Int J Food Microbiol, 2003 Jan 25, 80(2), 171 - 6 Beta-glucosidase activity in a Saccharomyces cerevisiae wine strain; Hernandez LF et al.; Beta-glucosidase activity contributes to aroma formation during the winemaking process . This study investigated whether beta-glucosidase activity was expressed by wild Saccharomyces cerevisiae strains and by a laboratory strain . beta-Glucosidase activity was assayed on several culture media and under various growth conditions . The highest activities were obtained in Yeast Extract Peptone medium, but activity was also detected using grape juice as the growth medium, although a 25% drop activity was observed when anaerobic conditions were employed . A number of parameters affecting beta-glucosidase activity were evaluated . Optimal conditions for activity were pH 4 and a temperature of 40-50 degrees C . The results showed beta-glucosidase activity to be present during the process of winemaking, although different from the optimal conditions. J Exp Bot, 2002 Nov, 53(378), 2151 - 8 Recovery of tobacco cells from cadmium stress is accompanied by DNA repair and increased telomerase activity; Fojtova M et al.; It has been shown previously that apoptosis of tobacco cells induced by cadmium ions shows a relatively long lag period between exposure and cell death . This lag phase lasts for 3 d in TBY-2 cell cultures and is characterized by the maintenance of full cell viability despite extensive fragmentation of DNA into pieces of chromatin loop size . Experiments reported here demonstrate that cell death can be prevented if 50 micro M CdSO(4) is removed from the growth medium during the lag phase, suggesting that an irreversible apoptotic trigger is delivered within 24 h, between the third and fourth days of cadmium treatment . The post-cadmium recovery phase was characterized by DNA repair at the level of 50-200 kb and increased telomerase activity . Analysis of high-molecular-weight DNA by pulsed-field-gel electrophoresis revealed that the majority of DNA strand breaks was repaired within 48 h after cadmium withdrawal . Telomerase activity increased 2.5-fold in the recovery phase, but elevated levels were also found in cell extracts from apoptotic cells suggesting that telomerase might be associated with DNA repair, but it is not capable of inhibiting ongoing apoptosis . Limited exposure of TBY-2 cells to cadmium elicits non-random DNA damage of relatively high magnitude that can be repaired . It is proposed that plants might have developed a highly efficient DNA repair system to cope with transient genotoxic stress. Proc Natl Acad Sci U S A, 2002 Oct 15, 99(21), 13413 - 8 Epub 2002 Oct 07. Structure and function in rhodopsin: a tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants; Reeves PJ et al.; Tetracycline-inducible HEK293S stable cell lines have been prepared that express high levels (up to 10 mg/liter) of WT opsin and its mutants only in response to the addition of tetracycline and sodium butyrate . The cell lines were prepared by stable transfection of HEK293S-TetR cells with expression plasmids that contained the opsin gene downstream of a cytomegalovirus promoter containing tetO sequences as well as the neomycin resistance gene under control of the weak H(2)L(d) promoter . The inducible system is particularly suited for overcoming problems with toxicity either due to the addition of toxic compounds, for example, tunicamycin, to the growth medium or due to the expressed protein products . By optimization of cell growth conditions in a bioreactor, WT opsin, a constitutively active opsin mutant, E113Q/E134Q/M257Y, presumed to be toxic to the cells, and nonglycosylated WT opsin obtained by growth in the presence of tunicamycin have been prepared in amounts of several milligrams per liter of culture medium. Water Res, 2002 Sep, 36(15), 3727 - 38 Applying whole-water samples directly to fish cell cultures in order to evaluate the toxicity of industrial effluent; Dayeh VR et al.; Methodology was developed for presenting to fish cells in culture whole-water samples without extraction and used to evaluate the toxicity to a rainbow trout gill cell line, RTgill-W1, of more than 30 whole-water samples collected from a paper mill over approximately a year of operation . Presentation to cells was achieved by adding to water samples the amounts of salts, galactose and sodium pyruvate, as solids, that were necessary to give concentrations and osmolality of the basal growth medium, Leibovitz's L-15 . Cell viability was measured with three fluorescent indicator dyes: alamar Blue for metabolism, 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) for membrane function, and neutral red for lysosomal activity . Eighteen samples were tested with the Daphnia lethality bioassay and 11 of these were toxic . None of these were judged cytotoxic to RTgill-W1 . Sixteen samples were tested with the rainbow trout lethality bioassay and only one was toxic . This sample was also the only sample that was cytotoxic to RTgill-W1 . Therefore, these methods for presenting water samples and measuring their cytotoxicity to RTgill-W1 are a promising substitute for toxicity tests of industrial effluent with rainbow trout but not with Daphnia. Antibiot Khimioter, 2002, 47(4), 7 - 12 {L-Glutamate oxidase from Streptomyces cremeus 510 MGU: growth media optimization for the enhancement of the producer fermentative activity}; Vinogradova KA et al.; The investigation was devoted to culture conditions optimization aimed to maximum secretion of extracellular L-glutamate oxidase by Streptomyces cremeus 510 MGU . It was shown that Ca ions at the concentration 5-20 mM and 0.1% ammonium sulphate enhanced activity of extracellular enzyme to 4 folds . L-glutamate acid supplement had no effect on enzyme activity . Influence of some bivalent cations and aeration regimes on L-glutamate oxidase activity was investigated . Growth media optimization along with screening of active variants resulted with isolation of the strain with L-glutamate oxidase activity about 2 U/mL Rate of peroxide degradation in the presence of filtrated culture of S . cremeus was determined by chemiluminescence method. Microbiology, 2002 Oct, 148(Pt 10), 2937 - 49 Azole antifungals are potent inhibitors of cytochrome P450 mono-oxygenases and bacterial growth in mycobacteria and streptomycetes; McLean KJ et al.; The genome sequence of Mycobacterium tuberculosis has revealed the presence of 20 different cytochrome P450 mono-oxygenases (P450s) within this organism, and subsequent genome sequences of other mycobacteria and of Streptomyces coelicolor have indicated that these actinomycetes also have large complements of P450s, pointing to important physiological roles for these enzymes . The actinomycete P450s include homologues of 14alpha-sterol demethylases, the targets for the azole class of drugs in yeast and fungi . Previously, this type of P450 was considered to be absent from bacteria . When present at low concentrations in growth medium, azole antifungal drugs were shown to be potent inhibitors of the growth of Mycobacterium smegmatis and of Streptomyces strains, indicating that one or more of the P450s in these bacteria were viable drug targets . The drugs econazole and clotrimazole were most effective against M . smegmatis (MIC values of <0.2 and 0.3 micro M, respectively) and were superior inhibitors of mycobacterial growth compared to rifampicin and isoniazid (which had MIC values of 1.2 and 36.5 micro M, respectively) . In contrast to their effects on the actinomycetes, the azoles showed minimal effects on the growth of Escherichia coli, which is devoid of P450s . Azole drugs coordinated tightly to the haem iron in M . tuberculosis H37Rv P450s encoded by genes Rv0764c (the sterol demethylase CYP51) and Rv2276 (CYP121) . However, the azoles had a higher affinity for M . tuberculosis CYP121, with K(d) values broadly in line with the MIC values for M . smegmatis . This suggested that CYP121 may be a more realistic target enzyme for the azole drugs than CYP51, particularly in light of the fact that an S . coelicolor DeltaCYP51 strain was viable and showed little difference in its sensitivity to azole drugs compared to the wild-type . If the azole drugs prove to inhibit a number of important P450s in M . smegmatis and S . coelicolor, then the likelihood of drug resistance developing in these species should be minimal . This suggests that azole drug therapy may provide a novel antibiotic strategy against strains of M . tuberculosis that have already developed resistance to isoniazid and other front-line drugs. Genome Res, 2002 Oct, 12(10), 1556 - 63 Draft sequencing and comparative genomics of Xylella fastidiosa strains reveal novel biological insights; Bhattacharyya A et al.; Draft sequencing is a rapid and efficient method for determining the near-complete sequence of microbial genomes . Here we report a comparative analysis of one complete and two draft genome sequences of the phytopathogenic bacterium, Xylella fastidiosa, which causes serious disease in plants, including citrus, almond, and oleander . We present highlights of an in silico analysis based on a comparison of reconstructions of core biological subsystems . Cellular pathway reconstructions have been used to identify a small number of genes, which are likely to reside within the draft genomes but are not captured in the draft assembly . These represented only a small fraction of all genes and were predominantly large and small ribosomal subunit protein components . By using this approach, some of the inherent limitations of draft sequence can be significantly reduced . Despite the incomplete nature of the draft genomes, it is possible to identify several phage-related genes, which appear to be absent from the draft genomes and not the result of insufficient sequence sampling . This region may therefore identify potential host-specific functions . Based on this first functional reconstruction of a phytopathogenic microbe, we spotlight an unusual respiration machinery as a potential target for biological control . We also predicted and developed a