J Immunol Methods, 2003 Dec, 283(1-2), 307 - 15 Isolation and transplantation of allogeneic pulmonary endothelium derived from GFP transgenic mice; Ewing P et al.; The isolation of primary endothelial cells from murine tissues has long been a challenge and remains a difficult task . Using GFP transgenic C57/BL6 mice as donors, we describe a reliable method to isolate pulmonary endothelial cells by flow cytometry after staining with DiI-Ac-low density lipoprotein (LDL) . After mechanical dissociation of murine lung tissue and enzymatic digestion, adherent cells can be quickly stained and sorted by flow cytometry . The isolated cells express endothelial cell markers such as CD31, MECA32 and CD106 and stained positive for Isolectin B4 . After 50-fold expansion using standard endothelial growth media, cells could be transplanted into lethally irradiated allogeneic hosts and were detectable using fluorescence microscopy up to 24-h post-transplantation in pulmonary tissue. Mech Ageing Dev, 2003 Dec, 124(10-12), 1059 - 63 Biodemographic trajectories of age-specific reproliferation from stationary phase in the yeast Saccharomyces cerevisiae seem multiphasic; Gendron CM et al.; Ageing is usually seen as a monotonic decline of functions and survival . However, recent studies reported that age-specific mortality rates increased and then leveled off or even declined at later ages in several species including humans . Preliminary data using the yeast, Saccharomyces cerevisiae, demonstrated an even more complicated, non-monotonic pattern of reproliferation after stationary phase (i.e . the ability of a cell to exit stationary phase and form a colony) . In the present article, we conducted a study of the age-specific reproliferation rates of yeast populations . Stationary phase yeast cells were maintained in water and the reproliferation rates were estimated by the number of yeast able to exit stationary phase on rich growth media . We showed that the age-specific reproliferation rates in yeast seem to rise, fall and rise again . Furthermore, we observed this pattern in different experiments and in different genotypes and established that this pattern was not due to genetic heterogeneity of the populations. FEMS Microbiol Lett, 2003 Dec 5, 229(1), 15 - 21 The glutamine synthetase of Prevotella bryantii B(1)4 is a family III enzyme (GlnN) and glutamine supports growth of mutants lacking glutamate dehydrogenase activity; Wen ZT et al.; Prevotella spp . are believed to play a central role in ruminal nitrogen metabolism, but little is understood about the genetics and biochemistry of nitrogen assimilation and regulation in these bacteria . The gene encoding a family III glutamine synthetase (GSIII, glnN) in Prevotella bryantii B(1)4 was cloned by Escherichia coli mutant complementation, and enzyme assays as well as Northern blot analysis showed that maximal enzyme activity and glnN transcription occurred in cells grown under nitrogen-limiting conditions . Addition of methionine sulfoximine (MSX), a GS inhibitor, terminated bacterial growth when ammonium was provided as the sole nitrogen source, but the inhibitory effect could be overcome by the inclusion of either L-glutamine or trypticase in the growth medium . A P . bryantii mutant lacking glutamate dehydrogenase (GdhA) activity was isolated by ethylmethylsulfonate mutagenesis . Growth studies with different nitrogen sources showed that the mutant strain was still capable of growth with ammonium as the sole nitrogen source, albeit at a decreased growth rate . The mutant strain could also grow with L-glutamine as a nitrogen source in the presence of MSX . These data suggest that GlnN provides an effective route of ammonium assimilation for P . bryantii, in addition to that afforded by the glutamate dehydrogenase pathway. Pain, 2003 Dec, 106(3), 347 - 56 Alterations in dorsal horn neurones in a rat model of cancer-induced bone pain; Urch CE et al.; Cancer-induced bone pain is a major clinical problem . A rat model based on intra-tibial injection of MRMT-1 mammary tumour cells was used to mimic progressive cancer-induced bone pain . At the time of stable behavioural changes (decreased thresholds to mechanical and cold stimuli) and bone destruction, in vivo electrophysiology was used to characterize natural (mechanical, thermal, and cold) and electrical-evoked responses of superficial and deep dorsal horn neurones in halothane-anaesthetized rats . Receptive field size was significantly enlarged for superficial neurones in the MRMT-1 animals . Superficial cells were characterised as either nociceptive specific (NS) or wide dynamic range (WDR) . The ratio of WDR to NS cells was substantially different between sham operated (growth media alone) (26:74%) and MRMT-1 injected rats (47:53%) . NS cells showed no significant difference in their neuronal responses in MRMT-1-injected compared to sham rats . However, superficial WDR neurones in MRMT-1-injected rats had significantly increased responses to mechanical, thermal and electrical (A beta-, C fibre-, and post-discharge evoked response) stimuli . Deep WDR neurones showed less pronounced changes to the superficial dorsal horn, however, the response to thermal and electrical stimuli, but not mechanical, were significantly increased in the MRMT-1-injected rats . In conclusion, the spinal cord is significantly hyperexcitable with previously superficial NS cells becoming responsive to wide-dynamic range stimuli possibly driving this plasticity via ascending and descending facilitatory pathways . The alterations in superficial dorsal horn neurones have not been reported in neuropathy or inflammation adding to the evidence for cancer-induced bone pain reflecting a unique pain state. EMBO J, 2003 Dec 15, 22(24), 6505 - 15 Growth inhibition by the mammalian SWI-SNF subunit Brm is regulated by acetylation; Bourachot B et al.; In mammalian cells, the SWI-SNF chromatin-remodeling complex is a regulator of cell proliferation, and overexpression of the catalytic subunit Brm interferes with cell cycle progression . Here, we show that treatment with histone deacetylase (HDAC) inhibitors reduces the inhibitory effect of Brm on the growth of mouse fibroblasts . This observation led to the identification of two carboxy-terminal acetylation sites in the Brm protein . Mutation of these sites into non-acetylatable sequences increased both the growth-inhibitory and the transcriptional activities of Brm . We also show that culture in the presence of HDAC inhibitors facilitates the isolation of clones overexpressing Brm . Removal of the HDAC inhibitors from the growth medium of these clones leads to downregulation of cyclin D1 . This downregulation is absent in cell transformed by oncogenic ras. FEMS Yeast Res, 2003 Dec, 4(3), 285 - 96 The Saccharomyces cerevisiae alcohol acetyl transferase gene ATF1 is a target of the cAMP/PKA and FGM nutrient-signalling pathways; Verstrepen KJ et al.; The ATF1-encoded Saccharomyces cerevisiae yeast alcohol acetyl transferase I is responsible for the formation of several different volatile acetate esters during fermentations . A number of these volatile esters, e.g . ethyl acetate and isoamyl acetate, are amongst the most important aroma compounds in fermented beverages such as beer and wine . Manipulation of the expression levels of ATF1 in brewing yeast strains has a significant effect on the ester profile of beer . Northern blot analysis of ATF1 and its closely related homologue, Lg-ATF1, showed that these genes were rapidly induced by the addition of glucose to anaerobically grown carbon-starved cells . This induction was abolished in a protein kinase A (PKA)-attenuated strain, while a PKA-overactive strain showed stronger ATF1 expression, indicating that the Ras/cAMP/PKA signalling pathway is involved in this glucose induction . Furthermore, nitrogen was needed in the growth medium in order to maintain ATF1 expression . Long-term activation of ATF1 could also be obtained by the addition of the non-metabolisable amino acid homologue beta-L-alanine, showing that the effect of the nitrogen source did not depend on its metabolism . In addition to nutrient regulation, ATF1 and Lg-ATF1 expression levels were also affected by heat and ethanol stress . These findings help in the understanding of the effect of medium composition on volatile ester synthesis in industrial fermentations . In addition, the complex regulation provides new insights into the physiological role of Atf1p in yeast. Plant Cell Rep, 2003 Dec, 22(5), 320 - 7 Epub 2003 Aug 29. Somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye ( Secale cereale L.); Ma R et al.; A method for somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (Secale cereale L . cv . Auvinen) was developed . Fast-growing and friable embryogenic calli with a high regeneration capacity were induced from immature rye inflorescences using modified MS medium . These friable embryogenic calli were used for suspension culture initiation in liquid AA medium . A high yield of protoplasts was obtained from suspension cell clumps after 3-5 days of subculture . Isolated protoplasts were cultured in KM8p medium . The frequency of protoplast cell divisions and colony formations in liquid culture medium were similar to those on agarose-solidified medium . Compact embryogenic calli were developed from protoplast-derived microcalli in growth medium mMS . Approximately 7% of the transferred embryogenic calli produced green shoots on N6 regeneration medium . Of 33 green plants, 28 were fertile with normal flowering and seed set . The ratio of green and albino plantlets was 1:4 . Rye protoplast-derived green plants showed normal diploid characters as determined by flow cytometer analysis and chromosome counting. J Magn Reson, 2003 Dec, 165(2), 237 - 47 NMR assignment of protein side chains using residue-correlated labeling and NOE spectra; Mueller GA et al.; A new approach for the isotopic labeling of proteins is proposed that aims to facilitate side chain resonance assignments . Residue-correlated (RC) labeling is achieved by the expression of a protein on a medium containing a mixture of labeled, e.g., {U-13C,15N}amino acids, and NMR silent, {U-2H}amino acids . De novo synthesis of amino acids was suppressed by feedback inhibition by the amino acids in the growth medium and by the addition of beta-chloro-L-alanine, a transaminase inhibitor . Incorporation of these amino acids into synthesized proteins results in a relative diminution of inter-residue NOE interactions and a relative enhancement of intra-residue NOEs . Comparison of the resulting NOE spectra with those obtained from a uniformly labeled sample allows identification of intra-residue NOE peaks . Thus, this approach provides direct information for sidechain assignments in the NOE spectra, which are subsequently used for structural analysis . We have demonstrated the feasibility of this strategy for the 143 amino acid nuclease inhibitor NuiA, both at 35 degrees C, corresponding to a rotational correlation time of 9.5 ns, and at 5 degrees C, corresponding to a rotational correlation time of 22 ns. Anal Bioanal Chem, 2004 Jan, 378(2), 391 - 5 Epub 2003 Nov 28. Fermentation monitoring using multisensor systems: feasibility study of the electronic tongue; Esbensen K et al.; The electronic tongue based on an array of 30 non-specific potentiometric chemical sensors has been applied to qualitative and quantitative monitoring of a batch fermentation process of starting culture for light cheese production . Process control charts were built by using PLS regression and data from fermentations run under "normal" operating conditions . Control charts allow discrimination of samples from fermentation batches run under "abnormal" operating conditions from "normal" ones at as early as 30-50% of fully evolved fermentations . The capability of the electronic tongue to quantify concentrations of important organic acids (citric, lactic and orotic) in the present type of fermentation media was demonstrated . Average prediction errors were assessed in the range 5-13% based on test set validation . Correlation between peptide profiles determined using HPLC and the electronic tongue output was also established . The electronic tongue was demonstrated to be a promising tool for fermentation process monitoring and quantitative analysis of growth media. Hum Reprod, 2003 Dec, 18(12), 2672 - 7 Prolactin acts as a potent survival factor against C2-ceramide-induced apoptosis in human granulosa cells; Perks CM et al.; BACKGROUND: The role of prolactin in the regulation of ovarian folliculogenesis and corpus luteal function and in particular its relationship to atresia in these structures is as yet unclear . We established a model of apoptosis in which to examine the actions of prolactin . METHOD: Granulosa cells collected from IVF-flush were cultured at 0.1-0.3 x 10(6) cells/well in growth media for 48 h, placed into serum-free media for 24 h prior to dosing for 24 h . Dose responses to C2-ceramide and prolactin were performed . Cells were then treated with an apoptotic dose of C2-ceramide alone, prolactin (100 ng/ml) alone or a combination of the two . Cell death was assessed by Trypan Blue cell counting and MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl Blue} assay and apoptosis confirmed by morphological assessment and flow cytometry . RESULTS: C2-ceramide (0-40 micro mol/l) induced a dose-dependent increase in cell death (63.8% increase at 40 micro mol/l) and, morphologically, cells exhibited classical features of apoptosis . Prolactin alone had no effect on metabolic activity or total cell number . On co- incubation, prolactin alone had no effect on cell death, whereas C2-ceramide induced an approximately 62.6% increase in apoptosis, which was inhibited in the presence of prolactin . CONCLUSIONS: Prolactin may contribute significantly to early corpus luteum formation and survival by acting as a potent antiapoptotic factor for human granulosa cells. FEMS Microbiol Lett, 2003 Nov 21, 228(2), 167 - 73 Indole-3-butyric acid (IBA) production in culture medium by wild strain Azospirillum brasilense; Martinez-Morales