insensitive human prostate cancer cells . However, the effects of EGCG on PCA cells depended on (1) the relative concentrations of added EGCG and Cu(2+) and (2) their order of addition . Our results indicated that few free radicals may be generated in vitro . If so, free radicals generated intracellularly may be a major factor behind apoptosis and growth inhibition observed in the PCA cells . Thus, EGCG might exert its effects intracellularly.

J Exp Bot, 2004 Mar, 55(397), 685 - 93 Epub 2004 Jan 30.
Developmental anatomy and auxin response of lateral root formation in Ceratopteris richardii; Hou G et al.; The homosporous fern Ceratopteris richardii exhibits a homorhizic root system where roots originate from the shoot system . These shoot-borne roots form lateral roots (LRs) that arise from the endodermis adjacent to the xylem poles, which is in contrast to flowering plants where LR formation arises from cell division in the pericycle . A detailed study of the fifth shoot-borne root showed that one lateral root mother cell (LRMC) develops in each two out of three successive merophytes . As a result, LRs emerge alternately in two ranks from opposite positions on a parent root . From LRMC initiation to LR emergence, three developmental stages were identified based on anatomical criteria . The addition of auxins (either indole-3-acetic acid or indole-3-butyric acid) to the growth media did not induce additional LR formation, but exogenous applications of both auxins inhibited parent root growth rate . Application of the polar auxin-transport inhibitor N-(1-naphthyl)phthalamic acid (NPA) also inhibited parent root growth without changing the LR initiation pattern . The results suggest that LR formation does not depend on root growth rate per se . The result that exogenous auxins do not promote LR formation in C . richardii is similar to reports for certain species of flowering plants, in which there is an acropetal LR population and the formation of the LRs is insensitive to the application of auxins . It also may indicate that different mechanisms control LR development in non-seed vascular plants compared with angiosperms, taking into consideration the long and independent evolutionary history of the two groups.

Int J Phytoremediation, 2003, 5(3), 235 - 44
Nickel and cobalt phytoextraction by the hyperaccumulator Berkheya coddii: implications for polymetallic phytomining and phytoremediation; Keeling SM et al.; We investigated the potential of the South African high-biomass Ni hyperaccumulator Berkheya coddii to phytoextract Co and/or Ni from artificial metalliferous media . Plant accumulation of both metals from single-element substrates indicate that the plant/media metal concentration quotient (bioaccumulation coefficient) increases as total metal concentrations increase . Cobalt was readily taken up by B . coddii with and without the presence of Ni . Nickel uptake was, however, inhibited by the presence of an equal concentration of Co . Bioaccumulation coefficients of Ni and Co for the single element substrates (total metal concentration of 1000 micrograms g-1) were 100 and 50, respectively . Cobalt phytotoxicity was observed above a total Co concentration in plant growth media of 20 micrograms g-1 . Elevated Co concentrations significantly decreased the biomass production of B . coddii without affecting the bioaccumulation coefficients . The mixed Ni-Co substrate produced bioaccumulation coefficients of 22 for both Ni and Co . Cobalt phytotoxicity in mixed Ni-Co substrate occurred above a total Co concentration of 15 micrograms g-1 . When grown in the presence of both Ni and Co, the bioaccumulation coefficients of each metal were reduced, as compared to single-element substrate . This may indicate competition for binding sites in the root zone . The interference relationship between Ni and Co uptake demonstrated by B . coddii suggests a significant limitation to phytoextraction where both metals are present.

Am J Obstet Gynecol, 2004 Jan, 190(1), 239 - 45
Immunologic properties of human fetal mesenchymal stem cells; Gotherstrom C et al.; OBJECTIVE: Mesenchymal stem cells (MSCs) can be isolated from adult bone marrow and fetal liver . We investigated the immunologic properties of undifferentiated and differentiated human fetal MSCs . STUDY DESIGN: Expression of HLA class I and II was investigated by flow cytometry and Western blot on undifferentiated fetal MSC and after in vitro differentiation to adipocytes and osteocytes . Alloreactivity was studied after adding fetal MSCs to allogeneic lymphocytes in mixed lymphocyte cultures . RESULTS: Fetal MSCs expressed HLA class I but not HLA class II . The presence of interferon gamma (IFN-gamma) in the growth medium for 2 days initiated the intracellular synthesis of HLA class II, but 7 days of exposure was required for cell surface expression . Neither undifferentiated nor differentiated fetal MSCs induced proliferation of allogenic lymphocytes . Fetal MSCs treated with IFN-gamma suppressed alloreactive lymphocytes . CONCLUSION: Undifferentiated and differentiated fetal MSCs do not elicit alloreactive lymphocyte proliferation . The results suggest that fetal MSCs have potentials for allogenic transplantation.

Infect Immun, 2004 Feb, 72(2), 766 - 73
Acid-responsive gene induction of ammonia-producing enzymes in Helicobacter pylori is mediated via a metal-responsive repressor cascade; van Vliet AH et al.; Although the adaptive mechanisms allowing the gastric pathogen Helicobacter pylori to survive acid shocks have been well documented, the mechanisms allowing growth at mildly acidic conditions (pH approximately 5.5) are still poorly understood . Here we demonstrate that H . pylori strain 26695 increases the transcription and activity of its urease, amidase, and formamidase enzymes four- to ninefold in response to growth at pH 5.5 . Supplementation of growth medium with NiCl2 resulted in a similar induction of urease activity (at low NiCl2 concentration) and amidase activity (at > or = 500 micro M NiCl2) but did not affect formamidase activity . Mutation of the fur gene, which encodes an iron-responsive repressor of both amidases, resulted in a constitutively high level of amidase and formamidase activity at either pH but did not affect urease activity at pH 7.0 or pH 5.5 . In contrast, mutation of the nikR gene, encoding the nickel-responsive activator of urease expression, resulted in a significant reduction of acid-responsive induction of amidase and formamidase activity . Finally, acid-responsive repression of fur transcription was absent in the H . pylori nikR mutant, whereas transcription of the nikR gene itself was increased at pH 5.5 in wild-type H . pylori . We hypothesize that H . pylori uses a repressor cascade to respond to low pH, with NikR initiating the response directly via the urease operon and indirectly via the members of the Fur regulon.