new defined growth medium for Xylella. Bioresour Technol, 2002 Dec, 85(3), 225 - 34 Optimal conditions for bio-oxidation of ferrous ions to ferric ions using Thiobacillus ferrooxidans; Malhotra S et al.; A chemo-biochemical process using Thiobacillus ferrooxidans for desulphurization of gaseous fuels and emissions containing hydrogen sulphide (H2S) has been developed . In the first stage, H2S present in fuel gas and emissions is selectively oxidized to elemental sulphur using ferric sulphate . The ferrous sulphate produced in the first stage of the process is oxidized to ferric sulphate using Thiobacillus ferrooxidans for recycle and reuse in the process . The effects of process variables, temperature, pH, total dissolved solids (TDS), elemental sulphur, ferric and magnesium ions on bio-oxidation of ferrous ions to ferric ions were investigated using flask culture experiments . The bio-oxidation of ferrous ions to ferric ions could be achieved efficiently in the temperature range of 20(+/-1)-44(+/-1) degrees C . A pH range of 1.8(+/-0.02)-2.2(+/-0.02) was optimum for the growth of culture and effective bio-oxidation of ferrous ions to ferric ions . The effect of TDS on bio-oxidation of ferrous ions indicated that a preacclimatized culture in a growth medium containing high dissolved solid was required to achieve effective bio-oxidation of ferrous ions . Elemental sulphur ranging from 1000 to 100,000 mg/l did not have any effect on efficiency of ferrous ion oxidation . The efficiency of bio-oxidation of ferrous ions to ferric ions was not affected in the presence of ferric ions up to a concentration of 500 mg/l while 3 mg/l of magnesium ion was optimal for achieving effective bio-oxidation. Dis Aquat Organ, 2002 Aug 29, 51(2), 139 - 47 Analysis of chitinase expression in the crayfish plague fungus Aphanomyces astaci; Andersson MG et al.; Chitinase, as determined by enzymatic activity in the growth medium and by transcription of the chitinase gene AaChi1, is expressed at a high level during vegetative growth of the crayfish pathogen Aphanomyces astaci and expression is not further stimulated by chitin . Expression is not detected in zoospores and it does not increase to high levels until late during germination . Upon sporulation, chitinase expression increases to levels comparable with those seen in fast-growing mycelium . This pattern of chitinase expression is a common feature of strains representing all currently known genotypes of A . astaci, suggesting that it is an adaptation to the exclusively parasitic life-style of this species . In contrast, other Aphanomyces spp . including the saprophytes, A . laevis and A . stellatus, produce significant amounts of chitinase only in the presence of chitin . The pattern of chitinase expression is one of very few qualitative physiological characteristics known which can distinguish A . astaci from other parasitic and saprophytic species and may thus be of practical use for identification. Indian J Med Res, 2002 May, 115, 189 - 93 Establishment of two new cell lines from Bombyx mori (L.) (Lepidoptera: Bombycidae) & their susceptibility to baculoviruses; Sudeep AB et al.; BACKGROUND & OBJECTIVES: Lepidopteran cell cultures and baculovirus expression vector systems are becoming popular due to their potential applications in biotechnology especially for the expression of foreign proteins . Efforts were made to develop new, indigenous, cell lines from Bombyx mori larvae and pupae . METHODS: Eight to ten B . mori larvae and 10-12 pupae were surface sterilized, dissected and ovaries were removed aseptically . Ovaries were chopped finely, washed and suspended in growth medium . When the cells formed monolayers, they were subcultured and experiments were carried out . RESULTS: Two new cell lines from larval and pupal ovaries of B . mori were established in Grace's insect tissue culture medium supplemented with 20 per cent FBS (foetal bovine serum) . The larval cell line consisted predominantly of epithelial-like cells (98.31%), whereas the pupal cell line had a mixed cell population of epithelial-like (71.8%) and fibroblast-like cells (27.8%) . Karyology indicated a typical lepidopteran pattern in both the cell lines and had chromosome numbers ranging from 35 to 150 and 60 to 180 for larval and pupal ovaries respectively . Four-fold increase in cell number was observed in these cell lines in 7 days . Both the cell lines were found susceptible to B . mori multiple nucleopolyhedrovirus and Autographa californica multiple nucleopolyhedrovirus, but not to Helicoverpa armigera single nucleopolyhedrovirus and Spodoptera litura multiple nucleopolyhedrovirus . INTERPRETATION & CONCLUSION: These well characterized cell lines may be of immense application in biotechnology and medicine for the production of biologically active recombinant proteins to use in vaccine studies as well as in therapeutic applications. Lett Appl Microbiol, 2002, 35(4), 343 - 6 Optimization of glycosidases production by Pseudoalteromonas issachenkonii KMM 3549(T); Alexeeva YV et al.; AIMS: The present work aimed to design an optimized medium to yield a higher production of glycosides by Pseudoalteromonas issachenkonii KMM 3549(T) . METHODS AND RESULTS: Higher levels of fucoidan hydrolase, alginase, laminaranase and b-N-acetylglucosaminidase production were obtained with peptone concentrations ranging from 2.5 g l(-1) to 10 g l(-1), while the presence of both yeast extract and glucose did not affect enzyme production . The activity of fucoidan hydrolase and laminaranase increased up to 4.83 microM h(-1) mg(-1) and 19.23 microM h(-1) mg(-1) protein, respectively, in growth media containing xylose (1.0 g l(-1)), laminarin (0.5 g l(-1)) or alginate (0.5 g l(-1)), and production of b-N-acetylglucosaminidase substantially increased in the presence of fucoidan (0.5 g l(-1)) or galactose (1 g l(-1)) . All polysaccharides tested in concentrations of 0.5 g l(-1) fucoidan and 0.2 g l(-1) fucose induced production of alginase (up to 5.06 microM h(-1) mg-1 protein) . CONCLUSIONS: The production of glycosidases is not only stimulated by the presence of algal polysaccharides, but may also be stimulated by monosaccharides (e.g . xylose) . SIGNIFICANCE AND IMPACT OF THE STUDY: The production of glycosidases by Pseudoalteromonas issachenkonii KMM 3549(T) was significantly improved by using a simple nutrient medium containing peptone (2.5 g l(-1)) and xylose (5.0 g l(-1)) in 100% natural seawater. Physiol Plant, 2002 Oct, 116(2), 231 - 237 Effect of anti-auxins on maturation of embryogenic tissue cultures of Nordmanns fir (Abies nordmanniana); Find J et al.; The present study was conducted to improve the transition from proliferation to maturation in embryogenic cultures of Nordmanns fir . For that reason, chemicals reported to affect endogenous levels or activity of auxin were included in the growth media during maturation . The auxin antagonist PCIB reduced proliferation and promoted the development of numerous high-quality mature embryos in the tested cell lines . PCIB could not substitute for exogenously supplied ABA and the positive effect was only found when PCIB and ABA were used in combination . The effect of PCIB was dependent on the concentration and the application period . The auxin transport inhibitor TIBA also reduced proliferation, but had no positive effect on maturation . The auxin synergist phloroglucinol had the opposite effect of PCIB; proliferation was increased and no maturation was initiated . A lowered concentration of boron had no effect on proliferation but had some positive effect on maturation . The optimum protocol for PCIB application was strongly genotype dependent, and a general scheme that covered the tested cell lines could not be found . Overexposure to PCIB during maturation caused abnormal development of the mature embryos, which was revealed by a reduced number of cotyledons . These results suggest that endogenously produced auxin may be one reason for low or failing maturation of embryogenic cultures of Nordmanns fir, but also imply that auxin may play a critical role for proper development of cotyledons during the later stages of embryo maturation. J Obstet Gynaecol, 1986 Jan, 6 Suppl 1, S50 - 2 In vitro testing of Today vaginal contraceptive sponge with bacteria; Hammill HA et al.; PIP: In vitro methods were used to test Today vaginal contraceptive sponges for sterility, contamination by handling, and inhibition of bacterial growth . Also tested was an in vitro vaginal model surrounded by growth medium that continually seeded the dialysis tubing with nutrient in an attempt to replicate vaginal secretions . A goal of this research was to investigate manufacturer claims of hostility of the sponge in the presence of Staph aureus . Sponges added in a sterile manner to brain-heart infusion broth produced no growth under aerobic or anaerobic conditions when no organisms were added . However, the experiments that involved contamination of the sponges by hadling in a nonsterile fashion resulted in 10.8 colony forming units of Staph epidermidis and Staph aureus, coagulese negative . In the in vitro vaginal model, 16 hours after an inoculum of Staph aureus colony forming units was placed on a sponge, 3.5 x 10.10 colony forming units were cultured and there was a similar profusion of E coli sludge . These results fail to confirm claims of hostility of the vaginal sponge to the bacteria tested . There is concern that the technique recommended by the manufacturer involves adding water and then inserting the sponge with 1 hand and leaving it in place for 24 hours . This procedure may facilitate the enhancement of vaginitis and perhaps pelvic inflammatory disease . Contracept Technol Update, 1983 Dec, 4(12), 153 - 4 Investigators unable to substantiate suspected link between sponge, TSS; Growth-dependent stable carbon isotope fractionation by basidiomycete fungi: delta(13)C pattern and physiological process; Ecosystem Sciences Division, Department of Environmental Science, Policy, and Management, University of California, Berkeley, California 94720-3110, USA . mh@nature.berkeley.edu