LJ et al.; Some microorganisms found in the soil are able to produce substances which regulate plant growth . In this study, we show the presence of a substance associated with auxin activity, identified as indole-3-butyric acid (IBA), in Azospirillum brasilense UAP 154 growth medium . A . brasilense was grown and indolic compounds were extracted from the supernatant . These were then analyzed by high performance liquid chromatography (HPLC), gas chromatography and gas chromatography mass spectrometry . The retention time was similar to those of the authentic IBA standard . The compound obtained from HPLC was collected and applied to maize seedlings (Zea mays), inducing biological activity along the roots, similar to that induced by an authentic IBA standard. J Cell Biochem, 2003 Dec 15, 90(6), 1112 - 27 BMP treatment of C3H10T1/2 mesenchymal stem cells induces both chondrogenesis and osteogenesis; Shea CM et al.; The molecular mechanisms by which bone morphogenetic proteins (BMPs) promote skeletal cell differentiation were investigated in the murine mesenchymal stem cell line C3H10T1/2 . Both BMP-7 and BMP-2 induced C3H10T1/2 cells to undergo a sequential pattern of chondrogenic followed by osteogenic differentiation that was dependent on both the concentration and the continuous presence of BMP in the growth media . Differentiation was determined by the expression of chondrogenesis and osteogenesis associated matrix genes . Subsequent experiments using BMP-7 demonstrated that withdrawal of BMP from the growth media led to a complete loss of skeletal cell differentiation accompanied by adipogenic differentiation of these cells . Continuous treatment with BMP-7 increased the expression of Sox9, Msx 2, and c-fos during the periods of chondrogenic differentiation after which point their expression decreased . In contrast, Dlx 5 expression was induced by BMP-7 treatment and remained elevated throughout the time-course of skeletal cell differentiation . Runx2/Cbfa1 was not detected by ribonuclease protection assay (RPA) and did not appear to be induced by BMP-7 . The sequential nature of differentiation of chondrocytic and osteoblastic cells and the necessity for continuous BMP treatment to maintain skeletal cell differentiation suggests that the maintenance of selective differentiation of the two skeletal cell lineages might be dependent on BMP-7-regulated expression of other morphogenetic factors . An examination of the expression of Wnt, transforming growth factor-beta (TGF-beta), and the hedgehog family of morphogens showed that Wnt 5b, Wnt 11, BMP-4, growth and differentiation factor-1 (GDF-1), Sonic hedgehog (Shh), and Indian hedgehog (Ihh) were endogenously expressed by C3H10T1/2 cells . Wnt 11, BMP-4, and GDF-1 expression were inhibited by BMP-7 treatment in a dose-dependent manner while Wnt 5b and Shh were selectively induced by BMP-7 during the period of chondrogenic differentiation . Ihh expression also showed induction by BMP-7 treatment, however, the period of maximal expression was during the later time-points, corresponding to osteogenic differentiation . An interesting phenomenon was that BMP-7 activity could be further enhanced twofold by growing the cells in a more nutrient-rich media . In summary, the murine mesenchymal stem cell line C3H10T1/2 was induced to follow an endochondral sequence of chondrogenic and osteogenic differentiation dependent on both dose and continual presence of BMP-7 and enhanced by a nutrient-rich media . Our preliminary results suggest that the induction of osteogenesis is dependent on the secondary regulation of factors that control osteogenesis through an autocrine mechanism . J Appl Microbiol, 2003, 95(5), 1124 - 33 Shewanella sp . GA-22, a psychrophilic hydrocarbonoclastic antarctic bacterium producing polyunsaturated fatty acids; Gentile G et al.; AIMS: The effects of different growth media and temperature on production of polyunsaturated fatty acids (PUFA) by Shewanella sp . GA-22 were investigated . The attempts to characterize the GA-22 genes, homologous to those of PUFA biosynthesis gene cluster, was performed . METHODS AND RESULTS: Physiological and phylogenetic characterization of new Antarctic isolate GA-22 was performed . Total fatty acids were isolated from the cells growing under different conditions and analysed by gas chromatography-mass spectrometry (GC-MS) . Using degenerated primers derived from the conserved regions within PUFA fatty acid synthase operons, five fragments of homological genes were amplified from GA-22 DNA, and two of them corresponding to pfaA and pfaC synthase subunits were sequenced . CONCLUSIONS: Strain GA-22 was shown to be able to produce three different PUFA: linoleic, arachidonic and eicosapentaenoic acids . The PUFA production was temperature- and carbon source-dependent . The deduced gene products exhibited high similarity to corresponding fatty acid synthases PfaA and PfaC . SIGNIFICANCE AND IMPACT OF STUDY: The PUFA production was detected on media supplemented with crude oil, gasoline and n-tetradecane . The apparent conservation of PUFA genes may point to the potential utilization of designed primers as functional markers in culture-independent ecological studies, and for initial screening in biotechnological fields. J Appl Microbiol, 2003, 95(6), 1334 - 42 Substrate-induced lipase gene expression and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus; Yu J et al.; AIMS: To establish a relationship between lipase gene expression and aflatoxin production by cloning the lipA gene and studying its expression pattern in several aflatoxigenic and nontoxigenic isolates of Aspergillus flavus and A . parasiticus . METHODS AND RESULTS: We have cloned a gene, lipA, that encodes a lipase involved in the breakdown of lipids from aflatoxin-producing A . flavus, A . parasiticus and two nonaflatoxigenic A . flavus isolates, wool-1 and wool-2 . The lipA gene was transcribed under diverse media conditions, however, no mature mRNA was detected unless the growth medium was supplemented with 0.5% soya bean or peanut oil or the fungus was grown in lipid-rich medium such as coconut medium . The expression of the lipase gene (mature mRNA) under substrate-induced conditions correlated well with aflatoxin production in aflatoxigenic species A . flavus (SRRC 1007) and A . parasiticus (SRRC 143) . CONCLUSIONS: Substrate-induced lipase gene expression might be indirectly related to aflatoxin formation by providing the basic building block 'acetate' for aflatoxin synthesis . No direct relationship between lipid metabolism and aflatoxin production can be ascertained, however, lipase gene expression correlates well with aflatoxin formation . SIGNIFICANCE AND IMPACT OF THE STUDY: Lipid substrate induces and promotes aflatoxin formation . It gives insight into genetic and biochemical aspects of aflatoxin formation. PLoS Biol . 2003 Nov;1(2):E28 . Epub 2003 Nov 17. Partially phosphorylated Pho4 activates transcription of a subset of phosphate-responsive genes; Springer M et al.; A cell's ability to generate different responses to different levels of stimulus is an important component of an adaptive environmental response . Transcriptional responses are frequently controlled by transcription factors regulated by phosphorylation . We demonstrate that differential phosphorylation of the budding yeast transcription factor Pho4 contributes to differential gene expression . When yeast cells are grown in high-phosphate growth medium, Pho4 is phosphorylated on four critical residues by the cyclin-CDK complex Pho80-Pho85 and is inactivated . When yeast cells are starved for phosphate, Pho4 is dephosphorylated and fully active . In intermediate-phosphate conditions, a form of Pho4 preferentially phosphorylated on one of the four sites accumulates and activates transcription of a subset of phosphate-responsive genes . This Pho4 phosphoform binds differentially to phosphate-responsive promoters and helps to trigger differential gene expression . Our results demonstrate that three transcriptional outputs can be generated by a pathway whose regulation is controlled by one kinase, Pho80-Pho85, and one transcription factor, Pho4 . Differential phosphorylation of Pho4 by Pho80-Pho85 produces phosphorylated forms of Pho4 that differ in their ability to activate transcription, contributing to multiple outputs. Biotechnol Adv, 2003 Dec, 22(1-2), 35 - 43 The concept of multiple-nutrient-limited growth of microorganisms and its application in biotechnological processes; Egli T et al.; The "law of the minimum" (Liebig's law) states that usually one nutrient restricts the maximum quantity of biomass that can be produced within a system, whereas all other nutrients are in excess . This general rule has been applied also to the growth of microorganisms, e.g., by adjusting the relative concentrations of the individual nutrients in growth media such that one of them, in the case of heterotrophic microbes, usually the carbon source, determines the maximum cell density that can be obtained in a culture . However, experimental data demonstrated that growth of microbial cultures can be limited simultaneously by two or more nutrients . These authors reported that during growth of bacteria and yeasts at a constant dilution rate in the chemostat, three distinct growth regimes were recognised as a function of the C:N ratio in the inflowing medium: (1) a clearly carbon-limited regime with the nitrogen source in excess, (2) a transition ("double-nutrient-limited") growth regime where both the carbon and the nitrogen source were below the detection limit, and (3) a clearly nitrogen-limited growth regime with the carbon source in excess . Subsequent calculations suggested that the extension and position of this double-nutrient-limited zone should be strongly dependent on the imposed growth rate: Whereas it is very narrow at high growth rates it should become very broad during slow growth . This pattern as a function of growth rate has now been confirmed for a number of different organisms . In industrial processes, microbial growth is always in some way controlled by the limited availability of nutrients, and limitation of specific nutrients is frequently used to force microbial cultures into a productive physiological state . This article will discuss what the consequences of multiple-nutrient-limited growth are for industrial processes and how the concept might be applied . Specific examples will be given that demonstrate the advantages and the potential of multiple nutrient-limited growth conditions for industrial production processes. Pharm Res, 2003 Oct, 20(10), 1690 - 6 Serially passaged human nasal epithelial cell monolayer for in vitro drug transport studies; Yoo JW et al.; PURPOSE: To evaluate the feasibility of using a serially passaged culture of human nasal epithelial cell monolayers on a permeable support for in vitro drug transport studies . The optimum conditions for passaged culture as well as the correlation between the transepithelial electrical resistance (TEER) value and drug permeability (Papp) were evaluated . METHODS: Fresh human nasal epithelial cells were collected from normal inferior turbinates and were subcultured repeatedly in serum-free bronchial epithelial cell growth media (BEGM) in petri dishes . The subcultured cells of each passage were seeded onto permeable supports at 5 x 10(5) cells/cm2 and grown in Dulbecco's modified Eagle medium (DMEM) . Morphologic characteristics were observed by scanning electron microscopy (SEM) . To verify the formation of tight junctions, actin staining and transmission electron microscopy (TEM) studies were conducted . In the drug transport study, {14C}mannitol and budesonide were selected as the paracellular and the transcellular route markers, respectively . RESULTS: Serially passaged cells were successfully cultured on a permeable support and showed significantly high TEER values up to passage 4 . After 14 days of seeding, SEM showed microvilli, and protrusions of cilia and mucin granules were detected by TEM . The paracellular marker {14C}mannitol showed a nearly constant permeability coefficient (Papp) when the TEER value exceeded 500 omega x cm2 regardless of the passage number . However, as expected, budesonide showed a higher permeability coefficient compared to {14C}mannitol and was less affected by the TEER value . CONCLUSIONS: Human nasal epithelial cell monolayers were successfully subcultured on a permeable support up to passage 4 . These cell culture methods may be useful in high-throughput screening of in vitro nasal transport studies of various drugs. Biofouling, 2003 Apr, 19 Suppl, 151 - 60 An evaluation of microbial growth and corrosion of 316L SS in glycol/seawater mixtures; Lee JS et al.; Glycol/seawater mixtures containing > 50% glycol inhibit corrosion of 316L stainless steel and do not support bacterial growth . The results indicate bacteria are able to use low concentrations of glycol (10%) as a growth medium, but bacterial growth decreased with increasing glycol concentration . Pitting potential, determined by anodic polarization, was used to evaluate susceptibility of 316L SS to corrosion in seawater-contaminated glycol . Mixture containing a minimum concentration of 50% propylene glycol-based coolant inhibited pitting corrosion . A slightly higher minimum concentration (55%) was needed for corrosion protection in ethylene glycol mixtures. Mol Microbiol, 2003 Nov, 50(3), 911 - 29 The Gap1 general amino acid permease acts as an amino acid sensor for activation of protein kinase A targets in the yeast Saccharomyces cerevisiae; Donaton MC et al.; Addition of a nitrogen source to yeast (Saccharomyces cerevisiae) cells starved for nitrogen on a glucose-containing medium triggers activation of protein kinase A (PKA) targets through a pathway that requires for sustained activation both a fermentable carbon source and a complete growth medium (fermentable growth medium induced or FGM pathway) . Trehalase is activated, trehalose and glycogen content as well as heat resistance drop rapidly, STRE-controlled genes are repressed, and ribosomal protein genes