Angiogenesis, 2003, 6(2), 143 - 9
Inhibition of angiogenic initiation and disruption of newly established human vascular networks by juice from Morinda citrifolia (noni); Hornick CA et al.; noni, the juice of the fruit from the Morinda citrifolia plant, has been used for centuries as a medicinal agent . We tested the effects of noni juice in a three-dimensional fibrin clot matrix model using human placental vein and human breast tumor explants as sources for angiogenic vessel development . Noni in concentrations of 5% (vol/vol) or greater was highly effective in inhibiting the initiation of new vessel sprouts from placental vein explants, compared with initiation in control explants in media supplemented with an equivalent amount of saline . These concentrations of noni were also effective in reducing the growth rate and proliferation of newly developing capillary sprouts . When used at a concentration of 10% in growth media, noni was able to induce vessel degeneration and apoptosis in wells with established capillary networks within a few days of its application . We also found that 10% noni juice in media was an effective inhibitor of capillary initiation in explants from human breast tumors . In tumor explants which did show capillary sprouting, the vessels rapidly degenerated (2-3 days) in those exposed to media supplemented with 10% noni.

Circ Res, 2004 Mar 19, 94(5), 617 - 25 Epub 2004 Jan 22.
Transforming growth factor-beta-induced inhibition of myogenesis is mediated through Smad pathway and is modulated by microtubule dynamic stability; Zhu S et al.; The expression of muscle-specific genes associated with myogenesis is controlled by several myogenic transcription factors, including myogenin and MEF2D . Transforming growth factor-beta (TGF-beta) has been shown to inhibit myogenesis, yet the molecular mechanisms underlying such inhibition are not known . In the present study, TGF-beta was shown to inhibit myogenin and MEF2D expression and myotube formation in C2C12 myoblasts cultured in differentiation medium in a cell density-dependent manner . Transfection of C2C12 cells with Smad7, an antagonist for TGF-beta/Smad signaling, restored the capacity of these cells to differentiate in the presence of TGF-beta or when cultured in growth medium at low confluence, conditions that hinder muscle differentiation . Moreover, nocodazole, a microtubule-destabilizing agent, enhanced the inhibition of myogenesis exerted by TGF-beta, an effect that could be restored by tubulin-polymerizing agent taxol, both of which have been shown to affect Smad-microtubule interaction and regulate TGF-beta/Smad signaling . Our results indicate that TGF-beta inhibits myogenesis, at least in part, via Smad pathway, and provide evidence that low-dose pharmacological agents taxol and nocodazole can be used as a means to modulate myogenesis without affecting cell survival.

J Immunol Methods, 2004 Jan, 284(1-2), 45 - 54
Efficient purification of unique antibodies using peptide affinity-matrix columns; Jensen LB et al.; Phage display technology was used to identify peptide ligands with unique specificity for a monoclonal model antibody, MK16, that recognises the human multiple sclerosis associated MHC class II molecule DR2 in complex with a myelin basic protein (MBP)-derived peptide corresponding to residue 85-99 . Several peptide epitopes were identified and all of them recognised specifically MK16 . One peptide, ER6.1, was selected and linked to beaded agarose and demonstrated excellent performance as a peptide affinity chromatography matrix . This epitope matrix was efficient in the purification of MK16 Fab fragments and had no affinity for other antibodies . Using this peptide matrix MK16 IgG could be purified from cell culture supernatants thereby separating MK16 IgG from bovine IgG normally present in the enriched growth media used for such cells.Investigations of the fine specificity of the ER6.1 peptide demonstrated that it recognised a unique epitope within the heavy chain CDR3 region of the MK16 antibody . Thus, variants of MK16 antibody, which had retained the specificity and affinity of the original antibody but had slightly different amino acid composition in the CDR3 region, were not recognised by the ER6.1 peptide.

Izv Akad Nauk Ser Biol, 2003 Sep-Oct, (5), 560 - 4
{Effect of oligosaccharides on adaptation of winter wheat seedlings to negative temperature}; Zabotina OA et al.; We studied the dynamics of endogenous content of bioactive oligosaccharides in the roots of winter wheat seedlings . Previously these oligosaccharides proved to mediate development of frost resistance during the first days of hardening (Zabotina et al., 1998) . The changes in their endogenous content can be described by a single-humped curve peaking 6 h after the onset of frost hardening . The capacity of these polysaccharides to increase frost resistance (LT50 was evaluated by leakage of electrolytes) when added to growth medium did not depend on the pretreatment duration (from 1.5 to 18 h) but decreased if they were introduced in the course of the adaptive response . Inhibition of the adaptive response by inhibitors of RNA and protein synthesis was ceased in the presence of the oligosaccharides . We believe that the oligosaccharides that are products of metabolism of the cell wall polysaccharides are involved in adaptation to low temperature.

J Biol Chem, 2004 Apr 2, 279(14), 14447 - 55 Epub 2004 Jan 16.
Yeast contain a non-proteinaceous pool of copper in the mitochondrial matrix; Cobine PA et al.; The yeast mitochondrion is shown to contain a pool of copper that is distinct from that associated with the two known mitochondrial cuproenzymes, superoxide dismutase (Sod1) and cytochrome c oxidase (CcO) and the copper-binding CcO assembly proteins Cox11, Cox17, and Sco1 . Only a small fraction of mitochondrial copper is associated with these cuproproteins . The bulk of the remainder is localized within the matrix as a soluble, anionic, low molecular weight complex . The identity of the matrix copper ligand is unknown, but the bulk of the matrix copper fraction is not protein-bound . The mitochondrial copper pool is dynamic, responding to changes in the cytosolic copper level . The addition of copper salts to the growth medium leads to an increase in mitochondrial copper, yet the expansion of this matrix pool does not induce any respiration defects . The matrix copper pool is accessible to a heterologous cuproenzyme . Co-localization of human Sod1 and the metallochaperone CCS within the mitochondrial matrix results in suppression of growth defects of sod2Delta cells . However, in the absence of CCS within the matrix, the activation of human Sod1 can be achieved by the addition of copper salts to the growth medium.

Microb Pathog, 2004 Mar, 36(3), 153 - 7
Requirement of hydD, hydE, hypC and hypE genes for hydrogenase activity in Helicobacter pylori; Benoit S et al.; Helicobacter pylori possesses a membrane-bound, nickel containing, hydrogen uptake hydrogenase enzyme; its synthesis requires structural as well as accessory proteins, the latter needed for the complete maturation of the enzyme . Our lab previously characterized mutants in the accessory hyp genes, hypA, hypB, hypD and hypF that were all severely affected for hydrogenase activity, and in some cases (hypA and hypB mutants) also affected for urease activity . This finding prompted us to disrupt the two remaining unstudied hyp genes of H . pylori, hypC and hypE, in order to see if the same pleiotropic effect would be observed . In both mutants hydrogenase activity was abolished but urease activity remained unaffected . Addition of 5 microM nickel into the growth medium partially restored the hydrogenase activity in the hypE mutant and to a lesser extent in the hypC mutant . In addition, we also disrupted the genes HP0634 (referred as hydD in the H . pylori 26695 genome database) and HP0635 (whose function was unknown, referred to here as hydE) to address their possible roles in the hydrogenase synthesis/maturation process . In both cases, hydrogenase activities were abolished and addition of nickel could not restore the activity, suggesting that these proteins are involved in the hydrogenase synthesis process rather than in nickel mobilization/insertion steps.