We grew 11 basidiomycetes in axenic culture to characterize their physiological capacities to fractionate stable C isotopes . Generally, delta(13)C values of the fungal biomass were (i) enriched in (13)C relative to the growth medium, (ii) variable among the isolates, and (iii) dependent on the growth rate and growth stage of the fungi . We found a multiphasic dynamic of fractionation for Cryptoporus volvatus and Marasmius androsaceus during various growth stages . The first phase, P1, corresponded to the exponential growth stage and was characterized by an increasing enrichment in (13)C content of the fungal biomass relative to the growth medium ranging between 4.6 and 6.9 per thousand . The second phase, P2, exhibited a continual depletion in (13)C of the fungal biomass, with the delta(13)C values of the fungal biomass asymptotically returning to the delta(13)C value of the growth medium at inoculation . The expression of the various fractionation phases was dependent on the amount of low-concentration micronutrients and growth factors added to the growth medium . The onset of P2 occurred at reduced concentrations of these elements . All of the sugars in the growth medium (sucrose, maltose, and glucose) were utilized for growth, indicating that the observed fractionation was not an artifact derived from the preferential use of (13)C-rich maltose, which was found at low concentrations in the growth medium . In this study, we establish a framework with which to explore the impact of physiological fractionations by fungal interfaces on natural distributions of stable C isotopes. Appl Environ Microbiol, 2002 Oct, 68(10), 4871 - 5 Isolation and identification of Aspergillus fumigatus mycotoxins on growth medium and some building materials; Nieminen SM et al.; Genotoxic and cytotoxic compounds were isolated and purified from the culture medium of an indoor air mold, Aspergillus fumigatus . One of these compounds was identified as gliotoxin, a known fungal secondary metabolite . Growth of A . fumigatus and gliotoxin production on some building materials were also studied . Strong growth of the mold and the presence of gliotoxin were detected on spruce wood, gypsum board, and chipboard under saturation conditions. Plant Cell, 1993 Jun, 5(6), 693 - 700 cAMP Regulates Infection Structure Formation in the Plant Pathogenic Fungus Magnaporthe grisea; Lee YH et al.; Magnaporthe grisea, the causal agent of rice blast, is one of the most destructive fungal pathogens of rice throughout the world . Infection of rice by M . grisea requires the formation of an appressorium, a darkly pigmented, dome-shaped structure . The germ tube tip differentiates into an appressorium following germination of conidia on a leaf surface . When conidia germinate on growth medium or other noninductive surfaces, the emerging germ tube does not differentiate and continues to grow vegetatively . Little is known about the endogenous or exogenous signals controlling the developmental process of infection structure formation . We show here that a hydrophobic surface was sufficient for the induction of the appressorium . Furthermore, we demonstrate that the addition of cAMP, its analogs (8-bromo cAMP and N6-monobutyryl cAMP), or 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase) to germinating conidia or to vegetative hyphae induced appressorium formation on noninductive surfaces . The identification of cAMP as a mediator of infection structure formation provides a clue to the regulation of this developmental process . Elucidation of the mechanism involved is not only of biological interest but may also provide the basis for new disease control strategies. Z Naturforsch {C}, 2002 Jul-Aug, 57(7-8), 634 - 9 Biosorption of cadmium ions by different yeast species; Breierova E et al.; Toxicity and accumulation of Cd2+ in yeasts were studied in eight different yeast species . The adaptation to toxic concentration of this metal was dependent on the production of extracellular yeast glycoproteins . The highest concentration of Cd2+ ions in the growth medium was tolerated by a Hansenula anomala, strain while the lowest tolerance was found by the strain of species Saccharomyces cerevisiae . Extracellular glycoproteins of Hansenula anomala absorbed nearly 90% of the total content of Cd2+ ions bound by yeast cells, while extracellular glycoproteins of Saccharomyces cerevisiae bound only 6% of the total amount of cadmium . This difference is caused by the variable composition of the saccharide moiety in the extracellular glycoproteins . The composition of extracellular glycoproteins changed during the adaptation of the yeast cells to the presence of Cd2+ ions. Plant Cell, 1997 Dec, 9(12), 2225 - 2241 Soluble Signals from Cells Identified at the Cell Wall Establish a Developmental Pathway in Carrot; McCabe PF et al.; Cells in a plant differentiate according to their positions and use cell-cell communication to assess these positions . Similarly, single cells in suspension cultures can develop into somatic embryos, and cell-cell communication is thought to control this process . The monoclonal antibody JIM8 labels an epitope on cells in specific positions in plants . JIM8 also labels certain cells in carrot embryogenic suspension cultures . We have used JIM8 and secondary antibodies coupled to paramagnetic beads to label and immunomagnetically sort single cells in a carrot embryogenic suspension culture into pure populations . Cells in the JIM8(+) population develop into somatic embryos, whereas cells in the JIM8(-) population do not form somatic embryos . However, certain cells in JIM8(+) cultures (state B cells) undergo asymmetric divisions, resulting in daughter cells (state C cells) that do not label with JIM8 and that sort to JIM8(-) cultures . State C cells are competent to form somatic embryos, and we show here that a conditioned growth medium from a culture of JIM8(+) cells allows state C cells in a JIM8(-) culture to go on and develop into somatic embryos . JIM8 labels cells in suspension cultures at the cell wall . Therefore, a cell with a role in cell-cell communication and early cell fate selection can be identified by an epitope in its cell wall. Bioconjug Chem, 2002 Sep-Oct, 13(5), 1044 - 53 Incorporation of reversibly cross-linked polyplexes into LPDII vectors for gene delivery; Gosselin MA et al.; LPDII vectors are synthetic vehicles for gene delivery composed of polycation-condensed DNA complexed with anionic liposomes . In this study, we evaluated the stability and transfection properties of polyethylenimine (PEI, 25 kDa)/DNA polyplexes before and after covalent cross-linking with dithiobis(succinimidylpropionate) (DSP) or dimethyl x 3,3'-dithiobispropionimidate x 2HCl (DTBP), either alone or as a component of LPDII vectors . We found that cross-linking PEI/DNA polyplexes at molar ratios > or =10:1 (DSP or DTBP:PEI) stabilized these complexes against polyanion disruption, and that this effect was reversible by reduction with 20 mM dithioerythritol (DTE) . Transfection studies with polyplexes cross-linked at molar ratios of 10:1-100:1 in KB cells, a folate receptor-positive oral carcinoma cell line, showed decreasing luciferase gene expression with increasing cross-linking ratio . Subsequently, polyplexes, cross-linked with DSP at a molar ratio of 10:1, were combined with anionic liposomes composed of diolein/cholesteryl hemisuccinate (CHEMS) (6:4 mol/mol), diolein/CHEMS/poly(ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE) (6:4:0.05 mol/mol), or diolein/CHEMS/folate-PEG-cholesterol (folate-PEG-Chol) (6:4:0.05 mol/mol) for LPDII formation . Transfection studies in KB cells showed that LPDII vectors containing cross-linked polyplexes mediated approximately 2-15-fold lower gene expression than LPDII prepared with un-cross-linked polyplexes, depending on the lipid:DNA ratio . Inclusion of PEG-DSPE at 0.5 mol % appeared to further decrease transfection levels approximately 2-5-fold . Compared with LPDII formulated with PEG-DSPE, LPDII incorporating 0.5 mol % folate-PEG-Chol exhibited higher luciferase activities at all lipid:DNA ratios tested, achieving an approximately 10-fold increase at a lipid:DNA ratio of 5 . Compared with cross-linked LPDII vectors without PEG-DSPE, inclusion of folate-PEG-Chol increased luciferase activities 3-4-fold between lipid:DNA ratios of 1 and 5 . Interestingly, inclusion of 1 mM free folate in the growth media during transfection increased transfection activity approximately 3-4-fold for cross-linked LPDII vectors and LPDII containing folate-PEG-Chol, but had no effect on the transfection activity of LPDII formulated with PEG-DSPE . However, in the presence of 5 mM free folate, the luciferase activity mediated by LPDII vectors containing folate-PEG-Chol was reduced approximately 6-fold . Transmission electron micrographs were also obtained to provide evidence of LPDII complex formation . Results showed that cross-linked LPDII vectors appear as roughly spherical aggregated complexes with a rather broad size distribution ranging between 300 and 800 nm. Mol Cell Biochem, 2002 Aug, 237(1-2), 47 - 53 In vitro lead-induced cell toxicity and cytoprotective activity of fetal calf serum in human fibroblasts; Dominguez C et al.; The underlying mechanisms by which lead ions produce their deleterious effects prior to the onset of clinical symptoms are incompletely understood . This study aimed to assess lead-induced cell toxicity mechanisms by focusing on the effects of the metal on cell growth, DNA synthesis, cellular ATP, intracellular hexosaminidase activity and lysosomal function, and examine the possible cytoprotective role of fetal calf serum (FCS) . Several human dermal cultured fibroblast lines were exposed to Pb (400 microM) for 1-6 days with 2, 5, and 10% FCS . The earliest toxic effect of Pb was significant inhibition of DNA synthesis after 24 h direct exposure; this harmful effect was not progressive during the first 3 days, but worsened clearly on the 4th day regardless of the FCS concentration . Atime-dependent depletion of intracellularATP content was also caused by