are induced . We show that the rapid effect of amino acids on these targets specifically requires the general amino acid permease Gap1 . In the gap1Delta strain, transport of high concentrations of l-citrulline occurs at a high rate but without activation of trehalase . Metabolism of the amino acids is not required . Point mutants in Gap1 with reduced or deficient transport also showed reduced or deficient signalling . However, two mutations, S391A and S397A, were identified with a differential effect on transport and signalling for l-glutamate and l-citrulline . Specific truncations of the C-terminus of Gap1 (e.g . last 14 or 26 amino acids) did not reduce transport activity but caused the same phenotype as in strains with constitutively high PKA activity also during growth with ammonium as sole nitrogen source . The overactive PKA phenotype was abolished by mutations in the Tpk1 or Tpk2 catalytic subunits . We conclude that Gap1 acts as an amino acid sensor for rapid activation of the FGM signalling pathway which controls the PKA targets, that transport through Gap1 is connected to signalling and that specific truncations of the C-terminus result in permanently activating Gap1 alleles. Mol Microbiol, 2003 Nov, 50(3), 771 - 80 Molecular characterization of thymidine kinase from Ureaplasma urealyticum: nucleoside analogues as potent inhibitors of mycoplasma growth; Carnrot C et al.; Ureaplasma urealyticum (U . urealyticum), belonging to the class Mollicutes, is a human pathogen colonizing the urogenital tract and causes among other things respiratory diseases in premature infants . We have studied the salvage of pyrimidine deoxynucleosides in U . urealyticum and cloned a key salvage enzyme, thymidine kinase (TK) from U . urealyticum . Recombinant Uu-TK was expressed in E . coli, purified and characterized with regards to substrate specificity and feedback inhibition . Uu-TK efficiently phosphorylated thymidine (dThd) and deoxyuridine (dUrd) as well as a number of pyrimidine nucleoside analogues . All natural ribonucleoside/deoxyribonucleoside triphosphates, except dTTP, served as phosphate donors, while dTTP was a feedback inhibitor . The level of Uu-TK activity in U . urealyticum extracts increased upon addition of dUrd to the growth medium . Fluoropyrimidine nucleosides inhibited U . urealyticum and M . pneumoniae growth and this inhibitory effect could be reversed by addition of dThd, dUrd or deoxytetrahydrouridine to the growth medium . Thus, the mechanism of inhibition was most likely the depletion of dTTP, either via a blocked thymidine kinase reaction and/or thymidylate synthesis step and these metabolic reactions should be suitable targets for antimycoplasma chemotherapy. Biomaterials, 2004 Mar, 25(6), 995 - 1001 The induction of MMP-9 release from granulocytes by vitamin E in UHMWPE; Reno F et al.; Ultra-high molecular weight polyethylene (UHMWPE) is a biopolymer widely used in orthopaedic implants and its oxidation is considered as major responsible for inflammation and the prosthesis failure . We have studied the effect on the activation of resting human granulocytes of the addition of Vitamin E (Vit.E, alpha-tocopherol), a natural biological antioxidant and antiinflammatory agent, to UHMWPE . We have measured changes in granulocytes morphology and respiratory burst by flow cytometry using Dihydrorhodamine 123 and matrix metalloproteinase 9 (MMP-9, gelatinase B) release and activity in the growth medium using substrate zymography following contact (60 min at 37 degrees C) with cell grade polystyrene (PS), normal UHMWPE (PE) and Vit.E added UHMWPE (PE Vit.E) . FTIR analyses showed that the surfaces of PE and PE-Vit.E were not significantly different . PS, PE and PE Vit.E did not alter granulocytes morphology and respiratory burst as showed by the mean fluorescence emitted (PS=12.0+/-0.1, PE=13.0+/-0.4, PE Vit.E=14.5+/-0.1) . PE Vit.E was able to increase MMP-9 release compared to PS and normal PE (215+/-16% of the control, p<0.001) . The PE Vit.E-induced MMP-9 release was abolished by okadaic acid (0.5 nM), suggesting a direct role of Vit.E in the phenomenon. Mutagenesis, 2003 Nov, 18(6), 549 - 60 Mechanisms of cell death associated with death-inducing factors from genomically unstable cell lines; Nagar S et al.; We recently described a unique non-targeted effect of ionizing radiation whereby growth medium from two clones of GM10115 cells exhibiting radiation-induced chromosomal instability was cytotoxic to parental GM10115 cells . We termed this the death-inducing effect (DIE) . The goal of the present study was to determine how DIE killed cells . Our hypothesis was that DIE medium contained either a secreted factor(s) from unstable clones or products from dead/dying cells that were cytotoxic to parental cells . First, we investigated the apoptotic characteristics of our unstable clones by Annexin V binding and TUNEL assays . Both the parental GM10115 cells and cells from the unstable clone LS12 had a low background (approximately 2%) level of apoptosis . The unstable Fe-10-3 clone showed a high spontaneous level of apoptosis, indicating major differences in the spontaneously occurring levels of apoptosis . We then analyzed how medium from these unstable clones killed cells by investigating the induction of DNA breaks, micronucleus formation and apoptosis induction in cells exposed to medium from unstable clones . Medium from unstable clones was capable of eliciting DNA double-strand breaks and increased apoptosis . Increased micronucleus frequencies were also observed in cells exposed to medium from either unstable clone, indicating a role of mitotis-linked cell death in DIE . These data suggest that DIE most likely results from cytotoxic factors secreted into the culture medium that can cause DNA double-strand breaks in recipient cells . These breaks can then lead to mitotis-linked cell death, as measured by micronuclei, or apoptosis, which accounts for the DIE. FEMS Microbiol Lett, 2003 Nov 7, 228(1), 129 - 35 Content and biosynthesis of polyamines in salt and osmotically stressed cells of Synechocystis sp . PCC 6803; Jantaro S et al.; The effects of various NaCl and sorbitol concentrations in the growth medium on polyamine content and on two enzymes of the polyamine biosynthesis pathway, arginine decarboxylase (ADC) and S-adenosyl methionine decarboxylase (SAMDC), were investigated in the unicellular cyanobacterium Synechocystis sp . PCC 6803 . Synechocystis cells showed no difference in growth rate when the concentration of NaCl was raised up to 550 mM . The growth rate decreased at 300 mM sorbitol, and complete inhibition of growth occurred at concentrations of > or =700 mM sorbitol . Salt stress induced a moderate increase in the total cellular polyamine content, spermine in particular . Osmotic stress caused an apparent increase in the total cellular polyamine content with a marked increase of spermidine induced by 700 mM sorbitol . Importantly, a low level of spermine, which so far has never been detected in cyanobacteria, could be found in Synechocystis sp . PCC 6803 . ADC, a key enzyme for putrescine synthesis, was unaffected by salt stress but showed a six-fold increase in enzyme activity upon osmotic stress imposed by 700 mM sorbitol . SAMDC, another important enzyme for spermidine and spermine synthesis, responded to salt and osmotic stresses similarly to the pattern observed for ADC . An analysis by reverse transcription-polymerase chain reaction revealed an increase of ADC mRNA level in cells under salt and osmotic stresses . Most importantly, the increase of ADC mRNA was attributed to its slower turnover rate under both stress conditions . Interestingly, the samdc gene(s) of Synechocystis appear to be unique since comparisons with known gene sequences from other organisms resulted in no homologous sequences identified in the Synechocystis genome. Appl Environ Microbiol, 2003 Nov, 69(11), 6954 - 8 Phenylacetic and phenylpropionic acids do not affect xylan degradation by Ruminococcus albus; Reveneau C et al.; Since the addition of either ruminal fluid or a combination of phenylacetic and phenylpropionic acids (PAA/PPA) has previously been shown to dramatically improve cellulose degradation and growth of Ruminococcus albus, it was of interest to determine the effects of these additives on xylan-grown cultures . Although cell-bound xylanase activity increased when either PAA/PPA or ruminal fluid was added to the growth medium, total xylanase did not change, and neither of these supplements affected the growth or xylan-degrading capacity of R . albus 8 . Similarly, neither PAA/PPA nor ruminal fluid affected xylan degradation by multiple strains of R . albus when xylan prepared from oat spelts was used as a carbohydrate source . These results show that the xylanolytic potential of R . albus is not conditional on the availability of PAA/PPA or other components of ruminal fluid. Appl Environ Microbiol, 2003 Nov, 69(11), 6833 - 40 Variation in resistance of Mycobacterium paratuberculosis to acid environments as a function of culture medium; Sung N et al.; Acid resistance of Mycobacterium paratuberculosis was examined as a function of growth conditions (i.e., in vitro growth medium and pH) . M . paratuberculosis was cultured in either fatty acid-containing medium (7H9-OADC) or glycerol-containing medium (WR-GD or 7H9-GD) at two culture pHs (pHs 6.0 and 6.8) . Organisms produced in these six medium and pH conditions were then tested for resistance to acetate buffer at pHs 3, 4, 5, and 6 at 20 degrees C . A radiometric culture method (BACTEC) was used to quantify viable M . paratuberculosis cell data at various acid exposure times, and D values (decimal reduction times, or the times required to kill a 1-log(10) concentration of bacteria) were determined . Soluble proteins of M . paratuberculosis grown under all six conditions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to identify proteins that may be associated with acid resistance or susceptibility . The culture medium affected growth rate and morphology: thin floating sheets of cells were observed in 7H9-OADC versus confluent, thick, waxy, and wrinkled pellicles in WR-GD . Culture medium pH affected growth rate (which was highest at pH 6.0), but it had little or no effect on D values for M . paratuberculosis at any test pH . When grown in 7H9-OADC, M . paratuberculosis was more acid resistant at all test pHs (higher D values) than when grown in WR-GD . Glycerol appeared to be the culture medium component most responsible for lower levels of M . paratuberculosis acid resistance . When glycerol was substituted for OADC in the 7H9 medium, D values were significantly lower than those of 7H9-OADC-grown M . paratuberculosis and were approximately the same as those for M . paratuberculosis grown in WR-GD medium . Comparison of the SDS-PAGE protein profiles for M . paratuberculosis cultures grown in 7H9-OADC, WR-GD, or 7H9-GD medium revealed that increased expression of 34.2- and 14.0-kDa proteins was associated with higher levels of acid resistance of M . paratuberculosis grown in 7H9-OADC medium and that 56.6- and 41.3-kDa proteins were associated with lower levels of acid resistance . This is the first report showing that in vitro culture conditions significantly affect growth characteristics, acid resistance, and protein expression of M . paratuberculosis, and the results emphasize the importance of culture conditions for in vitro susceptibility studies. Environ Sci Technol, 2003 Oct 15, 37(20), 4743 - 50 Development of a set of simple bacterial biosensors for quantitative and rapid measurements of arsenite and arsenate in potable water; Stocker J et al.; Testing for arsenic pollution is commonly performed with chemical test kits of unsatisfying accuracy . Bacterial biosensors are an interesting alternative as they are easily produced, simple, and highly accurate devices . Here, we describe the development of a set of bacterial biosensors based on a nonpathogenic laboratory strain of Escherichia coli, the natural resistance mechanism of E . coli against arsenite and arsenate, and three reporter proteins: bacterial luciferase, beta-galactosidase and Green Fluorescent Protein (GFP) . The biosensors were genetically optimized to reduce background expression in the absence of arsenic . In calibration experiments with the biosensors and arsenite-amended potable water, arsenite concentrations at 4 microg of As/L (0.05 microM) were routinely and accurately measured . The currently most quantitative system expressed the bacterial luciferase as reporter protein, responding proportional with a concentration range between 8 and 80 microg of As/L . Sensor cells could be stored as frozen batches, resuspended in plain media, and exposed to the aqueous test sample, and light emission was measured after 30-min incubation . Field testing for arsenite was achieved with a system that contained beta-galactosidase, producing a visible blue color at arsenite concentrations above 8 microg/L . For this sensor, a protocol was developed in which the sensor cells were dried on a paper strip and placed in the aqueous test solution for 30 min after which time color development was allowed to take place . The GFP sensor showed good potential for continuous rather than end point measurements . In all cases, growth of the biosensors and production of the strip test was achieved by very simple means with common growth media, and quality control of the sensors was performed by isolating the respective plasmids with the genetic constructs according to simple standard genetic technologies . Therefore, the biosensor cells and protocols may offer a realistic alternative for measuring arsenic contamination in potable water. Oncogene, 2003 Oct 30, 22(49), 7819 - 30 TGF-beta-induced apoptosis in human thyrocytes is mediated by p27kip1 reduction and is overridden in neoplastic thyrocytes by NF-kappaB activation; Bravo SB et al.; Millions of people worldwide suffer goiter, a proliferative disease of the follicular cells of the thyroid that may become neoplastic . Thyroid neoplasms have low proliferative index, low apoptotic index