Oncogene, 2004 Jan 15, 23(2), 524 - 34
Myostatin inhibits rhabdomyosarcoma cell proliferation through an Rb-independent pathway; Langley B et al.; Rhabdomyosarcoma (RMS) tumors are the most common soft-tissue sarcomas in childhood . In this investigation, we show that myostatin, a skeletal muscle-specific inhibitor of growth and differentiation is expressed and translated in the cultured RMS cell line, RD . The addition of exogenous recombinant myostatin inhibits the proliferation of RD cells cultured in growth media, consistent with the role of myostatin in normal myoblast proliferation inhibition . However, unlike normal myoblasts, upregulation of p21 was not observed . Rather, myostatin signalling resulted in the specific downregulation of both Cdk2 and its cognate partner, cyclin-E . The analysis of Rb reveals that there was no change in its phosphorylation status with myostatin treatment, consistent with D-type-cyclin-Cdk4/6 complexes being active in the absence of p21 . Moreover, the activity of Rb appeared to be unchanged between treated and nontreated RD cells, as determined by the ability of Rb to bind E2F1 . The examination of NPAT, a substrate of cyclin-E-Cdk2 involved in the transcriptional activation of replication-dependent histone gene expression, revealed that it undergoes a loss of phosphorylation with myostatin treatment . Supporting this, a downregulation in H4-histone gene expression was observed . These results suggest that myostatin could potentially be used as an inhibitor of RMS proliferation and define a previously uncharacterized, Rb-independent mechanism for the inhibition of muscle precursor cell proliferation by myostatin.

Curr Genet, 2004 Mar, 45(3), 140 - 8 Epub 2004 Jan 10.
Characterization of extracellular lytic enzymes produced by the yeast biocontrol agent Candida oleophila; Bar-Shimon M et al.; The yeast Candida oleophila, the base of the commercial product Aspire, is recommended for the control of postharvest decay of citrus and pome fruit . Competition for nutrients and space is believed to be the major mode of action . Involvement of fungal cell wall-degrading enzymes is also suggested to play a role in the mechanism of action of yeast antagonists . The present study showed that the yeast C . oleophila is capable of producing and secreting various cell wall-degrading enzymes, including exo-beta-1,3-glucanase, chitinase and protease . Exo-beta-1,3-glucanase and chitinase were produced and maximized in the early stages of growth, whereas protease reached a maximum level only after 6-8 days . Production of exo-beta-1,3-glucanase, chitinase and protease was stimulated by the presence of cell wall fragments of Penicillium digitatum in the growth medium, in addition to glucose . This study also provided evidence that C . oleophila is capable of secreting exo-beta-1,3-glucanase into the wounded surface of grapefruit . The role of exo-beta-1,3-glucanase ( CoEXG1) in the biocontrol activity of C . oleophila was tested using CoEXG1-knockouts and double- CoEXG1 over-producing transformants . In vitro bioassays showed that wild-type C . oleophila and exo-beta-1,3-glucanase over-expressing transformants had similar inhibitory effects on spore germination and germ-tube elongation; and both were more inhibitory to the fungus than the knockout transformant . In experiments conducted on fruit to test the biocontrol activity against infection by P . digitatum, no significant difference in inhibition was observed between transformants and untransformed C . oleophila cells at the high concentrations of cells used, whereas at a lower concentration of yeast cells the knockout transformants appeared to be less effective.

FASEB J, 2004 Mar, 18(3), 568 - 70 Epub 2004 Jan 08.
Platelet-activating factor (PAF) induces activation of matrix metalloproteinase 2 activity and vascular endothelial cell invasion and migration; Axelrad TW et al.; Tumor-induced angiogenic responses lead to complex phenotypic changes in vascular endothelial cells, which must coordinate the expression of both proteases and protease inhibitors prior to the proliferation and invasion of surrounding stroma . Matrix metalloproteinase 2 (MMP2), which degrades Type IV collagen, is produced as proMMP2 . proMMP2 is activated in part through its interactions with membrane Type 1 MMP (MT1-MMP) and tissue inhibitor of matrix metalloproteinase 2 (TIMP2) . In this study, we demonstrate that platelet-activating factor (PAF) is a potent inducer of human umbilical vein endothelial cell (HUVEC) migration and invasion, which is attenuated by PAF receptor antagonists, and that PAF receptor antagonists inhibit the migration and invasion of HUVEC mediated by medium conditioned by a prostatic carcinoma cell line . We confirm that PAF receptor antagonists inhibit proliferation of HUVEC grown in rich growth medium . We show that PAF increases mRNA levels for MT1-MMP and TIMP2, followed by increased temporal conversion of latent proMMP2 to MMP2 . Finally, we demonstrate that the ratio of MT1-MMP to TIMP2 in membrane preparations from PAF-stimulated HUVEC is 1.6:1, approximating the hypothesized ideal ratio of 2:1 necessary for the conversion of proMMP2 to MMP2 . Our data support the involvement of PAF in vascular endothelial cell migration and invasion.

Environ Toxicol Chem, 2003 Dec, 22(12), 2940 - 7
Toward detoxifying mercury-polluted aquatic sediments with rice genetically engineered for mercury resistance; Heaton AC et al.; Mercury contamination of soil and water is a serious problem at many sites in the United States and throughout the world . Plant species expressing the bacterial mercuric reductase gene, merA, convert ionic mercury, Hg(II), from growth substrates to the less toxic metallic mercury, Hg(0) . This activity confers mercury resistance to plants and removes mercury from the plant and substrates through volatilization . Our goal is to develop plants that intercept and remove Hg(II) from polluted aquatic systems before it can undergo bacterially mediated methylation to the neurotoxic methylmercury . Therefore, the merA gene under the control of a monocot promoter was introduced into Oryza sativa L . (rice) by particle gun bombardment . This is the first monocot and first wetland-adapted species to express the gene . The merA-expressing rice germinated and grew on semisolid growth medium spiked with sufficient Hg(II) to kill the nonengineered (wild-type) controls . To confirm that the resistance mechanism was the conversion of Hg(II) to Hg(0), seedlings of merA-expressing O . sativa were grown in Hg(II)-spiked liquid medium or water-saturated soil media and were shown to volatilize significantly more Hg(0) than wild-type counterparts . Further genetic manipulation could yield plants with increased efficiency to extract soil Hg(II) and volatilize it as Hg(0) or with the novel ability to directly convert methylmercury to Hg(0).