ionic lead, thereby compromising the cell energy charge which precedes cell death . Fibroblast growth was progressively and significantly inhibited from day 2 onwards; the greatest noxious effect was observed in the presence of 2% FCS: 49% reduction in cell proliferation after 5 days . Lead salts produced loss of cell adhesion to the culture dish which worsened from the 2nd day and was more pronounced when FCS in growth medium was decreased . Toxic actions on lysosomal membrane integrity provoked a decrease in neutral red uptake (NRU) which was exposure time-dependent and more marked with 2% FCS . In contrast, increased relative NRU (to 20% at 4 days), suggestive of endocytosis-induced lysosome enlargement, was observed in Pb-exposed cells . Intracellular hexosaminidase activity was not negatively affected until 5 days after exposure to Pb salts . FCS had a significant cytoprotective effect on Pb-induced toxicity. Plant Physiol, 1994 Dec, 106(4), 1335 - 1346 The rhd6 Mutation of Arabidopsis thaliana Alters Root-Hair Initiation through an Auxin- and Ethylene-Associated Process; Masucci JD et al.; Root-hair initiation in Arabidopsis thaliana provides a model for studying cell polarity and its role in plant morphogenesis . Root hairs normally emerge at the apical end of root epidermal cells, implying that these cells are polarized . We have identified a mutant, rhd6, that displays three defects: (a) a reduction in the number of root hairs, (b) an overall basal shift in the site of root-hair emergence, and (c) a relatively high frequency of epidermal cells with multiple root hairs . These defects implicate the RHD6 gene in root-hair initiation and indicate that RHD6 is normally associated with the establishment of, or response to, root epidermal cell polarity . Similar alterations in the site of root-hair emergence, although less extreme, were also discovered in roots of the auxin-, ethylene-, abscisic acid-resistant mutant axr2 and the ethylene-resistant mutant etr1 . All three rhd6 mutant phenotypes were rescued when either auxin (indoleacetic acid) or an ethylene precursor (1-aminocyclopropane-1-carboxylic acid) was included in the growth medium . The rhd6 root phenotypes could be phenocopied by treating wild-type seedlings with an inhibitor of the ethylene pathway (aminoethoxyvinylglycine) . These results indicate that RHD6 is normally involved in directing the selection or assembly of the root-hair initiation site through a process involving auxin and ethylene. Plant Physiol, 1994 Sep, 106(1), 151 - 158 Differential Exudation of Polypeptides by Roots of Aluminum-Resistant and Aluminum-Sensitive Cultivars of Triticum aestivum L . in Response to Aluminum Stress; Basu U et al.; Cultivars of Triticum aestivum differing in resistance to Al were grown under aseptic conditions in the presence and absence of Al and polypeptides present in root exudates were collected, concentrated, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Upon exposure to 100 and 200 {mu}M Al, root elongation in Al-sensitive cultivars was reduced by 30 and 65%, respectively, whereas root elongation in resistant cultivars was reduced by only 15 and 30% . Accumulation of polypeptides in the growth medium increased with time for 96 to 120 h, with little additional accumulation thereafter . This pattern of exudation was virtually unaffected by exposure to 100 {mu}M Al in the Al-resistant cultivars Atlas 66 and Maringa, whereas total accumulation was reduced in sensitive cultivars . Changes in exudation were consistent with alterations in root elongation . Al-induced or Al-enhanced polypeptide bands were detected in Atlas 66 and Maringa after 72 h of exposure to Al . Increased accumulation of 12-, 22-, and 33-kD bands was observed at 75 {mu}M Al in Atlas 66 and 12-, 23-, and 43.5-kD bands started to appear at 50 {mu}M Al in Maringa . In the Al-sensitive cultivars Roblin and Katepwa, no significant effect on polypeptide profiles was observed at values up to 100 {mu}M Al . When root exudates were separated by ultrafiltration and the Al content was measured in both high molecular mass (HMM; >10 kD) and ultrafiltrate (<10 kD) fractions, approximately 2 times more Al was detected in HMM fractions from Al-resistant cultivars than from Al-sensitive cultivars . Dialysis of HMM fractions against water did not release this bound Al;digestion with protease released between 62 and 73% of total Al, with twice as much released from exudates of Al-resistant than of Al-sensitive cultivars . When plants were grown in the presence of 0 to 200 {mu}M Al, saturation of the Al-binding capacity of HMM exudates occurred at 50 {mu}M Al in Al-sensitive cultivars . Saturation was not achieved in resistant cultivars . Differences in exudation of total polypeptides in response to Al stress, enhanced accumulation of specific polypeptides, and the greater association of Al with HMM fractions from Al-resistant cultivars suggest that root exudate polypeptides may play a role in plant response to Al. Plant Physiol, 1994 Sep, 106(1), 103 - 108 Regulation of Periplasmic Carbonic Anhydrase Expression in Chlamydomonas reinhardtii by Acetate and pH; Fett JP et al.; The effects of mixotrophic growth with acetate and growth medium pH on expression of extracellular carbonic anhydrase (CA) in Chlamydomonas reinhardtii were evaluated . Addition of 10 mM acetate to the culture medium resulted in reduction of CA activity that was parallel to the reduction generated by growth of the algae in high external CO2 concentrations . This reduction in activity is a consequence of lower level of the CA protein as determined by western analysis . Transcript abundance of cah-1, the gene encoding the low CO2-induced CA, is also reduced by the addition of acetate as verified by northern analysis . Measurements of photosynthesis and respiration suggest that the acetate-induced reduction of CA expression is not a function of lowered photosynthetic capacity, but may be the result of increased internal CO2 concentration generated by high, acetate-stimulated respiratory rates . Growth medium pH can also influence extracellular CA expression . The induction of CA activity, protein abundance, and transcript levels by exposure to limiting inorganic carbon (Ci) concentrations is much more pronounced at higher than at lower pH values . The relationship between pH regulation of CA expression and its role in the Ci-concentrating mechanism are discussed. Plant Physiol, 1994 Feb, 104(2), 339 - 347 A Role for Cytokinins in De-Etiolation in Arabidopsis (det Mutants Have an Altered Response to Cytokinins); Chory J et al.; When grown in the absence of light, Arabidopsis thaliana deetiolated (det) mutants develop many of the characteristics of light-grown plants, including the development of leaves and chloroplasts, the inhibition of hypocotyl growth elongation, and elevated expression levels of light-regulated genes . We show here that dark-grown wild-type seedlings exhibit similar phenotypic traits if any one of a variety of cytokinins are present in the growth medium . We further show that the striking phenotype of det mutants is unlikely to be caused by different levels of cytokinins in these mutants . The three major Arabidopsis cytokinins, zeatin, zeatin riboside, and isopentenyladenosine, accumulate to similar levels in wild-type seedlings grown in either the light or the dark . There is no consistently different pattern for the levels of these cytokinins in wild-type versus det1 or det2 mutants . However, det1 and det2 have an altered response to cytokinin in a detached leaf senescence assay and in tissue culture experiments . A model is proposed in which light and cytokinins act independently or sequentially through common signal transduction intermediates such as DET1 and DET2 to control the downstream light-regulated responses. Plant Physiol, 1993 Dec, 103(4), 1107 - 1114 Kinematics and Dynamics of Sorghum (Sorghum bicolor L.) Leaf Development at Various Na/Ca Salinities (I . Elongation Growth); Bernstein N et al.; In many salt-sensitive species, elevated concentrations of Ca in the root growth media ameliorate part of the shoot growth reduction caused by NaCl stress . The physiological mechanisms by which Ca exerts protective effects on leaf growth are still not understood . Understanding growth inhibition caused by a stress necessitates locating the leaf expansion region and quantifying the profile of the growth reduction . This will enable comparisons and correlations with spatial gradients of probable physiologically inhibiting factors . In this work we applied the methods of growth kinematics to analyze the effects of elevated Ca concentrations on the spatial and temporal distributions of growth within the intercalary expanding region of salinized sorghum (Sorghum bicolor {L.