and a high incidence of metastasis . TGF-beta is overexpressed in thyroid follicular tumor cells . To investigate the role of TGF-beta in thyroid tumor progression, we established cultures of human thyrocytes from different proliferative pathologies (Grave's disease, multinodular goiter, follicular adenoma, papillary carcinoma), lymph node metastasis, and a normal thyroid sample . All cultures maintained the thyrocyte phenotype . TGF-beta induced cell-cycle arrest in all cultures, in contrast with results reported for other epithelial tumors . In deprived medium, TGF-beta induced apoptosis in normal thyrocyte cultures and all neoplastic cultures except the metastatic cultures . This apoptosis was mediated by a reduction in p27kip1 levels, inducing cell-cycle initiation . Antisense p27 expression induced apoptosis in the absence of TGF-beta . By contrast, in cells in which p27 was overexpressed, TGF-beta had a survival effect . In growth medium, a net survival effect occurs in neoplastic thyrocytes only, not normal thyrocytes, due to activation of the NF-kappaB survival program . Together, these findings suggest that (a) thyroid neoplasms are due to reduced apoptosis, not increased division, in line with the low proliferative index of these pathologies, and (b) TGF-beta induces apoptosis in normal thyrocytes via p27 reduction, but that in neoplastic thyrocytes this effect is overridden by activation of the NF-kappaB program. Biomaterials, 2004 Jan, 25(2), 277 - 82 Biomimetic nucleation and growth of CaCO3 in hydrogels incorporating carboxylate groups; Grassmann O et al.; Poly-acrylamide hydrogels were modified by copolymerization with acrylic acid and used as growth medium for CaCO3 in a double-diffusion arrangement . The carboxylate functionalities in the gel network facilitate the nucleation of a multitude of small crystallites of vaterite and calcite, which are temporarily stabilized even while supersaturation is increasing within the hydrogel . After an extended induction period the rapid spherulitic growth of calcite crystals along their c-axis is observed yielding spheres with diameters exceeding 300 microm . In the center of those aggregates disordered, porous regions can be identified as starting point of this rapid crystallization . The results are compared to previous studies on native poly-acrylamide hydrogels and networks modified with -SO(3)H functional groups . The mineralization mechanism is significantly altered by specific interactions between the -COOH functionalized network and the evolving mineral phase. Mol Cell Biol, 1982 Dec, 2(12), 1481 - 91 Properties and possible functions of the adenylate cyclase in plasma membranes of Saccharomyces cerevisiae; Jaynes PK et al.; We have examined the possible role of adenosine 3',5'-phosphate (cAMP) in functions associated with the plasma membranes of Saccharomyces cerevisiae . Purified membranes from this source contained an adenylate cyclase which was insensitive to activation by fluoride or guanine nucleotides, only weakly responsive to changes of carbon source in the growth medium, and strongly stimulated by vanadate . They also contained at least two classes of receptor proteins for guanine nucleotides (as measured by binding of labeled 5'-guanylyl methylene diphosphate) with apparent dissociation constants equal to 1.0 x 10(-7) and 3 x 10(-6) M, a protein kinase capable of phosphorylating added histones, the activity of which was stimulated by cAMP, and cAMP receptors that may function as regulatory subunits for this kinase . Membrane proteins were also susceptible to phosphorylation by endogenous kinase(s), with polypeptides of apparent molecular weights equal to 160 x 10(3), 135 x 10(3), 114 x 10(3), and 58 x 10(3) as the major targets . Of these, the 114,000-molecular-weight polypeptide was probably identical to the proton-translocating ATPase of the membranes . However, the cAMP-dependent protein kinase did not appear to be involved in these reactions . Intact (rho+ or rho0) cells responded to dissipation of the proton electrochemical gradient across their plasma membranes by rapid and transient changes in their intracellular level of cAMP, as suggested earlier (J . M . Trevillyan and M . L . Pall, J . Bacteriol., 138:397-403, 1979) . Thus, although yeast plasma membranes contain all the essential components of a stimulus-responsive adenylate cyclase system, the precise nature of the coupling device and the targets involved remain to be established. Antonie Van Leeuwenhoek, 2003, 84(3), 229 - 38 Cell wall composition and structure of Yarrowia lipolytica transposon mutants affected in calcofluor sensitivity; Ruiz-Herrera J et al.; A collection of transposon-mutagenized strains of Yarrowia lipolytica was screened for wall defects by determination of their sensitivity to calcofluor white . A number of strains were hypersensitive, whereas others were resistant . Different non-allelic mutants displayed increased sensitivity to autolysis and lytic enzymes, independently of whether they were sensitive or resistant to calcofluor white . A thorough analysis of their cell walls revealed minor quantitative alterations, and no significant changes in chitin content . Electrophoretic analysis of wall-bound and excreted proteins proved to be a sensitive method that revealed defects in the cell wall structure of the mutants . Important alterations in the patterns of the wall proteins extracted by SDS or by enzymatic treatments were noticed for the mutants, as compared to the parental strain . Mutants released to the growth medium a larger number of protein species than the parental strain, suggesting impairment in wall assembly of certain polypeptides . Patterns of wall-bound and excreted proteins, as well as alterations in wall chemical composition were not diagnostic of calcofluor white sensitivity or resistance, but were specific for each mutant . Our data show that an increase in either sensitivity or resistance of Y . lipolytica to certain levels of calcofluor is equally indicative of alterations in cell wall structure, independent of chitin levels. Arch Insect Biochem Physiol, 2003 Nov, 54(3), 134 - 42 Xenorhabdus nematophilus inhibits p-bromophenacyl bromide (BPB)-sensitive PLA2 of Spodoptera exigua; Park Y et al.; Xenorhabdus nematophilus is a Gram-negative symbiotic bacterium of the entomopathogenic nematode, Steinernema carpocapsae . The bacteria delivered into the insect hemocoel by the nematodes cause immunodepression of the target insects to protect host nematodes and themselves from the cellular immune reaction . Previous reports suggest that the immunodepression is caused by inhibition of the eicosanoid pathway that is known to be critically important to mediate cellular immunity . This study focused on the inhibitory effect of X . nematophilus on PLA2 activity of Spodoptera exigua . The PLA2 activity was functionally associated with the activation cascade of prophenoloxidase (pPO) . Dexamethasone (DEX), a specific PLA2 inhibitor, inhibited pPO activation completely at the higher doses of approximately 2.4 muM in vitro condition . The inhibitory effect of DEX was reversed by the addition of arachidonic acid, the catalytic product of PLA2 . By means of this in vitro PLA2 inhibitor assay system, two different PLA2 inhibitors were used to compare their inhibitory effects on the hemolymph PLA2 of S . exigua . p-Bromophenacyl bromide (BPB), a specific inhibitor of secretory PLA2 (sPLA2), significantly inhibited pPO activation, but methylarachidonyl fluorophosphates (MAFP), a specific inhibitor of cytosolic PLA2 (cPLA2), did not show any inhibitory effect . BPB also inhibited pPO activation of the plasma, though much higher PO activation and its inhibition by BPB was found in the hemocytes . Growth medium of X . nematophilus at the stationary phase had a PLA2 inhibitory effect . Via the in vitro PLA2 inhibitor assay, it was shown that the ethyl ether extract of the medium contained significant PLA2 inhibitor activity . These results indicate that X . nematophilus produces and secretes PLA2 inhibitor, which acts on BPB-susceptible PLA2 of S . exigua . Biochem J, 2004 Feb 1, 377(Pt 3), 769 - 74 LEA (late embryonic abundant)-like protein Hsp 12 (heat-shock protein 12) is present in the cell wall and enhances the barotolerance of the yeast Saccharomyces cerevisiae; Motshwene P et al.; Yeast cells Saccharomyces cerevisiae, late embryogenic abundant-like stress response protein Hsp 12 (heat-shock protein 12) were found by immunocytochemistry to be located both in the cytoplasm and in the cell wall, from where they could be extracted with dilute NaOH solutions . Yeast cells with the Hsp 12 gene disrupted were unable to grow in the presence of either 12 mM caffeine or 0.43 mM Congo Red, molecules known to affect cell-wall integrity . The volume of yeast cells were less affected by rapid changes in the osmolality of the growth medium when compared with the wild-type yeast cells, suggesting a role for Hsp 12 in the flexibility of the cell wall . This was also suggested by subjecting the yeast cells to rapid changes in barometric pressure where it was found that wild-type yeast cells were more resistant to cellular breakage. FEMS Microbiol Lett, 2003 Oct 10, 227(1), 39 - 45 Regulation of the glutamate-dependent acid-resistance system of diarrheagenic Escherichia coli strains; Bhagwat AA; The ability to withstand an acid challenge of pH 2.5 or less by Escherichia coli strains is a trait generally believed to be restricted to their stationary phase of growth . Of the three distinct acid-resistance systems that have been identified in E . coli, the glutamate-dependent acid resistance (GAD) system provides the highest level of acid resistance . Earlier reports indicated that in the exponential growth phase of E . coli K-12 strains the GAD system is not active . The present study reports that when grown on minimal medium several diarrheagenic and K-12 strains of E . coli have a complete set of induced genes necessary for GAD in the exponential growth phase to overcome the acid challenge of pH 2.5 for several hours . A previously identified factor(s) specific to the GAD system in the stationary phase and predicted to undergo dilution during the exponential phase appears to be glutamate-decarboxylase isozyme(s) inactivated differentially in the rich vs . minimal growth media. Pathophysiology, 2003 Jan, 9(2), 103 - 109 Suppression of the release of type-1 plasminogen activator inhibitor from human vascular endothelial cells by Hawaii deep sea water; Ueshima S et al.; The effect of deep sea water on the fibrinolytic properties of human vascular endothelial cells was investigated . There was no difference in the growth ratio between human umbilical vein endothelial cells (HUVECs) cultured with growth medium (RPMI-1640 containing 20% fetal calf serum) prepared with Hawaii deep sea water (HDSW medium) and those with medium prepared with normal distilled water (control medium) . The secretion of type 1 plasminogen activator inhibitor (PAI-1) from HUVECs was significantly reduced by about twofold . However, the levels of PAI-1 mRNA in HUVECs cultured with HDSW medium did not change when compared with those cultured with control medium . Though HDSW medium also reduced the secretion of tissue-type plasminogen activator, the suppressive effect was more prominent for PAI-1 . Thus, the balance of fibrinolytic activity was turned toward anti-thrombotic in HUVECs . This was evidenced by the lysis of 125I-fibrin clot in the presence of plasminogen . That is, HUVECs cultured with HDSW medium degraded 125I-fibrin more efficiently than HUVECs with control medium . Such enhanced clot lysis was maintained as long as HDSW medium was present . The accelerated effect of HDSW medium on clot lysis disappeared after the exchange of HDSW medium to control medium . These findings suggest that HDSW may specifically and predominantly affect the process of molecular transfer of PAI-1 after its transcription, resulting in an enhanced fibrinolytic activity of HUVECs . Since HDSW reduces the thrombotic potential of cultured HUVECs, it is speculated that the materials contained in HDSW may prevent the incidence of thrombotic disorders. Cryo Letters, 2003 Sep-Oct, 24(5), 269 - 74 Substances which inhibit ice nucleation: a review; Holt CB; There are a number of substances described in the published literature which inhibit ice nucleation . Certain bacterial strains, mostly found among the nonfluorescent pseudomonade species, release material into the growth medium which reduces the nucleation temperature of water droplets to below that of distilled water . Extracts from the seeds of food crops including apricot, peach and plum can reduce the nucleation temperature of water droplets and dispersions of silver iodide . Antifreeze glycoproteins can reduce the nucleation temperature of saline solutions . Antifreeze proteins can inhibit the activity of some biological ice nucleators but not others . Certain novel polymers have been shown to inhibit the nucleation activity of dispersions of silver iodide and ice-nucleating bacteria. BMC Infect Dis . 