J Biol Chem, 2004 Mar 19, 279(12), 11042 - 50 Epub 2004 Jan 05.
An alternative pathway of oleate beta-oxidation in Escherichia coli involving the hydrolysis of a dead end intermediate by a thioesterase; Ren Y et al.; The degradation of 2-trans,5-cis-tetradecadienoyl-CoA, a metabolite of oleic acid, by the purified complex of fatty acid oxidation from Escherichia coli was studied to determine how much of the metabolite is converted to 3,5-cis-tetradecadienoyl-CoA and thereby diverted from the classical, isomerase-dependent pathway of oleate beta-oxidation . Approximately 10% of the 2,5-intermediate was converted to the 3,5-isomer . When the latter compound was allowed to accumulate, it strongly inhibited the flux through the main pathway . Since Delta(3,5),Delta(2,4)-dienoyl-CoA isomerase was not detected in E . coli cells grown on oleate, the 3,5-intermediate cannot be metabolized via the reductase-dependent pathway . However, it was hydrolyzed by a thioesterase, which was most active with 3,5-cis-tetradecadienoyl-CoA as substrate and which was induced by growth of E . coli on oleate . An analysis of fatty acids present in the medium after growth of E . coli on oleate revealed the presence of 3,5-tetradecadienoate, which was not detected after cells were grown on palmitate or glucose . Altogether, these data prompt the conclusion that oleate is mostly degraded via the classical, isomerase-dependent pathway in E . coli but that a small amount of 2-trans,5-cis-tetradecadienoyl-CoA is diverted from the pathway via conversion to 3,5-cis-tetradecadienoyl-CoA by Delta(3),Delta(2)-enoyl-CoA isomerase . The 3,5-intermediate, which would strongly inhibit beta-oxidation if allowed to accumulate, is hydrolyzed, and the resultant 3,5-tetradecadienoate is excreted into the growth medium . This study provides evidence for the novel function of a thioesterase in beta-oxidation.

Zhongguo Wei Zhong Bing Ji Jiu Yi Xue, 2004 Jan, 16(1), 13 - 8
{Effects of fibroblast growth factor-10 on the secretions of transforming growth factor-alpha, platelet-derived growth factor-AB and vascular endothelial growth factor by normal adult human keratinocytes in culture}; Sun XQ et al.; OBJECTIVE: To evaluate the effect of fibroblast growth factor-10 (FGF-10) on the secretion of transforming growth factor-alpha (TGF-alpha), platelet-derived growth factor-AB (PDGF-AB) and vascular endothelial growth factor (VEGF) by human keratinocytes . METHODS: Concentrations of FGF-10 used were 4, 16, 125 and 500 ng/ml . Serum-free keratinocyte growth medium without EGF or with EGF were as negative control and positive control, respectively . Cells were cultured at 2500, 37500 cells/cm 2 in dishes in serum-free medium and supernatants were collected at 24, 48 and 72 hours . The concentrations of TGF-alpha PDGF-AB and VEGF in cell culture supernatant were measured by using TGF-alpha, PDGF-AB and VEGF enzyme linked immunoadsorbent assay (ELISA) kits, respectively and cell numbers were counted by haemocytometer . RESULTS: For cells cultured at low density and cells were subconfluent, TGF-alpha in each group was low and there was no significant difference among them at 24 hours . At 48 hours, both in absolute concentrations and on a per-cell basis, FGF-10 enhanced the secretion of TGF-alpha by cells cultured at low density in a dose-dependent fashion . At 72 hours, the concentrations of TGF-alpha in all dose of FGF-10 were significantly higher than that in negative control (P<0.05) . The production of TGF-alpha in pg/10(6) cells in 500 ng/ml FGF-10 reached statistical significance compared with negative control (P<0.05) . The secretion of TGF-alpha in FGF-10 were lower than that in positive control at 48 and 72 hours . For cells cuttured at 37500 cells/cm(2), the secretion of TGF-alpha stimulated by FGF-10 was similar to that when cells cuttured at lower density . PDGF-AB in each group was undetectable . When cells cuttured at high density and reached 100 percent confluent, PDGF-AB could not be detected at 24 hours . At 48 hours, both in absolute concentrations and on a per-cell basis, PDGF-AB productions in 125 and 500 ng/ml of FGF-10 were markedly greater than those of negative control at 48 and 72 hours (P<0.05), and the secretion of PDGF-AB stimulated by FGF-10 was in a dose-dependent fashion . For cells cultured at low density, although the concentrations of VEGF in 16-500 ng/ml of FGF-10 were significantly higher than that in negative control (P<0.05) at 72 hours, on a per-cell basis, they were not greater than that of negative control . The secretion of VEGF in positive control was greater than that of FGF-10 at 24-72 hours . CONCLUSION: The effect of FGF-10 on increasing secretion of TGF-alpha and PDGF-AB might mediate the actions of FGF-10 on keratinocytes, fibroblasts and endothelial cells to promote re-epithelialization as well as granulation tissue deposition during wound healing.

Biotechnol Bioeng, 2004 Jan 20, 85(2), 130 - 7
Optimization of an in vivo plant P450 monooxygenase system in Saccharomyces cerevisiae; Jiang H et al.; Cytochrome P450s are heme-thiolate oxygenases involved in a wide number of reactions such as epoxidation, hydroxylation, and demethylation . Heterologously expressed eukaryotic P450s are potentially useful biocatalysts for stereospecific oxygenation reactions under mild conditions . Numerous factors, such as intracellular pH, cytochrome P450, cytochrome P450 reductase, NADPH, and oxygen concentration all influence the in vivo activity . To systematically examine these factors, we selected ferulate 5-hydroxylase (F5H), a plant P450, with the Saccharomyces cerevisiae WAT11 strain as an expression host . Two media compositions and two cultivation procedures were investigated to optimize the in vivo activity of F5H . We modified a previously published selective growth medium (Pompon et al . {1996} Methods Enzymol 272:51-64) that increased the specific growth rate and cell yield of the host strain . A cultivation procedure with separate growth and induction stages that each contained selective media resulted in a 45% increase of whole cell F5H specific activity . In a medium designed for simultaneous growth and induction, we observed a 2.6-fold higher specific F5H activity, but substantially lower cell yield . Surprisingly, in this medium the higher specific F5H activity did not correlate with a higher P450 concentration . The effects of addition of the first committed heme precursor, delta-aminolevulinic acid, and Fe(III) at the beginning of induction period were also studied for our two-stage procedure . A small, but significant (P < 0.05) increase in whole cell F5H activity was observed following ALA addition .