} Moench, cv NK 265) leaves . NaCl (100 mM) caused a decrease in leaf elongation rate by shortening the leaf growing zone by 20%, as well as reducing the peak value of the longitudinal relative elemental growth rate (REG rate) . Increasing the Ca concentrations from 1 to 10 mM restored the length of the growing zone of both emerged and unemerged salinized leaves and increased the peak value of the REG rate . The beneficial effects of supplemental Ca were, however, more pronounced in leaves after their appearance above the whorl of encircling older leaf sheaths . Elevated Ca then resulted in a peak value of REG rate higher than in the salinized leaves . The peak value of unemerged leaves was not increased, although it was maintained over a longer distance . The duration of elongation growth associated with a cell during its displacement from the leaf base was longer in salinized than control leaves, despite the fact that the elongation zone was shorter in salinity . Although partially restoring the length of the elongation region, supplemental Ca had no effect on the age of cessation of growth . Elongation of a tissue element, therefore, ceased when a cellular element reached a certain age and not a specific distance from the leaf base. Plant Physiol, 1993 Oct, 103(2), 621 - 627 Oxidative Stimulation of Glutathione Synthesis in Arabidopsis thaliana Suspension Cultures; May MJ et al.; A system based on Arabidopsis thaliana suspension cultures was established for the analysis of glutathione (GSH) synthesis in the presence of hydrogen peroxide . Mild oxidative stress was induced by use of the catalase inhibitor, aminotriazole, and its development was monitored by measurement of the oxidative inactivation of aconitase . Addition of 2 mM aminotriazole resulted in a 25% decrease in activity of aconitase over 4 h . During the subsequent 10 h, no further decrease in aconitase activity was measured despite a sustained inhibition of catalase . In combination with our failure to detect significant increases in the level of lipid peroxidation, another marker indicative of oxidative injury, these data suggest that although hydrogen peroxide initially leaked into the cytosol, its accumulation was limited by a cytosolic catalase-independent mechanism . A 4-fold increase in the level of GSH, which was almost exclusively in the reduced form, was observed under the same treatment . To determine to what extent this increase in reduced GSH played a role in limiting the accumulation of hydrogen peroxide in the cytosol, we inhibited GSH synthesis with buthionine sulfoximine (BSO), a specific inhibitor of {gamma}-glutamylcysteine synthetase . No significant oxidative injury was detected as a result of treatment with 50 {mu}M BSO alone, and furthermore, this treatment had no effect on cell viability, However, addition of 2 mM aminotriazole to cells preincubated with 50 {mu}M BSO for 15 h led to a rapid loss of aconitase activity (75% in 4 h), and significant accumulation of products of lipid peroxidation . Within 72 h, cell viability was lost completely . After removal of BSO from the growth medium, GSH levels recovered to normal over a period of 20 h . Addition of 2 mM aminotriazole to cells at different time points during this recovery period demonstrated a strong correlation between the level of reduced GSH and the degree of protection against oxidative injury . These data strongly suggest that the induction of GSH synthesis by an oxidative stimulus plays a crucial role in determining the susceptibility of cells to oxidative stress. Plant Physiol, 1993 Sep, 103(1), 243 - 249 Two Isoforms of Dihydroxyacetone Phosphate Reductase from the Chloroplasts of Dunaliella tertiolecta; Gee R et al.; Three isoforms of dihydroxyacetone phosphate reductase in extracts from Dunaliella tertiolecta have been separated by a diethylaminoethyl cellulose column chromatography with a shallow NaCl gradient . The chloroplasts contained the two major isoforms, and the third, minor form was in the cytosol . The isoforms are unstable in the absence of glycerol and they are cold labile, but they may be partially reactivated at 35{deg}C . The first chloroplast form to elute from the DEAE cellulose column was the major form when the cells were grown on high NaCl and it has been referred to as the form for glycerol production for osmoregulation or "osmoregulator form." The second form increased in specific activity when inorganic phosphate was increased in the growth media to stimulate growth, and it has been given the designation for the form for glyceride synthesis, "glyceride form." The osmoregulator form was stimulated by NaCl added to the enzyme assay, but not by reduced Escherichia coli thioredoxin . The glyceride form had properties similar to the enzyme in leaf chloroplast, such as inhibition by NaCl and by fatty acyl-coenzyme A derivatives and some stimulation by dithiothreitol, uridine diphosphate galactose, cyti-dine diphosphate dipalmatoyl diglyceride, and reduced E . coli thioredoxin . Thus, Dunaliella chloroplasts have a salt-stimulated osmoregulatory form of dihydroxyacetone phosphate reductase, which seems to have a role in glycerol production, and an isoform, which may be involved in glyceride synthesis and which has properties similar to the enzyme in chloroplasts of higher plants. Plant Physiol, 1995 Feb, 107(2), 485 - 490 Perception of Fungal Sterols in Plants (Subnanomolar Concentrations of Ergosterol Elicit Extracellular Alkalinization in Tomato Cells); Granado J et al.; Suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) reacted to spores and spore exudates of the pathogen Cladosporium fulvum with a rapid, transient alkalinization of their growth medium that resembled the previously described alkalinization response elicited by chitin fragments (G . Felix, M . Regenass, T . Boller {1993} Plant J 4: 307-316) and was likewise inhibited by the protein kinase inhibitor K-252a . However, the spore factor recognized by the cells differed from chitin fragments in that it was butanol soluble and active in cells refractory to stimulation by chitin fragments . The spore factor was purified and identified as ergosterol, the main sterol of most higher fungi . With pure ergosterol, half-maximal induction was reached at about 10 pm . After treatment with ergosterol, tomato cells became refractory to a subsequent stimulation by C . fulvum and vice versa, indicating that ergosterol was the principal component of the spores recognized by the plant cells . Most other sterols were inactive, including cholesterol, a range of animal steroid hormones, and all natural plant sterols tested, except for stigmasterol, which was about 106 times less active than ergosterol . Our data demonstrate that tomato cells perceive ergosterol with a selectivity and sensitivity that resembles the perception of steroid hormones in animals. Plant Physiol, 2002 Sep, 130(1), 347 - 61 Probing in vivo metabolism by stable isotope labeling of storage lipids and proteins in developing Brassica napus embryos; Schwender J et al.; Developing embryos of Brassica napus accumulate both triacylglycerols and proteins as major storage reserves . To evaluate metabolic fluxes during embryo development, we have established conditions for stable isotope labeling of cultured embryos under steady-state conditions . Sucrose supplied via the endosperm is considered to be the main carbon and energy source for seed metabolism . However, in addition to 220 to 270 mM carbohydrates (sucrose, glucose, and fructose), analysis of endosperm liquid revealed up to 70 mM amino acids as well as 6 to 15 mM malic acid . Therefore, a labeling approach with multiple carbon sources is a precondition to quantitatively reflect fluxes of central carbon metabolism in developing embryos . Mid-cotyledon stage B . napus embryos were dissected from plants and cultured for 15 d on a complex liquid medium containing (13)C-labeled carbohydrates . The (13)C enrichment of fatty acids and amino acids (after hydrolysis of the seed proteins) was determined by gas chromatography/mass spectrometry . Analysis of (13)C isotope isomers of labeled fatty acids and plastid-derived amino acids indicated that direct glycolysis provides at least 90% of precursors of plastid acetyl-coenzyme A (CoA) . Unlabeled amino acids, when added to the growth medium, did not reduce incorporation of (13)C label into plastid-formed fatty acids, but substantially diluted (13)C label in seed protein . Approximately 30% of carbon in seed protein was derived from exogenous amino acids and as a consequence, the use of amino acids as a carbon source may have significant influence on the total carbon and energy balance in seed metabolism . (13)C label in the terminal acetate units of C(20) and C(22) fatty acids that derive from cytosolic acetyl-CoA was also significantly diluted by unlabeled amino acids . We conclude that cytosolic acetyl-CoA has a more complex biogenetic origin than plastidic acetyl-CoA . Malic acid in the growth medium did not dilute (13)C label incorporation into fatty acids or proteins and can be ruled out as a source of carbon for the major storage components of B . napus embryos. Fungal Genet Biol, 2002 Oct, 37(1), 29 - 38 Visualization of vacuoles in Aspergillus oryzae by expression of CPY-EGFP; Ohneda M et al.; Vacuolar carboxypeptidase Y (CPY) from Aspergillus nidulans was used to construct a CPY-EGFP fusion protein and expressed in A . oryzae to study vacuolar morphology and functions in A . oryzae . While the fluorescence of EGFP was barely detectable in A . oryzae expressing CPY-EGFP grown under normal conditions at pH 5-6, the increase in pH of the growth medium towards alkalinity restored the fluorescence . In accordance with such an observation, the fluorescence of CPY-EGFP fusion protein in cell extract decreased in acidic pH condition, concomitant with lowered content of EGFP detected in A . oryzae grown under acidic pH conditions . The pH sensitivity of EGFP fluorescence and enhanced degradation of proteins in vacuoles under acidic pH conditions are thus proposed to result in the reduction of fluorescence in A . oryzae . Further, visualization of vacuoles revealed the presence of peculiar ring- or tube-like structures as distinct from normal spherical-shaped vacuoles. Nitric Oxide, 2002 Sep, 7(2), 127 - 31 Application of the nitric oxide donor SNAP to cardiomyocytes in culture provides protection against oxidative stress; Monastyrskaya E et al.; Multiple data indicates that nitric oxide (NO) donors retain immediate protective effects against different disturbances in cardiovascular system . The aim of the present study was to investigate delayed effects of nitric oxide donor S-nitroso-N-acetyl-l,l-penicillamine (SNAP) application in cardiac H9c2 cell line . Cardiomyocytes were treated with SNAP for 2h followed by 24h wash with fresh growth medium . The concentration curve was constructed in range from 0.5 to 2mM, toxicity was observed at 2mM concentration of SNAP . For the study of SNAP-induced protection against t-butyl hydroperoxide-induced oxidative injury 1mM concentration was used . Cell viability was assessed by MTT reductase activity assay; mitochondrial transmembrane potential (mdeltapsi) was measured by flow cytometry with fluorescent dye DiOC(6) . Synthesis of heat-shock proteins (hsps) was analyzed by Western blot . Analysis of the cell viability and mdeltapsi reflected delayed protective