2003 Oct 17;3(1):24. Determination of decimal reduction time (D value) of chemical agents used in hospitals for disinfection purposes; Mazzola PG et al.; BACKGROUND: Prior to the selection of disinfectants for low, intermediate and high (sterilizing) levels, the decimal reduction time, D-value, for the most common and persistent bacteria identified at a health care facility should be determined . METHODS: The D-value was determined by inoculating 100 mL of disinfecting solution with 1 mL of a bacterial suspension (10(4)-10(5) CFU/mL for vegetative and spore forms) . At regular intervals, 1 mL aliquots of this mixture were transferred to 8 mL of growth media containing a neutralizing agent, and incubated at optimal conditions for the microorganism . RESULTS: The highest D-values for various bacteria were determined for the following solutions: (i) 0.1% sodium dichloroisocyanurate (pH 7.0)--E . coli and A . calcoaceticus (D = 5.9 min); (ii) sodium hypochlorite (pH 7.0) at 0.025% for B . stearothermophilus (D = 24 min), E . coli and E . cloacae (D = 7.5 min); at 0.05% for B . stearothermophilus (D = 9.4 min) and E . coli (D = 6.1 min) and 0.1% for B . stearothermophilus (D = 3.5 min) and B . subtilis (D = 3.2 min); (iii) 2.0% glutaraldehyde (pH 7.4)--B . stearothermophilus, B . subtilis (D = 25 min) and E . coli (D = 7.1 min); (iv) 0.5% formaldehyde (pH 6.5)--B . subtilis (D = 11.8 min), B . stearothermophilus (D = 10.9 min) and A . calcoaceticus (D = 5.2 min); (v) 2.0% chlorhexidine (pH 6.2)--B . stearothermophilus (D = 9.1 min), and at 0.4% for E . cloacae (D = 8.3 min); (vi) 1.0% Minncare (peracetic acid and hydrogen peroxide, pH 2.3)--B . stearothermophilus (D = 9.1 min) and E . coli (D = 6.7 min) . CONCLUSIONS: The suspension studies were an indication of the disinfectant efficacy on a surface . The data in this study reflect the formulations used and may vary from product to product . The expected effectiveness from the studied formulations showed that the tested agents can be recommended for surface disinfection as stated in present guidelines and emphasizes the importance and need to develop routine and novel programs to evaluate product utility. Eur Cell Mater, 2001 Jul 24, 2, 10 - 20 Influence of cell isolation, cell culture density, and cell nutrition on differentiation of rat calvarial osteoblast-like cells in vitro; Gerber I et al.; The effects of various cell isolation procedures, growth media and the cell culture density on the in vitro differentiation of neonatal rat calvarial osteoblast-like cells were investigated . Cells were isolated by enzymatic treatment, or after explant culture and inoculated as a monolayer or micromass in serum containing BGJb, or Dulbecco's Modified Eagle Medium (DMEM) . The cells were kept for up to 3 weeks in culture and were then characterized, both morphologically and biochemically . The isolation technique appeared to have no effect on the differentiation process . The calvaria could be used several times as explant cultures for a reliable source of differentiating osteoblast-like cells . The cultures kept in DMEM had a significantly higher DNA content, but significantly less alkaline phosphatase activity (ALP) per DNA and protein per DNA content than the BGJb cultures . Monolayer cultures had a significantly higher DNA content than the micromass cultures, in both growth media . Furthermore, the micromass culture had a significantly higher ALP per DNA than monolayer cultures at 1 week . The morphology of all cell cultures at 3 weeks reflected the biochemical results . Only the cells grown in BGJb formed abundant ALP positive and mineralized nodules in monolayer cultures . In contrast, cells grown as micromasses formed a dense calcified area, independently of the growth medium used . DMEM promoted the proliferation, whereas BGJb stimulated the differentiation of osteoblast-like cells in monolayer cultures . Micromass cultures were less sensitive to nutritional conditions than monolayer cultures and promoted the differentiation of osteoblast-like cells. Protein Pept Lett, 2003 Oct, 10(5), 459 - 68 Direct screening of libraries of yeast clones for alpha-amylase activity on raw starch hydrolysis; Wong DW et al.; High-throughput screening for high-activity barley alpha-amylase mutants expressed in Saccharomyces cerevisiae is hampered by the interference of reducing agents, particularly the glucose used in yeast growth media . The present investigation employed colorimetric and chemiluminescent detection systems that enable direct and rapid screening of activities on raw starch substrate . Active clones could be separated into two groups, based on high total activity or high specific activity. J Biol Chem, 2003 Dec 26, 278(52), 53123 - 30 Epub 2003 Oct 14. Unique mechanism of action of the thiourea drug isoxyl on Mycobacterium tuberculosis; Phetsuksiri B et al.; The thiourea isoxyl (thiocarlide; 4,4'-diisoamyloxydiphenylthiourea) is known to be an effective anti-tuberculosis drug, active against a range of multidrug-resistant strains of Mycobacterium tuberculosis and has been used clinically . Little was known of its mode of action . We now demonstrate that isoxyl results in a dose-dependent decrease in the synthesis of oleic and, consequently, tuberculostearic acid in M . tuberculosis with complete inhibition at 3 microg/ml . Synthesis of mycolic acid was also affected . The anti-bacterial effect of isoxyl was partially reversed by supplementing growth medium with oleic acid . The specificity of this inhibition pointed to a Delta9-stearoyl desaturase as the drug target . Development of a cell-free assay for Delta9-desaturase activity allowed direct demonstration of the inhibition of oleic acid synthesis by isoxyl . Interestingly, sterculic acid, a known inhibitor of Delta9-desaturases, emulated the effect of isoxyl on oleic acid synthesis but did not affect mycolic acid synthesis, demonstrating the lack of a relationship between the two effects of the drug . The three putative fatty acid desaturases in the M . tuberculosis genome, desA1, desA2, and desA3, were cloned and expressed in Mycobacterium bovis BCG . Cell-free assays and whole cell labeling demonstrated increased Delta9-desaturase activity and oleic acid synthesis only in the desA3-overexpressing strain and an increase in the minimal inhibitory concentration for isoxyl, indicating that DesA3 is the target of the drug . These results validate membrane-bound Delta9-desaturase, DesA3, as a new therapeutic target, and the thioureas as anti-tuberculosis drugs worthy of further development. Eukaryot Cell, 2003 Oct, 2(5), 971 - 7 A Phytophthora infestans G-protein beta subunit is involved in sporangium formation; Latijnhouwers M et al.; The heterotrimeric G-protein pathway regulates cellular responses to a wide range of extracellular signals in virtually all eukaryotes . It also controls various developmental processes in the oomycete plant pathogen Phytophthora infestans, as was concluded from previous studies on the role of the G-protein alpha-subunit PiGPA1 in this organism . The expression of the P . infestans G-protein beta-subunit gene Pigpb1 was induced in nutrient-starved mycelium before the onset of sporangium formation . The gene was hardly expressed in mycelium incubated in rich growth medium . The introduction of additional copies of Pigpb1 into the genome led to silencing of the gene and resulted in transformants deficient in PiGPB1 . These Pigpb1-silenced mutants formed very few asexual spores (sporangia) when cultured in rye sucrose medium and produced a denser mat of aerial mycelium than the wild type . Partially Pigpb1-silenced mutants showed intermediate phenotypes with regard to sporulation, and a relatively large number of their sporangia were malformed . The results show that PiGPB1 is important for vegetative growth and sporulation and, therefore, for the pathogenicity of this organism. Eukaryot Cell, 2003 Oct, 2(5), 886 - 900 Chitin synthesis in Saccharomyces cerevisiae in response to supplementation of growth medium with glucosamine and cell wall stress; Bulik DA et al.; In Saccharomyces cerevisiae most chitin is synthesized by Chs3p, which deposits chitin in the lateral cell wall and in the bud-neck region during cell division . We have recently found that addition of glucosamine (GlcN) to the growth medium leads to a three- to fourfold increase in cell wall chitin levels . We compared this result to the increases in cellular chitin levels associated with cell wall stress and with treatment of yeast with mating pheromone . Since all three phenomena lead to increases in precursors of chitin, we hypothesized that chitin synthesis is at least in part directly regulated by the size of this pool . This hypothesis was strengthened by our finding that addition of GlcN to the growth medium causes a rapid increase in chitin synthesis without any pronounced change in the expression of more than 6,000 genes monitored with Affymetrix gene expression chips . In other studies we found that the specific activity of Chs3p is higher in the total membrane fractions from cells grown in GlcN and from mutants with weakened cell walls . Sucrose gradient analysis shows that Chs3p is present in an inactive form in what may be Golgi compartments but as an active enzyme in other intracellular membrane-bound vesicles, as well as in the plasma membrane . We conclude that Chs3p-dependent chitin synthesis in S . cerevisiae is regulated both by the levels of intermediates of the UDP-GlcNAc biosynthetic pathway and by an increase in the activity of the enzyme in the plasma membrane. Biotechnol Adv, 1984, 2(2), 217 - 32 Production of useful modified lignin polymers by bioconversion of lignocellulose with Streptomyces; Crawford DL et al.; Lignin degrading strains of Streptomyces were grown on lignocelluloses from a variety of plant sources . These actinomycetes readily degraded the lignin present in the residues and released a major portion of the lignin into the growth medium as a water soluble, modified polymer . The polymer, an acid precipitable polyphenolic lignin (APPL), was recovered from spent culture media by acid precipitation or dialysis/lyophilization . APPL's were shown to be mostly free of nonlignin components . As compared to native lignin they were more oxidized, were especially enriched in phenolic hydroxyl groups, and were significantly reduced in methoxyl groups . The yield of APPL from different lignocelluloses correlated with their biodegradability . Grasses such as corn stover were the optimal lignocellulose type for APPL production by Streptomyces . In contrast white-rot fungi produced only small amounts of APPL as they decomposed lignin . A solid state bioconversion process was developed using Streptomyces viridosporus T7A to produce APPL from corn stover lignocellulose in yields >or= 30% of the initial lignin present in the substrate . APPL produced by S . viridosporus was examined for its properties and possible use as an antioxidant . The APPL was shown to have good antioxidant properties after mild chemical treatment to reduce the alpha-carbonyl groups present in the APPL . Oxidation of the APPL with hydroxyl radical (OH(*)) further improved its antioxidant properties probably as the result of aromatic ring hydroxylation reactions . As compared with currently used commercial antioxidants, the modified APPL was thought to be competitive when economics of production was considered . Native lignin on the other hand was shown to exhibit no antioxidant properties, even after reduction and/or oxidation. Biotechnol Adv, 1987, 5(1), 1 - 27 Microbial desulphurization of heavy oils and bitumen; Bhadra A et al.; Most oil producing countries have extensive reserves of heavy oil and bitumen . As easily accessible sources of conventional crudes decline, these reserves will become more important in supplementing the energy requirements . Heavy oil and bitumen are highly viscous and contain 3 to 6% sulphur . These objectionable quantities of sulphur must be removed before being acceptable as refinery feedstock . This paper addresses the potential of biological desulphurization of heavy oil and bitumen . The aerobic and anaerobic processes to remove organic as well as inorganic sulphur have been reviewed . To date, most studies were performed with model substrates, particularly dibenzothiophene (DBT) in a synthetic medium . Early work concerned with the isolation of microorganisms, identification and characterization of intermediate metabolites, and the development of growth media . No commercially viable process has emerged since the engineering details of the process have not been addressed conclusively . Due to high utility and catalyst cost conventional hydrodesulphurization processes are reported to be uneconomic in case of high sulphur oils . Microbial desulphurization, on the other hand, appears to be promising due to the inherent low energy requirement . This process may become more attractive by the application of genetically modified bacteria and improvements in bioreactor design. Biotechnol Adv, 1985, 3(1), 65 - 83 Bio-surfactants; Parkinson M; Interest in microbially produced biosurfactants has increased recently, due mainly to their potential as agents in enhanced oil recovery . A variety of microbes and their products have been assessed for their surface-active properties, and it has been suggested that biosurfactants may prove useful in a broad spectrum of potential applications which presently utilise synthetic surfactants . The most commonly produced biosurfactants tend to be glycolipids, usually a mono- or di-saccharide attached to a fatty acid, but more complex molecules such as lipopeptides, lipoproteins, and lipo-heteropoly-saccharides have been isolated and studied . Biosurfactant production by microbes is often but not invariably enhanced by the addition of hydrocarbon to the growth medium, and needs to be optimised by controlling such factors as carbon source, nitrogen source and concentrations, aeration and metal ions . Biosurfactants have been shown to be as effective, if not more so, than many conventional synthetic surfactants and their future utilisation may depend utilimately upon the prevailing economics for their production. Biotechnol Adv, 2000 Jul, 18(4), 267 - 88 Are microbes intelligent beings?: An assessment of cybernetic modeling; Patnaik PR; Microorganisms growing in a multi-substrate medium have different and varying preferences for the various components of the medium . The preferences depend on the operating conditions and the substrates may be utilized sequentially or simultaneously . Sometimes an organism may change its preferences among substrates and/or switch between sequential and simultaneous utilization . These aspects are difficult to describe through models based on chemical and physical laws alone . Cybernetic modeling ascribes to microorganisms the ability to perceive their environment (i.e . the growth medium) and make 'intelligent' choices regarding substrate utilization to maximize an objective, which is usually the growth rate . This article reviews the development of cybernetic modeling since it began in 1982 . Different workers have suggested different perspectives of how microbes make optimal use of their resources . These are discussed and future directions for improvement are indicated. Appl Environ Microbiol, 2003 Oct, 69(10), 6272 - 9 Identification of peptides that mimic the pertussis toxin binding site on bovine fetuin; Bogdan JA et al.; The introduction of acellular pertussis vaccines has greatly enhanced the safety profile of vaccines to prevent whooping cough . Pertussis toxin (Ptx) is one component produced by Bordetella pertussis that is contained in all of these vaccines, either in combination with other known pertussis virulence factors or as the sole pertussis component, combined with tetanus and diphtheria toxoids . A hydrogen peroxide toxoid of Ptx has been shown to be efficacious in preventing pertussis infections in a mass vaccination trial and is presently licensed in the United States and Europe (B . Trollfors, J . Taranger, T . Lagergard, L . Lind, V . Sundh, G . Zackrisson, C . U . Lowe, W . Blackwelder, and J . B . Robbins, N . Engl . J . Med . 