Tree Physiol, 2004 Mar, 24(3), 355 - 9
Nitrogen mobilization, nitrogen uptake and growth of cuttings obtained from poplar stock plants grown in different N regimes and sprayed with urea in autumn; Dong S et al.; Nitrogen mobilization, nitrogen uptake and growth of cuttings obtained from poplar stock plants fertigated with different nitrogen (N) treatments and sprayed with urea in autumn were studied . Stock plants propagated from poplar cuttings were trained to a single shoot and fertigated with 0, 5, 10, 15 or 20 mmol l(-1) N during the first growing season . In October, a subset of stock plants from each N fertigation treatment was sprayed twice with either 3% urea or water, and overwintered outside . In March, total tree biomass and total N concentration and content of stems were estimated for stock plants in each treatment, and cuttings were taken from the middle of each stock plant and stored in plastic bags at 2 degrees C . In mid-April, cuttings were planted in 7.5-l pots containing N-free medium and grown outdoors with a weekly fertigation with nutrient solution containing 0 or 10 mmol l(-1) 15NH4 15NO3 . In mid-July, cuttings were harvested, and new shoot (new stems and leaves), shank (old cutting stem) and roots were analyzed for new biomass growth and total N and 15N content . Growth of stock plants was positively related to N supply in the previous growing season . Foliar urea application in autumn had no effect on subsequent stock plant growth even though urea sprays increased both N concentration and content in stem tissues . Biomass growth of cuttings obtained from stock plants was closely related to their N content when the cuttings were grown in an N-free medium regardless of previous treatments applied to the stock plants . When N was supplied in the growth medium, the strength of the relationship between regrowth and N content of cuttings was significantly reduced . Cuttings from stock plants treated with foliar urea and grown in a N-free medium remobilized between 75 and 82% of their total N for new growth, whereas cuttings from plants receiving no urea spray remobilized only between 60 and 69% of their total N for new growth . Current N fertilization of the cuttings reduced the percentage of N remobilized . We conclude that new growth of poplar cuttings in spring was more dependent on currently applied N than on reserve N, and urea N applied as a spray in autumn was more easily remobilized than N taken up by roots during the previous season.

Ann Surg Oncol, 2004 Jan, 11(1), 99 - 104
A new in vitro assay for human tumor angiogenesis: three-dimensional human tumor angiogenesis assay; Gulec SA et al.; BACKGROUND: A human tissue-based angiogenesis assay is needed to study the biology of angiogenesis in human tumor tissue and to tailor drug selection for patients . METHODS: Fragments of tumor tissue are embedded in fibrin gels containing medium 199, endothelial growth medium, fetal bovine serum, and epsilon-aminocaproic acid . Tumor implants sprout angiogenic vessels that progressively grow into the fibrin matrix . The differential growth pattern of tumor cells and angiogenic vessels in the fibrin gel matrix separates the angiogenic vessels and the tumor stroma into independently observable regions (vessel and tumor compartments) . The reproducibility of the assay was tested by using fresh tissue obtained from human tumor xenografts (IMR-32 {neuroblastoma}, MDA-MB-231 {breast cancer}, and LNCaP {prostate cancer}) grown in nude mice and from fresh surgical breast and thyroid cancer specimens . RESULTS: All tumor fragments studied showed angiogenic sprouting into the fibrin matrix . This created an angiogenic vessel compartment, which was separate from the tumor fragment . The capillary nature of sprouting was confirmed histologically by factor VIII immunohistochemistry . The angiogenic growth fraction was >80% in all groups studied . CONCLUSIONS: This assay may allow functional assessment of the angiogenic potential of human tumors and simultaneous evaluation of a therapeutic agent's antitumor and antiangiogenic effects by virtue of its dual-compartmental structure.

Aerosp Am, 2003 Dec, 41(12), 86 - 7
Life sciences; Martin-Brennan C et al.; NASA: Space life sciences research activities are reviewed for 2003 . Many life sciences experiments were lost with the tragic loss of STS-107 . Life sciences experiments continue to fly as small payloads to the International Space Station (ISS) via the Russian Progress vehicle . Health-related studies continue with the Martian Radiation Environment Experiment (MARIE) aboard the Odyssey spacecraft, collecting data on the radiation environment in Mars orbit . NASA Ames increased nanotechnology research in all areas, including fundamental biology, bioastronautics, life support systems, and homeland security . Plant research efforts continued at NASA Kennedy, testing candidate crops for ISS . Research included plant growth studies at different light intensities, varying carbon dioxide concentrations, and different growth media . Education and outreach efforts included development of a NASA/USDA program called Space Agriculture in the Classroom . Canada sponsored a project called Tomatosphere, with classrooms across North America exposing seeds to simulated Mars environment for growth studies . NASA's Office of Biological and Physical Research released an updated strategic research plan .

In Vitro Cell Dev Biol Anim, 2003 Sep-Oct, 39(8-9), 388 - 94
Bovine pituitary extract provides remarkable protection against oxidative stress in human prostate epithelial cells; Kent KD et al.; Bovine pituitary extract (BPE) is routinely used as a mitogenic supplement in serum-free growth medium . In addition to its mitogenic activity, BPE contains a variety of growth factors and hormones with reported antioxidant activity . This study examines the antioxidant potential of BPE in nontumorigenic human prostate epithelial cells (RWPE-1) . Treatment of RWPE-1 cells with BPE (50 microg/ml) provided significant protection against H(2)O(2)-induced cell death, deoxyribonucleic acid fragmentation, protein oxidation, and membrane damage . Treatment with heat (71 degrees C, 10 min) and proteolytic enzymes reduced the antioxidant activity of BPE, suggesting that proteins present in BPE may be responsible for the antioxidant activity . Residual catalase activity present in BPE was responsible for a portion (30%) of the antioxidant activity . Interestingly, RWPE-1 cells treated with BPE and H(2)O(2) rapidly accumulated intracellular reactive oxygen species (ROS) to a greater extent than cells receiving only H(2)O(2) . Pretreatment of RWPE-1 cells with tyrosine kinase inhibitors (genistein, tyrphostin 47, and AG-1296) before the addition of H(2)O(2) diminished BPE protection against H(2)O(2)-induced cell death, whereas treatment with purified mitogens commonly found in BPE, growth hormone and basic fibroblast growth factor, did not protect against oxidative damage . Taken together, these data suggest that BPE contains proteins or protein complexes with remarkable antioxidant activity . These yet unidentified compounds appear to confer protection against H(2)O(2)-induced cell death by tyrosine kinase-dependent pathways that increase intracellular ROS generation . The antioxidant activity of BPE may represent a confounding variable when studying oxidative stress in cells maintained in BPE-supplemented serum-free medium.