effect of 1mM SNAP application against oxidative injury . SNAP in 1mM concentration caused 70% induction of hsp75 synthesis in cardiomyocytes . However, the other analyzed hsps (hsp70, hsp27, hsp60, hsp10, and CyP A) did not display any significant induction after incubation with SNAP . Present work demonstrates that the NO donor SNAP causes delayed protection against oxidative stress in H9c2 cardiomyocyte cell line, reflected in cell viability increase and preservation of the mdeltapsi . We suppose the major pathway for the development of SNAP-induced protection is through mitochondria . Induction of hsp75 expression following SNAP pretreatment is one possible way to explanation the mechanisms of this protection. Brain Res Dev Brain Res, 2002 Aug 30, 137(2), 115 - 25 Enhanced viability and neuronal differentiation of neural progenitors by chromaffin cell co-culture; Schumm MA et al.; The transplantation of neural stem cells and progenitors has potential in restoring lost cellular populations following central nervous system (CNS) injury or disease, but survival and neuronal differentiation in the adult CNS may be insufficient in the absence of exogenous trophic support . Adrenal medullary chromaffin cells produce a trophic cocktail including basic fibroblast growth factor (FGF-2) and neurotrophins . The aim of this study was to evaluate whether chromaffin cells can provide a supportive microenvironment for neural progenitor cells . In order to assess this, the growth and differentiation of neural progenitor cell cultures from embryonic rat cortex were compared in standard FGF-2-supplemented neural progenitor growth media, in standard media but lacking FGF-2, or in media lacking FGF-2 but co-cultured with bovine chromaffin cells . Using bromodeoxyuridine (BrdU)-prelabeling, findings indicated poor survival of progenitor cultures in the absence of FGF-2 . In contrast, the addition of chromaffin cells in co-culture appeared to 'rescue' the progenitor cultures and resulted in robust neurospheres containing numerous BrdU-labeled cells interspersed with and closely apposed to chromaffin cells . As indicated by H3 labeling, cells in co-cultures continued to proliferate, but at a substantially reduced rate compared with standard FGF-2 supplemented growth media . The co-cultures contained more beta-tubulin III-positive processes than parallel cultures maintained in FGF-2-supplemented media and these cells displayed a more mature phenotype with numerous varicosities and complex processes . These findings indicate that chromaffin cells can provide a supportive environment for the survival and neuronal differentiation of neural progenitor cells and suggest that their addition may be useful as a sustained source of trophic support to improve outcomes of neural stem cell transplantation. Genome Res, 2002 Sep, 12(9), 1434 - 44 Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones; Wild J et al.; The widely used, very-low-copy BAC (bacterial artificial chromosome) vectors are the mainstay of present genomic research . The principal advantage of BACs is the high stability of inserted clones, but an important disadvantage is the low yield of DNA, both for vectors alone and when carrying genomic inserts . We describe here a novel class of single-copy/high-copy (SC/HC) pBAC/oriV vectors that retain all the advantages of low-copy BAC vectors, but are endowed with a conditional and tightly controlled oriV/TrfA amplification system that allows: (1) a yield of ~100 copies of the vector per host cell when conditionally induced with L-arabinose, and (2) analogous DNA amplification (only upon induction and with copy number depending on the insert size) of pBAC/oriV clones carrying >100-kb inserts . Amplifiable clones and libraries facilitate high-throughput DNA sequencing and other applications requiring HC plasmid DNA . To turn on DNA amplification, which is driven by the oriV origin of replication, we used copy-up mutations in the gene trfA whose expression was very tightly controlled by the araC-P(araBAD) promoter/regulator system . This system is inducible by L-arabinose, and could be further regulated by glucose and fucose . Amplification of DNA upon induction with L-arabinose and its modulation by glucose are robust and reliable . Furthermore, we discovered that addition of 0.2% D-glucose to the growth medium helped toward the objective of obtaining a real SC state for all BAC systems, thus enhancing the stability of their maintenance, which became equivalent to cloning into the host chromosome J Environ Sci (China), 2002 Jul, 14(3), 399 - 405 Differences of cadmium absorption and accumulation in selected vegetable crops; Ni WZ et al.; A pot experiment and a sandy culture experiment grown with three vegetable crops of Chinese cabbage (B . chinensis L., cv . Zao-Shu 5), winter greens (B . var . rosularis Tsen et Lee, cv . Shang-Hai-Qing) and celery (A . graveolens L . var . dulce DC., cv . Qing-Qin) were conducted, respectively . The initial soil and four incubated soils with different extractable Cd (0.15, 0.89, 1.38, 1.84 and 2.30 mg Cd/kg soil) were used for the pot experiment . Five treatments were designed (0, 0.0625, 0.125, 0.250 and 0.500 mg Cd/L) in nutrient solution in the sandy culture experiment . Each treatment in pot and sandy culture experiments was trireplicated . The objectives of the study were to examine Cd accumulation in edible parts of selected vegetable crops, its correlation with Cd concentrations in vegetable garden soil or in nutrient solution, and evaluate the criteria of Cd pollution in vegetable garden soil and in nutrient solution based on the hygienic limit of Cd in vegetables . Cadmium concentrations in edible parts of the three selected vegetable crops were as follows: 0.01-0.15 mg/kg fresh weight for Chinese cabbage, 0.02-0.17 mg/kg fresh weight for winter greens, and 0.02-0.24 mg/kg fresh weight for celery in the pot experiment, and 0.1-0.4 mg/kg fresh weight for Chinese cabbage, 0.1-1.4 mg/kg fresh weight for winter greens, and 0.05-0.5 mg/kg fresh weight for celery in the pot experiment (except no-Cd treatment) . The order of the three test vegetable crops for cadmium accumulation in the edible parts was celery > winter greens > Chinese cabbage in both the pot experiment and the sandy culture experiment . Cadmium accumulation in edible parts or roots of the vegetable crops increased with increasing of cadmium concentration in the medium (soil or nutrient solution) . And cadmium concentrations in edible parts of the test vegetable crops were significantly linearly related to the Cd levels in the growth media (soil and nutrient solution) . Based on the regression equations established and the limit of cadmium concentration in vegetable products, the thresholds of Cd concentration in the growth medium evaluated was as follows: 0.5 mg/kg soil of extractable Cd for soil and 0.02 mg/L for nutrient solution . The high capacity for cadmium accumulation in the edible parts of different vegetable crops together with the absence of visual symptoms implies a potential danger for humans. Yeast, 2002 Sep 15, 19(12), 1029 - 38 HXT5 expression is determined by growth rates in Saccharomyces cerevisiae; Verwaal R et al.; In the yeast Saccharomyces cerevisiae, hexose transporter (Hxt) proteins transport glucose across the plasma membrane . The Hxt proteins are encoded by a multigene family with 20 members, of which Hxt1-4p and Hxt6-7p are the major hexose transporters . The remaining Hxt proteins have other or unknown functions . In this study, expression of HXT5 under different experimental set-ups is determined . In glucose-grown batch cultures, HXT5 is expressed prior to glucose depletion . Independent of the carbon source used in batch cultures, HXT5 is expressed after 24 h of growth and during growth on ethanol or glycerol, which indicates that growth on glucose is not necessary for expression of HXT5 . Increasing the temperature or osmolarity of the growth medium also induces expression of HXT5 . In fed-batch cultures, expression of HXT5 is only observed at low glucose consumption rates, independent of the extracellular glucose concentration . The only common parameter in these experiments is that an increase of HXT5 expression is accompanied by a decrease of the growth rate of cells . To determine whether HXT5 expression is determined by the growth rate, cells were grown in a nitrogen-limited continuous culture, which enables modulation of only the growth rate of cells . Indeed, HXT5 is expressed only at low dilution rates . Therefore, our results indicate that expression of HXT5 is regulated by growth rates of cells, rather than by extracellular glucose concentrations, as is the case for the major HXTs . A possible function for Hxt5p and factors responsible for increased expression of HXT5 upon low growth rates is discussed . Circulation, 2002 Sep 3, 106(10), 1199 - 204 Smooth muscle progenitor cells in human blood; Simper D et al.; BACKGROUND: Recent animal data suggest that vascular smooth muscle cells within the neointima of the vessel wall may originate from bone marrow, providing indirect evidence for circulating smooth muscle progenitor cells (SPCs) . Evidence for circulating SPCs in human subjects does not exist, and the mechanism whereby such putative SPCs may home to sites of plaque formation is presently not understood but is likely to involve expression of specific surface adhesion molecules, such as integrins . In this study, we aimed to culture smooth muscle outgrowth cells (SOCs) from SPCs in human peripheral blood and characterize surface integrin expression on these cells . METHODS AND RESULTS: Human mononuclear cells isolated from buffy coat were seeded on collagen type 1 matrix and outgrowth cells selected in endothelial growth medium (EGM-2) or EGM-2 and platelet-derived growth factor BB . Selection in platelet-derived growth factor BB-enriched medium caused rapid outgrowth and expansion of SOC to >40 population doublings in a 4-month period . These SOCs were positive for smooth muscle cell-specific alpha actin (alphaSMA), myosin heavy chain, and calponin