333:1045-1050, 1995) . The industrial production of Ptx can be performed through the cultivation of B . pertussis in well-defined growth media, in which the components can be well characterized and their origins can be documented . Once the bacteria are removed from the culture, Ptx can be isolated from the supernatant and purified by using the technique described by Sekura et al . (R . D . Sekura, F . Fish, C . R . Manclark, B . Meade, and Y . L . Zhang, J . Biol . Chem . 258:14647-14651, 1983) . The only drawback of this procedure, which combines two affinity chromatography steps, one with Blue Sepharose and a second with matrix-bound bovine fetuin (BF), is the source and purity of the BF . Concern about vaccine preparations that may possibly risk contamination by material associated with bovine spongioform encephalopathy has continued to increase . We thus sought a replacement for the BF affinity chromatography and, more specifically, for the glycosidic moiety on BF . We describe here the identification of a seven-amino-acid peptide that mimics the glycosidic moiety on BF to which Ptx binds . Furthermore, we have constructed an affinity column containing this peptide that can be used to replace BF in Ptx purification . Finally, we used the X-ray crystallographic structure of Ptx bound to the oligosaccharide moiety of BF as a scaffold and replaced the oligosaccharide with the peptide. Gene, 2003 Sep 18, 314, 63 - 71 Functional characterization of two flap endonuclease-1 homologues in rice; Kimura S et al.; Flap endonuclease-1 (FEN-1) is an important enzyme involved in DNA replication and repair . Previously, we isolated and characterized a complementary DNA (cDNA) from rice (Oryza sativa) encoding a protein which shows homology with the eukaryotic flap endonuclease-1 (FEN-1) . In this report, we found that rice (O . sativa L . cv . Nipponbare) possessed two FEN-1 homologues designated as OsFEN-1a and OsFEN-1b . The OsFEN-1a and OsFEN-1b genes were mapped to chromosome 5 and 3, respectively . Both genes contained 17 exons and 16 introns . Alignment of OsFEN-1a protein with OsFEN-1b protein showed a high degree of sequence similarity, particularly around the N and I domains . Northern hybridization and in situ hybridization analysis demonstrated preferential expression of OsFEN-1a and OsFEN-1b in proliferating tissues such as the shoot apical meristem or young leaves . The levels of OsFEN-1a and OsFEN-1b expression were significantly reduced when cell proliferation was temporarily halted by the removal of sucrose from the growth medium . When the growth-halted cells began to regrow following the addition of sucrose to the medium, both OsFEN-1a and OsFEN-1b were again expressed at high level . These results suggested that OsFEN-1a and OsFEN-1b are required for cell proliferation . Functional complementation assay suggested that OsFEN-1a cDNA had the ability to complement Saccharomyces cerevisiae rad27 null mutant . On the other hand, OsFEN-1b cDNA could not complement the rad27 mutant . The roles of OsFEN-1a and OsFEN-1b in plant DNA replication and repair are discussed. J Bacteriol, 2003 Oct, 185(20), 5915 - 24 Production of high-quality particulate methane monooxygenase in high yields from Methylococcus capsulatus (bath) with a hollow-fiber membrane bioreactor; Yu SS et al.; In order to obtain particulate methane monooxygenase (pMMO)-enriched membranes from Methylococcus capsulatus (Bath) with high activity and in high yields, we devised a method to process cell growth in a fermentor adapted with a hollow-fiber bioreactor that allows easy control and quantitative adjustment of the copper ion concentration in NMS medium over the time course of cell culture . This technical improvement in the method for culturing bacterial cells allowed us to study the effects of copper ion concentration in the growth medium on the copper content in the membranes, as well as the specific activity of the enzyme . The optimal copper concentration in the growth medium was found to be 30 to 35 micro M . Under these conditions, the pMMO is highly expressed, accounting for 80% of the total cytoplasmic membrane proteins and having a specific activity as high as 88.9 nmol of propylene oxide/min/mg of protein with NADH as the reductant . The copper stoichiometry is approximately 13 atoms per pMMO molecule . Analysis of other metal contents provided no evidence of zinc, and only traces of iron were present in the pMMO-enriched membranes . Further purification by membrane solubilization in dodecyl beta-D-maltoside followed by fractionation of the protein-detergent complexes according to molecular size by gel filtration chromatography resulted in a good yield of the pMMO-detergent complex and a high level of homogeneity . The pMMO-detergent complex isolated in this way had a molecular mass of 220 kDa and consisted of an alphabetagamma protein monomer encapsulated in a micelle consisting of ca . 240 detergent molecules . The enzyme is a copper protein containing 13.6 mol of copper/mol of pMMO and essentially no iron (ratio of copper to iron, 80:1) . Both the detergent-solubilized membranes and the purified pMMO-detergent complex exhibited reasonable, if not excellent, specific activity . Finally, our ability to control the level of expression of the pMMO allowed us to clarify the sensitivity of the enzyme to NADH and duroquinol, the two common reductants used to assay the enzyme. Tsitologiia, 2003, 45(6), 535 - 48 {Basic types and vital forms of human peripheral and PHA-stimulated lymphocytes studied in vitro}; Demin SIu; Natural diversity in peripheral and PHA-stimulated lymphocytes seen in the same donors was studied using digitized streak photo of living cells in observational camera . Cells were monitored for 5-8 h at the superior limit of optical resolution by means of phase-contrast microscopy . Intact lymphocytes were observed in autological blood plasma, and PHA-stimulated lymphocytes were examined in self-conditioned centrifuged growth medium . The majority of intact cells were small- and middle-sized floating lymphocytes with microvilli, and middle-sized caudate lymphocytes capable of stick-slip motion . The lesser part consisted of "spread-eagle" or movable forms of both large granular lymphocytes and middle-sized lymphocytes of several types: narrow-plasm lymphocytes with lamellipodia, wide-plasm lymphocytes without cytoplasmic processes, lymphocytes with single pseudopodia, and lymphocytes with single lobopodia of complex shape . On the contrary, the minor fraction of PHA-stimulated lymphocytes of 3 day old cultures contained floating cells with microvilli or floating cells with microvilli and two pseudopodia, whereas the majority of these lymphocytes were spread-eagle or movable forms of cells of different type . These substrate-adhesive PHA-stimulated lymphocytes had well defined apical and basal cell surfaces, but upon mechanical stress are easily pinched off to become ball-shaped . At least 6 different cell types were distinguished among substrate-adhesive PHA-stimulated lymphocytes, with more than half of these being heavily vacuolated spheroid lymphocytes prone to forming cell clusters . The rest PHA-stimulated lymphocytes were represented by signet-ring lymphocytes with dark or light cytoplasm, narrow-plasm lymphocytes with large prolonged nuclei and lamellipodia, lymphocytes with single lobopodia, and lymphocytes with single spiral structures in the cytoplasm . The spiral structure is 10-11 microns in length and 0.5-0.7 micron in width, being presumably a mitochondrion or a group of butt-joined mitochondria . Since some of the caudate middle-sized lymphocytes also contain this structure, these may be regarded as putative precursors of respective type of PHA-stimulated lymphocytes . Under the conditions of observation, interphase nuclei of all live PHA-stimulated lymphocytes were seen to contain numerous globular or fiber structures of condensed chromatin made of 0.3-0.8 micron beads . These beads are doubtless interphase chromomeres. Tsitologiia, 2003, 45(4), 418 - 21 {Myocardium of mdx mice contains factor(s) that damage DNA structure and retard DNA reparation after gamma-irradiation (experiences in modeling system)}; Mikhailov VM et al.; We studied the action of saline extracts of ventricle myocard (EM) of C57BL and mdx mice on DNA structure and repair of one-strand breaks of DNA in a modelling system . The system involves DNA repair in E . coli WP2 cells after gamma-irradiation . Using standard technique, DNA reparation was estimated on measuring the speed of E . coli DNA sedimentation in alkaline sucrose gradients . It was shown, that EM of C57BL or mdx mice exerted no influence on DNA repair, which was completely declined within 60 min with EM present in the growth medium of permeabilized E . coli . Addition of C57BL mice EM into lytic solution does not accelerate DNA sedimentation of nonirradiated E . coli . At the same time, EM of mdx mice sharply accelerates DNA sedimentation of nonirradiated E . coli reducing DNA molecular weight from 200 x 10(6) to 135 x 10(6) Da . At entering in the lytic solution the EM of mdx mice also slows down E . coli DNA repair after gamma-irradiation . It is supposed, that EM of mdx mice may contain a factor(s) damaging DNA in the E . coli lysate and presumably slowing down DNA reparation after gamma-irradiation . Russian Foundation of Basic Research Grants 99-04-49390, 02-04-49870 and 00-04-49390. Angiogenesis, 1999, 3(4), 335 - 44 An in vitro model of angiogenesis: basic features; Bishop ET et al.; This report describes a model of angiogenesis which develops in admixtures (co-cultures) of human umbilical vein endothelial cells (HUVEC) and human diploid fibroblasts of dermal origin from adult patients . The system does not require the addition of further growth factors other than those normally present in endothelial growth medium (EGM), nor matrix proteins, and cell growth and proliferation are allowed to occur in a standard low (2%) concentration of fetal calf serum . Angiogenesis was specifically stimulated in response to vascular endothelial growth factor (VEGF), resulting in an increased development of structures resembling a microvasculature bed . Alternatively, angiogenesis was inhibited by addition of an excess of neutralising anti-VEGF antibodies, and the anti-angiogenic drugs such as suramin . We briefly show that stimulatory and inhibitory activities can be easily and quickly quantified by image analysis . Tubule formation was confirmed by confocal and electron microscopy, and the development and disposition of these structures within the co-cultures has been analysed immunochemically to show expression of specific endothelial cell determinants, such as PECAM-1 . On this and a number of other criteria, the findings validate this in vitro process as a model of in vivo angiogenesis that can be quantified to assay stimulatory and inhibitory agents, signals and drugs. Angiogenesis, 1998, 1(2), 192 - 200 On the mechanism of thrombin-induced angiogenesis: inhibition of attachment of endothelial cells on basement membrane components; Tsopanoglou NE et al.; Human umbilical vein endothelial cells (HUVECs) placed on plastic plates coated with collagen type IV or laminin adhered within 60 min to an extent of about 32 and 39%, respectively . Brief exposure of HUVECs to thrombin caused a marked dose-dependent inhibition of adhesion . Thrombin at 1 IU/ml caused 50% inhibition even after 5 min of exposure of HUVECs . This effect was reversible since reincubation of thrombin-treated HUVECs with fresh growth medium for 15 min restored their ability for attachment . This short-term inhibitory effect of thrombin on the adhesion of HUVECs to extracellular matrix components was specific and depended on the activation of thrombin receptor . Hirudin abolished this effect of thrombin . Similarly, the proteolytically inactive PPACK-thrombin had no effect, but when used in combination with thrombin prevents the inhibitory effect of thrombin . In addition, the thrombin receptor agonist peptide (TRAP) mimicked the effect of thrombin on HUVEC adhesion . The transduction mechanism involved in this action of thrombin seems to be via cAMP, since forskolin or the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine restored the ability of HUVECs that had been exposed to thrombin to adhere . This novel cellular action of thrombin on endothelial cells may represent an important early event in activation of the normally quiescent endothelial cells and initiation of the angiogenic cascade. Mol Cell Biol, 2003 Oct, 23(20), 7256 - 70 c-Myc augments gamma irradiation-induced apoptosis by suppressing Bcl-XL; Maclean KH et al.; Alterations in MYC and p53 are hallmarks of cancer . p53 coordinates the response to gamma irradiation (gamma-IR) by either triggering apoptosis or cell cycle arrest . c-Myc activates the p53 apoptotic checkpoint, and thus tumors overexpressing MYC often harbor p53 mutations . Nonetheless, many of these cancers are responsive to therapy, suggesting that Myc may sensitize cells to gamma-IR