Transfus Med Rev, 2004 Jan, 18(1), 11 - 24
Improving the bacteriological safety of platelet transfusions; Blajchman MA et al.; Despite the increased application of aseptic techniques for blood collection and the preparation of platelet concentrates, morbidity and mortality arising from the transfusion of bacterially contaminated allogeneic platelet products persist . This problem exists because stored platelet concentrates represent a nearly ideal growth medium for bacteria and because they are stored at temperatures (22 degrees +/- 2 degrees C) that facilitate bacterial growth . The presence of bacteria in blood components including platelets has been a problem for many decades and currently is the most common microbiological cause of transfusion-associated morbidity and mortality . A variety of strategies have been devised and/or proposed in an attempt to try to reduce the risk of transfusion-associated sepsis . These include pretransfusion bacterial detection, efforts to reduce the likelihood of bacterial contamination, the optimization of blood product processing and storage, reducing recipient exposure, and the introduction of pathogen inactivation methodology . With regard to doing bacterial detection, a number of automated detection systems have become available to test for contaminated platelet components, but their utility to some extent is restricted by the time they take to indicate the presence of bacteria and/or their lack of sensitivity to detect initially low bacterial loads . A variety of other approaches has been shown to reduce the risk of bacterial contamination and include filtration to remove leukocytes and bacteria, diversion of the initial aliquot of blood during donation, and improved donor skin disinfection . Platelet pathogen inactivation methods under investigation include the addition of L-carnitine, gamma-irradiation, riboflavin plus UVA irradiation, and amotosalen HCl plus UVA irradiation . The latter process is licensed for clinical use with platelets in some countries in Europe . All of these approaches, either collectively or individually, hold considerable promise that the prevalence of adverse events associated with bacteria in platelet products will decline significantly in the very foreseeable future.

J Mol Model (Online), 2004 Feb, 10(1), 76 - 84 Epub 2003 Dec 20.
A quantitative structure-antifungal activity relationship study of oxygenated aromatic essential oil compounds using data structuring and PLS regression analysis; Voda K et al.; Twenty two oxygenated aromatic essential oil compounds were chosen for the study of the antifungal activity against two wood-decaying fungi, the white-rot Trametes versicolor, which mainly metabolizes lignin, and the brown-rot Coniophoha puteana, which digests cellulose in plant cell walls . Minimal inhibitory concentrations (MICs) were determined by the agar dilution method, using dimethyl sulfoxide (DMSO) as the solvent for the selected compounds and potato-dextrose agar (PDA) as the growth medium for both fungi . The MICs were then used to generate a tree structure, which represents the structuring of the essential oil compounds by the nature and position of the substituents in their aromatic rings, and as dependent variables (log(1/MIC)) in the QSAR analysis . Data structuring proved that a relationship between the molecular structures of the essential oil compounds and their antifungal activity exists, and the hypotheses derived therefrom were complemented by performing a QSAR analysis using the partial least squares (PLS) method . Statistically significant PLS models were obtained with the 1-octanol-water partition coefficient ( C log P), the energy of the highest occupied molecular orbital ( E(HOMO)), and the number of hydrogen-bond donor atoms in the molecules of the compounds studied (Donor) for T . versicolor and with C log P and the fractional negative surface area (FNSA1) for C . puteana.Figure Tree structure representing the structuring of the oxygenated aromatic essential-oil compounds by the position and nature of their substituents in the aromatic ring

J Immunol, 2004 Jan 1, 172(1), 274 - 81
Colorectal cancer cells induce lymphocyte apoptosis by an endothelial monocyte-activating polypeptide-II-dependent mechanism; Murray JC et al.; Endothelial monocyte-activating polypeptide-II (EMAP-II) was first isolated from cell growth medium conditioned by tumor cells, and is closely related or identical with the p43 component of the mammalian multisynthase complex . In its secreted form, EMAP-II has multiple cytokine-like activities in vitro, inducing procoagulant activity on the surface of endothelial cells, increasing expression of E- and P-selectins and TNF-R1, and directing migration of monocytes and neutrophils . EMAP-II has also been shown to induce apoptosis in endothelial cells, leading to the suggestion that it is a proinflammatory polypeptide with antiangiogenic activity . The role of secreted EMAP-II in tumors remains poorly understood, and we hypothesized that EMAP-II may play a role in immune evasion by tumor cells . We investigated its effects on lymphocytes, using recombinant protein, or colorectal cancer cell lines, as a source of native EMAP-II . Recombinant EMAP-II inhibits DNA synthesis and cell division, and induces apoptosis in mitogen-activated lymphocytes in PBMC preparations, and in Jurkat T cells . Native EMAP-II, released by or expressed on the surface of colorectal carcinoma cells, also induces activation of caspase 8 and apoptosis of PBLs and Jurkat cells, which are partially blocked by addition of Abs against EMAP-II . Thus, activated lymphocytes, along with proliferating endothelial cells, are targets for the cytotoxic activity of EMAP-II . Membrane-bound and soluble EMAP-II appear to play multiple roles in the tumor microenvironment, one of which is to assist in immune evasion.

Neurochem Int, 2004 May, 44(6), 459 - 67
Involvement of histone phosphorylation in apoptosis of human astrocytes after exposure to saline solution; Enomoto R et al.; We have previously found using inhibitors of protein phosphatase that phosphorylation of histones may be involved in thymocyte apoptosis . In this study, we examined whether histone modification occurs in astrocyte apoptosis induced by a pathological condition in the absence of drug . Incubation of cultured human astrocytes with growth medium for 24 h after exposure to saline solution for 30 min induced an increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and nuclear condensation, biochemical and morphological hallmarks of apoptotic cell death . Acetic acid-urea-Triton X-100 (AUT) gel electrophoresis of the nuclear histone fraction and N-terminal peptide analysis showed that the treatment with saline solution caused rapid changes in phosphorylation of H2A subfamilies, but not in histone acetylation . The phosphorylation of the two subtypes increased markedly, whereas the phosphorylation of one subtype decreased . In contrast, exposure to ACF-95, an artificial cerebrospinal fluid (CSF), was associated with little induction of apoptotic cell death and induced less changes in histone phosphorylation . These results support the previous idea that chemical modification of histones is involved in the DNA fragmentation in astrocytes undergoing apoptosis.