on immunofluorescence and Western blotting and were also positive for CD34, Flt1, and Flk1 receptor but negative for Tie-2 receptor expression, suggesting a potential bone marrow angioblastic origin . In contrast, endothelial outgrowth cells (EOCs) grown in EGM-2 alone and the initial MNC population were negative for these smooth muscle-specific markers . Integrin alpha5beta1 expression by FACS and Western blotting was significantly increased in SOCs compared with EOCs, and this was confirmed by 8-fold greater adhesion of SOC to fibronectin (P<0.001), an effect that could be decreased using an alpha5beta1 antibody . Finally, SOC showed a significantly greater in vitro proliferative potential compared with EOCs of similar passage (P<0.001) . CONCLUSIONS: This study demonstrates for the first time outgrowth of smooth muscle cells with a specific growth, adhesion, and integrin profile from putative SPC in human blood . These data have implications for our understanding of adult vascular smooth muscle cell differentiation, proliferation, and homing. Free Radic Biol Med, 2002 Sep 1, 33(5), 691 - 702 DNA damage and apoptosis in hydrogen peroxide-exposed Jurkat cells: bolus addition versus continuous generation of H(2)O(2); Barbouti A et al.; Aspects of the molecular mechanism(s) of hydrogen peroxide-induced DNA damage and cell death were studied in the present investigation . Jurkat T-cells in culture were exposed either to low rates of continuously generated H(2)O(2) by the action of glucose oxidase or to a bolus addition of the same agent . In the first case, steady state conditions were prevailing, while in the latter, H(2)O(2) was removed by the cellular defense systems following first order kinetics . By using single-cell gel electrophoresis (also called comet assay), an initial increase in the formation of DNA single-strand breaks was observed in cells exposed to a bolus of 150 microM H(2)O(2) . As the H(2)O(2) was exhausted, a gradual decrease in DNA damage was apparent, indicating the existence of an effective repair of single-strand breaks . Addition of 10 ng glucose oxidase in 100 microl growth medium (containing 1.5 x 10(5) cells) generated 2.0 +/- 0.2 microM H(2)O(2) per min . This treatment induced an increase in the level of single-strand breaks reaching the upper limit of detection by the methodology used and continued to be high for the following 6 h . However, when a variety of markers for apoptotic cell death (DNA cell content, DNA laddering, activation of caspases, PARP cleavage) were examined, only bolus additions of H(2)O(2) were able to induce apoptosis, while the continuous presence of this agent inhibited the execution of the apoptotic process no matter whether the inducer was H(2)O(2) itself or an anti-Fas antibody . These observations stress that, apart from the apparent genotoxic and proapoptotic effects of H(2)O(2), it can also exert antiapoptotic actions when present, even at low concentrations, during the execution of apoptosis. Biochim Biophys Acta, 2002 Aug 15, 1572(1), 143 - 8 A rapid method for measuring intracellular pH using BCECF-AM; Ozkan P et al.; A rapid intracellular pH (pH(i)) measurement method based on initial rate of increase of fluorescence ratio of 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein upon dye addition to a cell suspension in growth medium is reported . A dye transport model that describes dye concentration and fluorescence values in intracellular and extracellular spaces provides the mathematical basis for the approach . Experimental results of ammonium chloride challenge response of the two suspension cells, Spodoptera frugiperda and Chinese hamster ovary (CHO) cells, successfully compared with results obtained using traditional perfusion method . Since the cell suspension does not require any preparation, measurement of pH(i) can be completed in about 1 min minimizing any potential errors due to dye leakage. Chembiochem, 2002 Aug 2, 3(8), 717 - 25 Prion-protein-specific aptamer reduces PrPSc formation; Proske D et al.; The critical initial event in the pathophysiology of transmissible spongiform encephalopathies (TSEs) appears to be the conversion of the cellular prion protein (PrP(C)) into the abnormal isoform PrP(Sc) . This isoform forms high-molecular-weight protease K (PK) resistant aggregates that accumulate in the central nervous system of affected individuals . We have selected nuclease-resistant 2'-amino-2'-deoxypyrimidine-modified RNA aptamers which recognize a peptide comprising amino acid residues 90-129 of the human prion protein with high specificity . This domain of prion proteins is thought to be functionally important for the conversion of PrP(C) into its pathogenic isoform PrP(Sc) and is highly homologous among prion proteins of various species including mouse, hamster, and man . Consequently, aptamer DP7 binds to the full-length human, mouse, and hamster prion protein . At low concentrations in the growth medium of persistently prion-infected neuroblastoma cells, aptamer DP7 significantly reduced the relative proportion of de novo synthesized PK-resistant PrP(Sc) within only 16 h . These findings may open the door towards a rational development of a new class of drugs for the therapy or prophylaxis of prion diseases. J Biol Inorg Chem, 2002 Sep, 7(7-8), 891 - 6 Epub 2002 Jun 15. The iron-binding properties of aminochelin, the mono(catecholamide) siderophore of Azotobacter vinelandii; Khodr HH et al.; Azotobacter vinelandii produces siderophores with different metal-binding properties, depending on the concentration of Fe(III) and molybdate in the growth medium . The three protonation constants of the mono(catecholamide) siderophore aminochelin were determined by simultaneous spectrophotometric and potentiometric titrations as log K(1)=12.1, log K(2)=10.22 and log K(3)=7.04 . Based on the two catechol protonation constants, log K(1) and log K(3), the overall stability constant of the aminochelin iron 3:1 complex was found to be log beta(3)=41.3, resulting in a pFe(3+) value of 17.6 at pH 7.45 . In order to further investigate the properties of the siderophore, the solubilization of Fe(III) hydroxide by a 8x10(-4) M solution of aminochelin at pH 7 and 25 degrees C was followed spectrophotometrically in the absence and in the presence of molybdate . It was observed that the addition of molybdate resulted in a significant delay in the solubilization. Tissue Eng, 2002 Aug, 8(4), 541 - 50 Derivation of type II alveolar epithelial cells from murine embryonic stem cells; Ali NN et al.; Embryonic stem (ES) cell pluripotency is being investigated increasingly to obtain specific cell lineages for tissue engineering . However, the possibility that ES cells can give rise to lung tissue has not been tested . We hypothesized that lung epithelial cells (type II pneumocytes) can be derived in vitro from murine ES cells . After withdrawal of leukemia inhibitory factor (LIF) and formation of embryoid bodies in maintenance medium for 10, 20, and 30 days, differentiating ES cells were kept in the same medium or transferred to serum-free small airway growth medium (SAGM) for a further 3 or 14 days of culture . The presence of type II pneumocytes in the resulting mixed cultures was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) of surfactant protein C (SPC) mRNA, immunostaining of SPC, and electron microscopy of osmiophilic lamellar bodies only at 30 days sampling time . SAGM appeared to be more favorable for type II cell formation than ES medium . No SPC transcripts were found in differentiating cells grown under the same conditions without formation of embryoid bodies . These findings could form the basis for the enrichment of ES cell-derived cultures with type II pneumocytes, and provide an in vitro system for investigating mechanisms of lung repair and regeneration. Curr Microbiol, 2002 Oct, 45(4), 277 - 80 Growth under alkaline conditions of the salt-tolerant yeast Debaryomyces hansenii IFO10939; Kurita O et al.; A salt-tolerant yeast Debaryomyces hansenii IFO 10939, which is able to grow at pH 10.0, was isolated and characterized . IFO 10939 had the ability of maintaining intracellular pH . The in vivo activation of plasma membrane ATPase was observed in cells grown at pH 6.2 during conditioning in buffer at pH 9.0 . Alkalification of growth medium exhibited a significant increase in acetate and propionate production . The results suggested that the regulation of intracellular pH was involved in plasma membrane ATPase pumping protons out of the cells and weak acid formation for the source of the protons in cells growing at high pH. J Immunol Methods, 2002 Jun 1, 264(1-2), 59 - 68 Dialysis-based bioreactor systems for the production of monoclonal antibodies--alternatives to ascites production in mice; Bruce MP et al.; Two commercially available bioreactor systems, CELLine and miniPERM, were evaluated for their ability to support the production of monoclonal antibody (mAb) from a variety of murine hybridoma cell lines . Production and purity of mAbs were compared between the two systems and with mouse ascites tumour fluid generation . The quality and purity of the mAb generated by each method was analysed on SDS-PAGE gels and the antibody immunoreactivity in each case was quantified by indirect ELISA tests . The relative benefits of conventional growth medium (Dulbecco's modified Eagle's media, DMEM) and serum-free medium (hybridoma serum-free media, H-SFM) using the miniPERM system were also analysed, in terms of the amount of antibody produced, cell concentration and specific antibody titre . In all cases, the CELLine units tested gave higher protein concentrations compared to the miniPERM system under the same conditions (means and 95% confidence limits are 4.2+/-0.8 and 2.1+/-0.8 mg/ml, respectively), yet the miniPERM system yielded greater total amounts over a similar culture period (428.7+/-243.3 mg compared to 183.3+/-100.9 mg in the CL-350 CELLine unit) . When defined by specific ELISA titre, both bioreactor systems yielded mAb levels that compared favourably with those derived from ascites . In