independent of p53 . In mouse embryo fibroblasts (MEFs) and in E micro -myc transgenic B cells in vivo, c-Myc acts in synergy with gamma-IR to trigger apoptosis, but alone, when cultured in growth medium, it does not induce a DNA damage response . Surprisingly, c-Myc also sensitizes p53-deficient MEFs to gamma-IR-induced apoptosis . In normal cells, and in precancerous B cells of E micro -myc transgenic mice, this apoptotic response is associated with the suppression of the antiapoptotic regulators Bcl-2 and Bcl-X(L) and with the concomitant induction of Puma, a proapoptotic BH3-only protein . However, in p53-null MEFs only Bcl-X(L) expression was suppressed, suggesting levels of Bcl-X(L) regulate the response to gamma-IR . Indeed, Bcl-X(L) overexpression blocked this apoptotic response, whereas bcl-X-deficient MEFs were inherently and selectively sensitive to gamma-IR-induced apoptosis . Therefore, MYC may sensitize tumor cells to DNA damage by suppressing Bcl-X. Fungal Genet Biol, 2003 Nov, 40(2), 103 - 14 The Aspergillus nidulans alcA promoter drives tightly regulated conditional gene expression in Aspergillus fumigatus permitting validation of essential genes in this human pathogen; Romero B et al.; Aspergillus fumigatus causes invasive aspergillosis, a mycosis that is usually fatal in immunocompromised patients . Functional genomics in this fungus will aid the discovery of novel antifungal drugs to treat invasive aspergillosis . However, there is still a need for appropriate molecular genetic tools to facilitate such functional studies . Here, we describe the use of a conditional gene expression system allowing the identification of novel therapeutic targets through validation of essential genes in A . fumigatus . This system is based on the capacity of the Aspergillus nidulans alcA promoter (alcA(p)) to tightly regulate gene expression in this fungus . Conditionally regulated gene expression in A . fumigatus was demonstrated by transcriptional and phenotypic analyses of strains expressing a nuclear migration gene with a terminal phenotype, the A . fumigatus nudC gene, under control of this promoter . This conditional expression system, the first one described in A . fumigatus, will also be useful for investigating the function of essential genes by altering the threonine/glucose ratio in the growth medium. Am J Physiol Lung Cell Mol Physiol, 2004 Jan, 286(1), L189 - 97 Epub 2003 Sep 26. Latent adenoviral infection induces production of growth factors relevant to airway remodeling in COPD; Ogawa E et al.; Previous studies showed an association between latent adenoviral infection with expression of the adenoviral E1A gene and chronic obstructive pulmonary disease (COPD) . The present study focuses on how the adenoviral E1A gene could alter expression of growth factors by human bronchial epithelial (HBE) cells . The data show that connective tissue growth factor (CTGF) and transforming growth factor (TGF)-beta 1 mRNA and protein expression were upregulated in E1A-positive HBE cells . Upregulation of CTGF in this in vitro model was independent of TGF-beta secreted into the growth medium . Comparison of E1A-positive with E1A-negative HBE cells showed that both expressed cytokeratin but only E1A-positive cells expressed the mesenchymal markers vimentin and alpha-smooth muscle actin . We conclude that latent infection of epithelial cells by adenovirus E1A could contribute to airway remodeling in COPD by the viral E1A gene, inducing TGF-beta 1 and CTGF expression and shifting cells to a more mesenchymal phenotype. Environ Microbiol, 2003 Oct, 5(10), 986 - 96 Involvement of rpoS in the survival of Escherichia coli in the viable but non-culturable state; Boaretti M et al.; When exposed to stress-provoking environmental conditions such as those of ground waters, many medically important bacteria have been shown to be capable of activating a survival strategy known as the viable but non-culturable (VBNC) state . In this state bacteria are no longer culturable on conventional growth media, but the cells maintain their viability and pathogenicity genes/factors and can start dividing again, in a part of the cell population, upon restoration of favourable environmental conditions . Little is known about the genetic mechanisms underlying the VBNC state . In this study we show evidence of involvement of the rpoS gene in persistence of Escherichia coli in the VBNC state . The kinetics of entry into the non-culturable state and duration of cell viability were measured in two E . coli mutants carrying an inactivated rpoS gene and compared with those of the parents . For these experiments, laboratory microcosms consisting of an artificial oligotrophic medium incubated at 4 degrees C were used . The E . coli parental strains reached the non-culturable state in 33 days when the plate counts were evaluated on Luria-Bertani agar containing sodium pyruvate, whereas cells of the rpoS mutants lost their culturability in only 21 days . Upon reaching unculturability the parents yielded respiring cells and cells with intact membranes for at least the next three weeks and resuscitation was allowed during this time . In contrast, the RpoS- mutant cells demonstrated intact membranes for only two weeks and a very restricted (<7 days) resuscitation capability . Guanosine 3',5'-bispyrophosphate (ppGpp) acts as a positive regulator during the production and functioning of RpoS . A mutant deficient in ppGpp production behaved like the rpoS mutants, while overproducers of ppGpp displayed a vitality at least comparable to that of RpoS+ strains . These results suggest that the E . coli parental strains enter the VBNC state which lasts for, at least, three weeks, after which apparently all the cells die . The rpoS mutants do not activate this survival strategy and early die . This implies involvement of the rpoS gene in E . coli persistence in the VBNC state. Biocell, 2003 Aug, 27(2), 173 - 9 Effect of sugars on the association between cowpea vicilin (7S storage proteins) and fungal cells; Rose TL et al.; Vicilins (7S storage proteins) found in various legume seeds have been previously shown to interfere with the germination of spores or conidia of phytopathogenic fungi and inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells . In the present work vicilins from cowpea (Vigna unguiculata) seeds were added to the growth medium of Saccharomyces cerevisiae cells and Fusarium oxysporum conidia . Helix pomatia lectin, wheat germ agglutinin and Ulex europaeus lectin were used to identify differences in the binding of the vicilins to the surface of cells of S . cerevisiae and F . oxysporum treated with this protein . After the growth period, the material in suspension (yeast cells) was centrifuged and the final pellet was also treated with different sugar (glucose, sucrose, glucosamine, N-acetyl-glucosamine) concentrations and 0.1 M HCl for extraction of vicilins associated to chitinous structures present in yeast cells . Our results showed that vicilin sub-units were present in the different sugar extracts of yeast cells pretreated with the vicilins and these proteins were eluted by 0.5 M solutions of sugars in the following order of efficiency of elution: N-acetyl-glucosamine, sucrose/glucose and glucosamine. J Inorg Biochem, 2003 Sep 15, 97(1), 59 - 68 An intracellular mechanism of aluminum tolerance associated with high antioxidant status in cultured tobacco cells; Devi SR et al.; An aluminum (Al) tolerance mechanism, together with oxidative stress tolerance, was investigated in an Al tolerant cell line (ALT301) and the parental Al sensitive cell line (SL) of tobacco . During Al exposure in a simple calcium solution for 24 h, Al triggered the evolution of a reactive oxygen species (ROS) in SL much higher than ALT301 {Plant Physiol . 128 (2002) 63} . Under the conditions, Al enhanced comparable rates of citrate secretion from both cell lines to the same extent . Al enhanced the gene expression of manganese superoxide dismutase (MnSOD) in both cell lines, but at a significantly higher rate in SL than in ALT301, and also enhanced the enzyme activity of MnSOD in both cell lines to nearly the same level . These results suggest that the extracellular chelation of Al with organic acids and MnSOD is not involved in the mechanism of Al tolerance of ALT301 . ALT301 contained ascorbate (ASA) and glutathione (GSH) levels that were higher than SL under normal growth conditions . During 24 h of post-Al treatment culture in growth medium, but not during 24-h Al exposure in a simple Ca(2+) solution, lipid peroxidation was enhanced in SL much higher than in ALT301, and the average SL amounts of ASA and GSH were exhausted compared to ALT301 . Pre-loading of ASA prior to Al treatment improved the growth of SL during the post-Al treatment culture . ALT301 also exhibited cross-tolerance to H(2)O(2), Fe(2+) and Cu(2+) . Under these oxidant exposures, ALT301 contained lower levels of intracellular H(2)O(2) or lipid peroxides, and maintained higher amounts of ASA and GSH than SL . Taken together, we conclude that the accumulation of Al in cells enhances the peroxidation of lipids exclusively under growing conditions, and that the higher content of ASA and GSH in ALT301 than in SL seems to be in part responsible for the tolerance mechanism of ALT301 to Al by protecting cells from either lipid peroxidation or H(2)O(2) commonly enhanced by Al or other oxidants. J Biol Chem, 2003 Dec 5, 278(49), 49625 - 35 Epub 2003 Sep 23. ATP generation in the Trypanosoma brucei procyclic form: cytosolic substrate level is essential, but not oxidative phosphorylation; Coustou V et al.; Trypanosoma brucei is a parasitic protist responsible for sleeping sickness in humans . The procyclic form of this parasite, transmitted by tsetse flies, is considered to be dependent on oxidative phosphorylation for ATP production . Indeed, its respiration was 55% inhibited by oligomycin, which is the most specific inhibitor of the mitochondrial F0/F1-ATP synthase . However, a 10-fold excess of this compound did not significantly affect the intracellular ATP concentration and the doubling time of the parasite was only 1.5-fold increased, suggesting that oxidative phosphorylation is not essential for procyclic trypanosomes . To further investigate the sites of ATP production, we studied the role of two ATP producing enzymes, which are involved in the synthesis of pyruvate from phosphoenolpyruvate: the glycosomal pyruvate phosphate dikinase (PPDK) and the cytosolic pyruvate kinase (PYK) . The parasite was not affected by PPDK gene knockout . In contrast, inhibition of PYK expression by RNA interference was lethal for these cells . In the absence of PYK activity, the intracellular ATP concentration was reduced by up to 2.3-fold, whereas the intracellular pyruvate concentration was not reduced . Furthermore, we show that this mutant cell line still excreted acetate from d-glucose metabolism, and both the wild type and mutant cell lines consumed pyruvate present in the growth medium with similar high rates, indicating that in the absence of PYK activity pyruvate is still present in the trypanosomes . We conclude that PYK is essential because of its ATP production, which implies that the cytosolic substrate level phosphorylation is essential for the growth of procyclic trypanosomes. In Vitro Cell Dev Biol Anim, 2003 Mar-Apr, 39(3-4), 110 - 3 Establishment and characterization of a new epithelial cell line, KC-1, from koala (Phascolarctos cinereus) conjunctiva; Girjes AA et al.; A novel, untransformed koala cell line (KC-1) was established by culturing koala conjunctival tissue in growth medium, which has permitted the study of the cell biology of this unique system . After the establishment of the KC-1 cell line, the cells were characterized by light microscopy, doubling time, and Western blot analysis . Light microscopy revealed that the cells have an epithelial morphology . Doubling times were significantly different (P < 0.015) depending on fetal calf serum (FCS) concentration (16.5 h in 10% FCS and 26.5 h in 2% FCS) . Cells constricted while in suspension but were shown to attach to the coverslip (or flask) and flatten rapidly, less than 1 h after seeding . To confirm the epithelial nature of the cells, protein was extracted and Western blot analysis was performed . Subsequent probing with primary and secondary antibodies (monoclonal anticytokeratin clone C-11 IgG1 and anti-mouse IgG) revealed two bands at 45 and 52 kDa (compared against a protein molecular weight marker) that correspond to primary type I keratin and major type II keratin, respectively, expressed in simple epithelial cells . The koala cell line was adapted to grow continuously in Dulbecco modified Eagle medium containing 10% FCS for at least 30 passages . This unique cell line is an ideal tool for further investigation on koala cell biology and cytogenetics and for exploration of the pathophysiological mechanism of eye infections caused by different pathogens in koalas. In Vitro Cell Dev Biol Anim, 2003 Mar-Apr, 39(3-4), 163 - 9 Enhanced myogenic differentiation by extracellular matrix is regulated at the early stages of myogenesis; Langen RC et al.; Myogenic cell lines have been used extensively in the study of skeletal muscle development, regeneration, and homeostasis . To induce myogenic differentiation, culture media composed of a wide variety of growth factors and other additives have been used . Because the diversity in these components may modulate the differentiation process differentially, we describe a differentiation protocol that does not require the introduction of any factors to the differentiation media (DM) other than those present in the growth media . By culturing C2C12 skeletal myocytes on a coating of diluted Matrigel, a soluble basement membrane, consisting of collagen IV, laminin, heparan sulfate proteoglycans, and entactin, myogenic differentiation was accomplished by mere serum reduction . Assessment of myotube formation, creatine kinase activity, myosin heavy chain-fast, and myogenin demonstrated that the kinetics and extent of myogenic differentiation were superior