Plant Physiol, 2004 Jan, 134(1), 452 - 9 Epub 2003 Dec 18.
Regulation of K+ transport in tomato roots by the TSS1 locus . Implications in salt tolerance; Rubio L et al.; The tss1 tomato (Lycopersicon esculentum) mutant exhibited reduced growth in low K+ and hypersensitivity to Na+ and Li+ . Increased Ca2+ in the culture medium suppressed the Na+ hypersensitivity and the growth defect on low K+ medium of tss1 seedlings . Interestingly, removing NH4+ from the growth medium suppressed all growth defects of tss1, suggesting a defective NH4(+)-insensitive component of K+ transport . We performed electrophysiological studies to understand the contribution of the NH4(+)-sensitive and -insensitive components of K+ transport in wild-type and tss1 roots . Although at 1 mm Ca2+ we found no differences in affinity for K+ uptake between wild type and tss1 in the absence of NH4+, the maximum depolarization value was about one-half in tss1, suggesting that a set of K+ transporters is inactive in the mutant . However, these transporters became active by raising the external Ca2+ concentration . In the presence of NH4+, a reduced affinity for K+ was observed in both types of seedlings, but tss1 at 1 mm Ca2+ exhibited a 2-fold higher Km than wild type did . This defect was again corrected by raising the external concentration of Ca2+ . Therefore, membrane potential measurements in root cells indicated that tss1 is affected in both NH4(+)-sensitive and -insensitive components of K+ transport at low Ca2+ concentrations and that this defective transport is rescued by increasing the concentration of Ca2+ . Our results suggest that the TSS1 gene product is part of a crucial pathway mediating the beneficial effects of Ca2+ involved in K+ nutrition and salt tolerance.

Acta Otorhinolaryngol Ital, 2003 Jun, 23(3), 168 - 74
Allergic fungal sinusitis . A naso-sinusal specific hyperreactivity for an infectious disease?
Corradini C, Del Ninno M, Schiavino D, Patriarca G, Paludetti G.
Allergic fungal sinusitis (AFS) is a rare disease of naso-sinusal complex affecting mainly young, immunocompetent adults who complain of chronic rhinitis and/or recurrent nasal polyposis despite medical and/or surgical treatment . Aim of the study is to analyse, from an allergological and otorhinolaryngological point of view, patients affected by the so-called "allergic fungal sinusitis" in order to better define the relationship between fungi present in naso-sinusal secretions and the host's immunoreactivity . From February 2001 to January 2002, 24 selected patients (13 male 11 female) age range 25-65 years (mean 45), with chronic rhinosinusitis, with a positive fungal examination of nasal secretion, underwent allergological evaluation . All patients were positive for diagnostic criteria of allergic fungal sinusitis and, in all patients, nasal lavage was performed for microscopic examination by fluorescence . Samples were then cultured on Sabouraud growth media for identification of the fungus . Skin prick tests (SPT) were then performed with the 15 main inhalant allergens and twelve fungal allergens (Bracco) . The total IgE serum level (PRIST), the specific fungal IgE and the eosinophilic cationic protein were then investigated by means of an immuno-fluorine enzymatic method . Finally, a nasal provocation test was carried out with diluted solutions (1/100, 1/10) and with a pure solution of fungal allergens, selected according to microbiological examination of nasal secretion of each subject . Prick tests were positive for seasonal and perennial allergens in 5 patients (21%), while prick tests with fungi were positive in only 4 patients (16.6%) . Total IgE levels were higher than in normals (200 KU/l) in 6 patients (25%) (mean 364.74 KU/l) . In another 18 patients, total IgE were normal . Specific IgE levels for the tested fungi and eosinophilic cationic protein levels were within normal range in all patients . Nasal provocation test was negative in all patients . Presence of fungi in nasal secretions of patients with AFS does not appear to be correlated with an allergic status to the isolated fungus . A role for IgE in either the aetiology or the pathophysiology of allergic fungal sinusitis in unlikely, and probably the diagnostic criteria for allergic fungal sinusitis should not include type I hypersensitivity, since no confirmed evidence exists that IgE-mediated type I hypersensitivity is involved in the pathophysiology of allergic fungal sinusitis.

J Dairy Sci, 2003 Nov, 86(11), 3416 - 22
Assimilation of cholesterol by yeast strains isolated from infant feces and Feta cheese; Psomas EI et al.; Eight yeast strains isolated from infant feces and the traditional Greek Feta cheese, selected for their probiotic properties, were tested along with a commercially available strain of Saccharomyces boulardii for their ability to remove cholesterol from a growth medium (yeast extract glucose peptone broth) supplemented with 0.3% Oxgall . The amount of cholesterol removed during 72 h of growth at 37 degrees C revealed significant variations among the yeast strains examined . Two isolates from infant feces, namely Saccharomyces cerevisiae KK1 and Isaatchenkia orientalis KK5.Y.1 and one isolate from Feta cheese, namely S . cerevisiae 832, along with the commercial strain S . boulardii, were able to remove cholesterol from the growth medium after 48 h of incubation at 37 degrees C . However, Saccharomyces strains proved to be able to remove cholesterol even after 24 h of growth at 37 degrees C . The cholesterol removed from the growth medium was not metabolically degraded but was rather assimilated into the yeast cells . The ability to assimilate cholesterol in vitro and to tolerate low pH levels, gastric juice, and bile indicate that S . cerevisiae 832, and especially S . cerevisiae KK1 and I . orientalis KK5.Y.1 (being more bile and gastric juice tolerant because of their human origin) may be promising candidate strains for use as probiotics.

J Chromatogr B Analyt Technol Biomed Life Sci, 2004 Jan 25, 799(2), 343 - 7
Separation of manganese peroxidase isoenzymes on strong anion-exchange monolithic column using pH-salt gradient; Podgornik H et al.; Different chromatographic methods including chromatofocusing are used for separation of manganese peroxidase (MnP) isoforms and their isolation from the fungal growth medium . We tested strong anion exchange methacrylate based monolithic columns as a stationary phase for fast separation of MnP's . Sodium acetate buffers of two different pH values (6 and 4) were used for formation of reproducible pH gradient . The entire cycle, involving analysis and column regeneration, was completed in 3 min . Use of pH gradient showed better MnP isoform separation comparing to the salt gradient, while application of combined pH-salt gradient, resulted in further improvement.