using this protocol, compared with a commonly used differentiation protocol, in which an extracellular matrix is not provided and the DM contains horse serum . In addition, the elevated transactivation of a troponin-I promoter reporter construct suggested that myogenesis was enhanced at the transcriptional level . Finally, assessment of genomic deoxyribonucleic acid content revealed that the Matrigel differentiation protocol resulted in lowered proliferation . This protocol may aid studies aimed at elucidating mechanisms of myogenic differentiation, where a homogeneous population of myotubes is preferred. Genetics, 2003 Sep, 165(1), 23 - 33 Analysis of the generation and segregation of propagons: entities that propagate the {PSI+} prion in yeast; Cox B et al.; The propagation of the prion form of the yeast Sup35p protein, the so-called {PSI(+)} determinant, involves the generation and partition of a small number of particulate determinants that we propose calling "propagons." The numbers of propagons in {PSI(+)} cells can be inferred from the kinetics of elimination of {PSI(+)} during growth in the presence of a low concentration of guanidine hydrochloride (GdnHCl) . Using this and an alternative method of counting the numbers of propagons, we demonstrate considerable clonal variation in the apparent numbers of propagons between different {PSI(+)} yeast strains, between different cultures of the same {PSI(+)} yeast strain, and between different cells of the same {PSI(+)} culture . We provide further evidence that propagon generation is blocked by growth in GdnHCl and that it is largely confined to the S phase of the cell cycle . In addition, we show that at low propagon number there is a bias toward retention of propagons in mother cells and that production of new propagons is very rapid when cells with depleted numbers of propagons are rescued into normal growth medium . The implications of our findings with respect to yeast prion propagation mechanisms are discussed. Mar Biotechnol (NY), 2003 May-Jun, 5(3), 302 - 10 Sustainable, high-yielding outdoor mass cultures of Chaetoceros muelleri var . subsalsum and Isochrysis galbana in vertical plate reactors; Zhang CW et al.; Continuous cultures of Chaetoceros muelleri and Isochrysis galbana were grown outdoors in flat plate-glass reactors in which light-path length (LPL) varied from 5 to 30 cm . High daily productivity (13 to 16 g cell mass per square meter of irradiated reactor surface) for long periods of time was obtained in reactors in which the optical path as well as cell density were optimized . 'Twenty centimeters was the optimal LPL, yielding the highest areal productivity of cell mass (g m(-2)d(-1)), eicosapentaenoic acid, and docosahexaenoic acid, which was identical with that previously found for polysaccharide production of Porphyridium and not far from the optimal LPL affecting maximal productivity in Nannochloropsis species . Relating the energy impinging on a given reactor surface area to the appropriate number of cells showed that the most efficient light dose per cell, obtained with the 20-cm LPL reactor, was approximately 2.5 times lower than the light dose available per cell in the 5-cm LPL reactor, in which a significant decline in areal cell density accompanied the lowest areal output of cell mass . The most effective harvesting regimen was in the range of 10% to 15% of culture volume harvested daily and replaced with fresh growth medium, resulting in a sustainable culture density of 24 x 10(6) and 28 x 10(6) cells/ml of C . muelleri and I . galbana, respectively. Curr Genet, 2003 Dec, 44(4), 224 - 30 Epub 2003 Sep 17. The application of PCR for the isolation of a lipase gene from the genomic DNA of an Antarctic microfungus; Bradner JR et al.; We successfully isolated a lipase gene (designated lipPA) directly from the genomic DNA of an Antarctic isolate of Penicillium allii using PCR and a suite of degenerate primers specifically designed to target two conserved regions of fungal lipase genes . We applied the biolistic transformation system to successfully integrate the lipPA gene into a heterologous fungal host, Trichoderma reesei, one of the most powerful secretors of extracellular proteins, and induced the transformant to secrete an active lipase into the growth medium . The recombinant lipase had a temperature optimum of 25 degrees C at pH 7.9 and retained greater than 50% of the maximum activity from 10 degrees C to 35 degrees C and over a pH range from 4.0 to 8.5. FEMS Microbiol Lett, 2003 Sep 12, 226(1), 39 - 43 Effects of purine nucleosides on the in vitro growth of Cryptosporidium parvum; Lawton P et al.; The effect of purine nucleosides on the in vitro growth of Cryptosporidium parvum was studied . Culturing the parasite in THP-1 cells for 72 h in growth medium supplemented with adenosine or inosine improved the parasite yields especially in the first 48 h . Similar results were obtained with parasites cultured in Madin-Darby bovine kidney cells and incubated for 24 h with inosine . The addition of inosine to 72-h cultures enhanced the growth of C . parvum in THP-1 cells, especially the trophic stages, whereas the analogue formycin B was toxic to the parasites and induced a marked decrease in the gamont stages . The monitoring of the added purine nucleosides by high performance liquid chromatography showed that at 37 degrees C in the presence of THP-1 cells, a rapid uptake of inosine occurred with hypoxanthine being the main purine present after 2 h in the medium. Chest, 2003 Sep, 124(3 Suppl), 58S - 68S Tissue factor, thrombin, and cancer; Rickles FR et al.; In addition to its primary role in hemostasis and blood coagulation, thrombin is a potent mitogen capable of inducing cellular functions . Therefore, it should come as no surprise that thrombin has proved to be of importance in the behavior of cancer . In this review, we focus on the ability of tissue factor (TF) and thrombin to influence tumor angiogenesis . Both exert their influence on angiogenesis through clotting-dependent and clotting-independent mechanisms: (1) . directly affecting signaling pathways that mediate cell functions, and (2) . mediating clot formation, thereby providing a growth media for tumor cells . Therefore, anticoagulant drugs may prove efficacious in cancer treatment due to their ability to reduce the characteristic hypercoagulability of cancer and alter the fundamental biology of cancer. J Appl Microbiol, 2003, 95(4), 799 - 806 Beijerinckia derxii releases plant growth regulators and amino acids in synthetic media independent of nitrogenase activity; Thuler DS et al.; AIMS: This study aims at evaluating the ability of Beijerinckia derxii, a free-living nitrogen (N)-fixing bacterium frequently isolated from tropical soils, to release certain plant growth regulators {indoleacetic acid (IAA), ethylene, polyamines} and amino acids into the growth medium . METHODS AND RESULTS: The production of those substances was compared using both cultures in which nitrogenase was active (N-free medium) and cultures in which nitrogenase was repressed (combined-N cultures) . Those cultures were grown under agitation and in absence of agitation . Total IAA production was higher in agitated, N-free cultures but specific production was greater in combined-N cultures under agitation . Putrescine and spermidine were detected under all conditions tested . Ethylene was produced in both N-free and combined-N cultures . A greatest diversity of amino acids was released in N-free cultures . CONCLUSIONS: There was no inhibition of the production of the analysed substances under conditions where nitrogenase was inactive . SIGNIFICANCE AND IMPACT OF THE STUDY: Beijerinckia derxii is potentially a producer of plant-active substances; its presence in the natural environment suggests that this bacterium may contribute to the development of other living organisms. Biochem Biophys Res Commun, 2003 Sep 26, 309(3), 702 - 8 Growth arrest by octanoate is required for porcine preadipocyte differentiation; Nakajima I et al.; A preadipocyte clonal line has been established from porcine subcutaneous tissue . This line, designated PSPA, showed a fibroblastic phenotype and kept on growing under a preadipose condition even after reaching confluence . When confluent cultures were stimulated with insulin, dexamethasone, biotin, pantothenate, and octanoate, growth was arrested, and the cells exhibited a marked increase in lipogenesis . However, adipose conversion was not induced upon exposure of PSPA cells to a standard hormonal mixture of mouse 3T3-L1 cells, and they continued dividing as did the preadipocytes in growth medium . By serially omitting each individual adipogenic agent from the PSPA differentiation medium, it was determined that octanoate was one of the most essential but the only factor able to induce growth arrest . Octanoate supplementation to 3T3-L1 medium increased the triglyceride accumulation of PSPA cells accompanied by growth arrest . Both RT-PCR and Western blot analysis supported the idea of octanoate as a potential agent with the antiproliferative activity requisite for porcine preadipocytes to enter terminal differentiation. J Clin Microbiol, 2003 Sep, 41(9), 4252 - 8 Modification of rapid susceptibility assay for antifungal susceptibility testing of Aspergillus fumigatus; Wetter TJ et al.; To improve objectivity and speed of current antifungal mold susceptibility testing, the yeast Rapid Susceptibility Assay (RSA) was adapted for Aspergillus species . The RSA is based on glucose utilization in the presence of an antifungal drug . Aspergillus fumigatus conidia were incubated in 0.2% glucose RPMI 1640 containing 0.03 to 16 micro g of amphotericin B or itraconazole/ml . Drug-related inhibition of glucose utilization correlated with suppression of conidial germination . Following incubation of conidia with various concentrations of antifungal drug, the percentage of residual glucose in the growth medium was determined colorimetrically and plotted against drug concentration to determine the MIC (MIC(RSA)) . National Committee for Clinical Laboratory Standards (NCCLS) M38-P testing was also performed to obtain NCCLS MICs (MIC(NCCLS)) for direct comparison with MIC(RSA)s . Conidial inocula of an optical density at 530 nm (OD(530)) of 0.11 facilitated determination of amphotericin B and itraconazole MIC(RSA)s at 16 h equal to or within a single twofold dilution of MIC(NCCLS)s obtained at 48 h . Preliminary testing with a 0.11-OD(530) conidial inoculum of the slower-growing Aspergillus terreus resulted in itraconazole and amphotericin B MIC(RSA)s at 16 h equal to or within a single twofold dilution of MIC(NCCLS)s obtained at 48 h . These data indicate that the mold RSA provides a more objective and rapid method for Aspergillus spp . susceptibility testing than the NCCLS M38-P assay. Appl Environ Microbiol, 2003 Sep, 69(9), 5643 - 7 Stereoselective microbial dehalorespiration with vicinal dichlorinated alkanes; De Wildeman S et al.; The suspected carcinogen 1,2-dichloroethane (1,2-DCA) is the most abundant chlorinated C(2) groundwater pollutant on earth . However, a reductive in situ detoxification technology for this compound does not exist . Although anaerobic dehalorespiring bacteria are known to catalyze several dechlorination steps in the reductive-degradation pathway of chlorinated ethenes and ethanes, no appropriate isolates that selectively and metabolically convert them into completely dechlorinated end products in defined growth media have been reported . Here we report on the isolation of Desulfitobacterium dichloroeliminans strain DCA1, a nutritionally defined anaerobic dehalorespiring bacterium that selectively converts 1,2-dichloroethane and all possible vicinal dichloropropanes and -butanes into completely dechlorinated end products . Menaquinone was identified as an essential cofactor for growth of strain DCA1 in pure culture . Strain DCA1 converts chiral chlorosubstrates, revealing the presence of a stereoselective dehalogenase that exclusively catalyzes an energy-conserving anti mechanistic dichloroelimination . Unlike any known dehalorespiring isolate, strain DCA1 does not carry out reductive hydrogenolysis reactions but rather exclusively dichloroeliminates its substrates . This unique dehalorespiratory biochemistry has shown promising application possibilities for bioremediation purposes and fine-chemical synthesis. J Chem Ecol, 2003 Aug, 29(8), 1919 - 37 Laboratory assessment of the allelopathic effects of fine leaf fescues; Bertin C et al.; Laboratory screening studies were conducted to evaluate the allelopathic potential of fine leaf fescues . Of the seven accessions selected from prior field evaluations for weed-suppressive ability, all inhibited root growth of large crabgrass and curly cress in laboratory assays . Grown in agar as a growth medium and in the presence of living fescue seedlings for 14 or 21 days, test species were sensitive depending on the fescue cultivars . Growth inhibition increased when fescue was grown for increasing periods of time in agar . Seedling fescues produced significant quantities of bioactive root exudates, which were released into the agar medium . Bioactive root exudates were extracted from living fescue roots by using methylene chloride . Shoot tissue was extracted in water and the aqueous extract was partitioned against hexane, ethyl acetate, and methylene chloride . Extracts were tested for inhibitory activity on seedling growth as measured by inhibition of curly cress germination and radicle elongation . Root exudates were more toxic (70% inhibition) than shoot extracts (up 40% inhibition), when formulated at 0.25 mg/ml concentration . Light microscopy and transmission electron microscopy were utilized in an attempt to identify the cellular location of production of secondary products contained in bioactive root exudates . Ultrastructural analysis indicated that the exudate is produced in actively dividing tips of fibrous root cells . The mode of release of these exudates into the environment remains unknown. Curr Genet, 2003 Nov, 44(3), 155 - 63 Epub 2003 Sep 04. Pro