Genetics, 2003 Nov, 165(3), 1045 - 58
Cellular differentiation in response to nutrient availability: The repressor of meiosis, Rme1p, positively regulates invasive growth in Saccharomyces cerevisiae; van Dyk D et al.; In the yeast Saccharomyces cerevisiae, the transition from a nutrient-rich to a nutrient-limited growth medium typically leads to the implementation of a cellular adaptation program that results in invasive growth and/or the formation of pseudohyphae . Complete depletion of essential nutrients, on the other hand, leads either to entry into a nonbudding, metabolically quiescent state referred to as G0 in haploid strains or to meiosis and sporulation in diploids . Entry into meiosis is repressed by the transcriptional regulator Rme1p, a zinc-finger-containing DNA-binding protein . In this article, we show that Rme1p positively regulates invasive growth and starch metabolism in both haploid and diploid strains by directly modifying the transcription of the FLO11 (also known as MUC1) and STA2 genes, which encode a cell wall-associated protein essential for invasive growth and a starch-degrading glucoamylase, respectively . Genetic evidence suggests that Rme1p functions independently of identified signaling modules that regulate invasive growth and of other transcription factors that regulate FLO11 and that the activation of FLO11 is dependent on the presence of a promoter sequence that shows significant homology to identified Rme1p response elements (RREs) . The data suggest that Rme1p functions as a central switch between different cellular differentiation pathways.

J Biol Chem, 2004 Feb 20, 279(8), 6337 - 44 Epub 2003 Dec 08.
Photo-oxidative stress in a xanthophyll-deficient mutant of Chlamydomonas; Baroli I et al.; When there is an imbalance between the light energy absorbed by a photosynthetic organism and that which can be utilized in photosynthesis, photo-oxidative stress can damage pigments, proteins, lipids, and nucleic acids . In this work we compared the wild type and a xanthophyll-deficient mutant of Chlamydomonas reinhardtii in their response to high amounts of light . Wild-type Chlamydomonas cells were able to acclimate to high amounts of light following transfer from low light conditions . In contrast, the npq1 lor1 double mutant, which lacks protective xanthophylls (zeaxanthin and lutein) in the chloroplast, progressively lost viability and photosynthetic capacity along with destruction of thylakoid membrane protein-pigment complexes and accumulation of reactive oxygen species and membrane lipid peroxides . Loss of viability was partially rescued by lowered oxygen tension, suggesting that the high sensitivity of the mutant to light stress is caused by the production of reactive oxygen species in the chloroplast . Cell death was not prevented by the addition of an organic carbon source to the growth medium, demonstrating that the photo-oxidative damage can target other essential chloroplast processes besides photosynthesis . From the differential sensitivity of the mutant to exogenously added pro-oxidants, we infer that the reactive oxygen species produced during light stress in npq1 lor1 may be singlet oxygen and/or superoxide but not hydrogen peroxide . The bleaching phenotype of npq1 lor1 was not due to enhanced photodamage to photosystem II but rather to a less localized phenomenon of accumulation of photo-oxidation products in chloroplast membranes.

Biotechnol Adv, 2004 Jan, 22(3), 189 - 259
Fungal morphology and metabolite production in submerged mycelial processes; Papagianni M; The use of fungi for the production of commercial products is ancient, but it has increased rapidly over the last 50 years . Fungi are morphologically complex organisms, differing in structure at different times in their life cycle, differing in form between surface and submerged growth, differing also with the nature of the growth medium and physical environment . Many genes and physiological mechanisms are involved in the process of morphogenesis . In submerged culture, a large number of factors contribute to the development of any particular morphological form . Factors affecting morphology include the type and concentration of carbon substrate, levels of nitrogen and phosphate, trace minerals, dissolved oxygen and carbon dioxide, pH and temperature . Physical factors affecting morphology include fermenter geometry, agitation systems, rheology and the culture modes, whether batch, fed-batch or continuous . In many cases, particular morphological forms achieve maximum performance . It is a very difficult task to deduce unequivocal general relationships between process variables, product formation and fungal morphology since too many parameters influence these interrelationships and the role of many of them is still not fully understood . The use of automatic image analysis systems during the last decade proved an invaluable tool for characterizing complex mycelial morphologies, physiological states and relationships between morphology and productivity . Quantified morphological information can be used to build morphologically structured models of predictive value . The mathematical modeling of the growth and process performance has led to improved design and operation of mycelial fermentations and has improved the ability of scientists to translate laboratory observations into commercial practice . However, it is still necessary to develop improved and new experimental techniques for understanding phenomena such as the mechanisms of mycelial fragmentation and non-destructive measurement of concentration profiles in mycelial aggregates . This would allow the establishment of a process control on a physiological basis . This review is focused on the factors influencing the fungal morphology and metabolite production in submerged culture.

J Am Chem Soc, 2003 Dec 17, 125(50), 15666 - 70
Unusual salt stability in highly charged diblock co-polypeptide hydrogels; Nowak AP et al.; The stability and properties of dilute solution hydrogels, synthesized by transition metal mediated polymerization of amino acid N-carboxyanhydrides (NCAs), have been studied in deionized (DI) water as well as various ionic media . These hydrogels are diblock amphiphilic copolymers of hydrophilic, charged segments of poly(l-lysine HBr) or poly(l-glutamic acid sodium salt), and helical, hydrophobic segments of poly(l-leucine) . While many of these samples are able to form strong gels in deionized water at polymer concentrations as low as 0.25 wt %, stability in salt or buffer solutions was found to be only achieved at moderately higher polymer concentrations ( approximately 3.0 wt %) . We have adjusted relative copolymer compositions and molecular weights to optimize hydrogel strength and polymer solubility in salt concentrations up to 0.5 M NaCl, as well as in cell growth media and aqueous buffers of varying pH . These materials are unique since they do not collapse in high ionic strength media, even though gel formation is contingent upon the presence of highly charged polyelectrolyte segments . The remarkable properties of these hydrogels make them excellent candidates for use as scaffolds in biomedical applications, such as tissue regeneration.

What Is Cell Biology?, What Is Protein?, What Is Yeast?, What Is Activated Sludge?, What Is Bioremediation?, e, Bacterium, a, Microorganism, e, Microbe, e, Microbiology, r, Bacteriology, c, Streptococcal, a, Streptococci, e, Antibiotics, c, Lactobacillus, n, Antibiotics, e, Micrococci, e, Streptococci, e, Antibiotics, c, Micrococci, s, Sepsis, i, Bioremediation, c, Antimicrobials, c, Ps. putida, e, Growth media, o, Bacillus subtilis, a, Enterobacters, c, Cryptococci, n, Rhizobacterium, e, Escherichia coli, i, Enterococci, a, Escherichia coli

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005