addition, SDS-PAGE analysis indicated that the bioreactor antibody product was relatively free of contaminating protein, whereas ascites tumour fluid preparations displayed significant levels of extraneous protein . This study has shown that both bioreactor systems are acceptable in vitro alternatives to the in vivo production of mAbs in mice. Yeast, 2002 Jun 15, 19(8), 659 - 70 Mutations in the Lcb2p subunit of serine palmitoyltransferase eliminate the requirement for the TSC3 gene in Saccharomyces cerevisiae; Monaghan E et al.; Serine palmitoyltransferase catalyses the committed step in sphingolipid synthesis, the condensation of serine with palmitoyl-CoA to form 3-ketosphinganine . Two proteins, Lcb1p and Lcb2p, are essential for enzyme activity and a third protein, the 80-amino acid Tsc3p, stimulates the activity of serine palmitoyltransferase several-fold . Tsc3p physically associates with a complex of Lcb1p-Lcb2p and stimulates enzyme activity posttranslationally, but its precise function is not known . Tsc3p is essential for cell viability only at elevated temperatures, although serine palmitoyltransferase activity is reduced in the tsc3 delta mutant, even at permissive growth temperatures . Tsc3p is apparently not required for any essential process besides stimulation of serine palmitoyltransferase at 37 degrees C, since providing sphingoid bases to the growth medium reverses the temperature-sensitive growth phenotype of the tsc3 delta mutant . To gain further insight into the function of Tsc3p, suppressor mutants that eliminate the Tsc3p requirement for growth at 37 degrees C were isolated and characterized . These studies show that dominant mutations in the Lcb2p subunit of serine palmitoyltransferase suppress the temperature-sensitive growth phenotype of the tsc3 delta null mutant by increasing the Tsc3p-independent serine palmitoyltransferase activity. Protein Expr Purif, 2002 Aug, 25(3), 472 - 80 Expression and one-step purification of a developmentally regulated protein from Dictyostelium discoideum; Ubeidat M et al.; To overexpress Dictyostelium 5NT, a 1506bp fragment of the cDNA encoding the gene was cloned into a pET32c+ vector and expressed in the Escherichia coli expression host BL21-CodonPlus(DE3)-RIL by Isopropyl-beta-D-thiogalactoside (IPTG) induction . Maximum induction of insoluble recombinant protein was reached after incubation of the culture for 3h with 1.0mM IPTG . High level of 5NT expression was confirmed by SDS-PAGE and immunoblotting analysis . The recombinant 5NT was purified to homogeneity by a one-step purification using continuous-elution electrophoresis . Ten mg recombinant 5NT was purified per liter of growth medium . To achieve one of the goals of this study, polyclonal antibody against the recombinant 5NT was produced in a rabbit . We have shown previously by Northern blot and reporter gene analyses that 5nt is developmentally regulated . In this report, we used polyclonal antibody against the recombinant protein in Western blot analysis of membrane protein extracts from different developmental stages of Dictyostelium . The 5NT protein levels were first detected at the tight aggregation stage of development . Thus, there is no significant delay between transcription and translation of 5nt. Pharm Res, 2002 Jul, 19(7), 933 - 8 Cationic cholesterol promotes gene transfection using the nuclear localization signal in protamine; Noguchi A et al.; PURPOSE: The purpose of this study was to evaluate protamine-mediated gene transfection by liposomes with a novel cationic cholesterol derivative (I) compared to those with DC-Chol or DOTMA (Lipofectin) . METHOD: Plasmid pGL3 DNA was complexed to the cationic liposomes with the derivative (I), DC-Chol, or DOTMA in SFM101(Nissui) at room temperature for 15 min, and thereafter the complex was incubated with target cells (NIH3T3) for 4 h at 37 degrees C . The cells then were washed and cultured for another 40 h in the growth medium at 37 degrees C before luciferase assay . RESULTS: The transfection efficiency by the liposomes with the derivative (I) was much higher than that by the liposomes with DC-Chol or DOTMA . In addition, its transfection efficiency was enhanced greatly by the addition of protamine . Atomic force microscopy showed clearly how the size of the DNA-liposome complex was changed by protamine . Furthermore, fluorescence microscopic images showed that Cy5-labeled antisense DNAs were transferred quicker into the nucleus of the target cells by the liposomes with the derivative I in the presence of protamine . CONCLUSION: Although there exist several possible mechanisms, such as improved protection of DNA intracellularly by derivative (I), one possibility is that the DNA-protamine-liposome complex with the derivative (I) promoted gene transfection more significantly into the nucleus of the target cells using the nuclear localization signal of protamine. J Biol Chem, 2002 Oct 18, 277(42), 39649 - 54 Epub 2002 Aug 09. The yeast iron regulon is induced upon cobalt stress and crucial for cobalt tolerance; Stadler JA et al.; To identify yeast genes involved in cobalt detoxification, we performed RNA expression profiling experiments and followed changes in gene activity upon cobalt stress on a genome-wide scale . We found that cobalt stress specifically results in an immediate and dramatic induction of genes involved in iron uptake . This response is dependent on the Aft1 protein, a transcriptional factor known to regulate a set of genes involved in iron uptake and homeostasis (iron regulon) . Like iron starvation, cobalt stress induces accumulation of the Aft1 protein in the nucleus to activate transcription of its target genes . Cells lacking the AFT1 gene (aft1) are hypersensitive to cobalt as well as to other transition metals, whereas expression of the dominant AFT1-1(up) allele, which results in up-regulation of AFT1-controlled genes, confers resistance . Cobalt resistance correlates with an increase in intracellular iron in AFT1-1(up) cells, and sensitivity of aft1 cells is associated with a lack of iron accumulation . Furthermore, elevated iron levels in the growth medium suppress the cobalt sensitivity of the aft1 mutant cells, even though they increase cellular cobalt . Results presented indicate that yeast cells acquire cobalt tolerance by activating the Aft1p-dependent iron regulon and thereby increasing intracellular iron levels. Biochem Biophys Res Commun, 2002 Aug 23, 296(3), 584 - 8 Inhibition of beta-catenin/Tcf activity by white tea, green tea, and epigallocatechin-3-gallate (EGCG): minor contribution of H(2)O(2) at physiologically relevant EGCG concentrations; Dashwood WM et al.; Epigallocatechin-3-gallate (EGCG) is the major polyphenol present in white tea and green tea . Recently, it was reported that the addition of EGCG and other tea polyphenols to cell culture media, minus cells, generated significant levels of H(2)O(2), with the corollary that this might represent an "artifact" in cell culture studies which seek to examine the chemopreventive mechanisms of tea . We show here that in cell growth media with and without serum, and in growth media containing human embryonic kidney 293 (HEK293) cells plus serum, physiologically relevant concentrations of EGCG (< or =25 microM) generated H(2)O(2) with a peak concentration of the order of 10-12 microM . However, addition of 20 microM H(2)O(2) directly to HEK293 cells transiently transfected with wild-type or mutant beta-catenin constructs and TCF-4 had no significant effect on beta-catenin/TCF-4 reporter activity or beta-catenin expression levels . In contrast, 2-25 microM EGCG inhibited beta-catenin/TCF-4 reporter activity in a concentration-dependent fashion and there was a concomitant reduction in beta-catenin protein levels in the cell lysates without changes in TCF-4 expression . The inhibition of reporter activity was recapitulated by white tea and green tea, each tested at a 25 microM EGCG equivalent concentration in the assay, and this was unaffected by the addition of exogenous catalase . The results indicate that physiologically relevant concentrations of tea and EGCG inhibit beta-catenin/TCF-4 reporter activity in HEK293 cells due to reduced expression of beta-catenin and that this is unlikely to be an artifact of H(2)O(2) generation under the assay conditions used here . These data are consistent with the findings from in vivo studies, showing the suppression of intestinal polyps by tea, via an apparent down-regulation of beta-catenin and Wnt target genes. J Biomed Opt, 2002 Jul, 7(3), 398 - 403 Contrast agents for confocal microscopy: how simple chemicals affect confocal images of normal and cancer cells in suspension; Zuluaga AF et al.; Normal and malignant human cervical cancer cells were imaged in vivo with confocal, phase contrast, and brightfield microscopies . Results were compared between cells in growth medium before and after addition of acetic acid, hypertonic saline solution, toluidine blue, and Lugol's iodine . The exogenous agents changed the backscattering characteristics of the cells when measured with confocal microscopy at 808 nm . A tendency toward higher scattering was observed in treated cells . Acetic acid and toluidine blue increased the brightness of the nucleus with respect to the cytoplasm in normal and cancer cells . Hypertonic saline solution made the cytoplasm brighter than the nucleus in both types of cells . The results indicate that simple chemicals can be used to enhance confocal microscopy's ability to differentiate intracellular components, such as nuclear size and shape . This can further confocal microscopy's ability to assess disease in cells and tissues. J Appl Microbiol, 2002, 93(3), 492 - 6 Nitrite inhibits hydrogen production and kills the cattle parasite Tritrichomonas foetus; Lloyd D et al.; AIMS: To investigate the effects of NaNO2 on the microaerophilic flagellated protozoan, Tritrichomonas foetus KV1, an economically important cattle parasite that inhabits the vagina and can spread rapidly through herds of animals by sexual transmission and leads to abortion of foetal calves . METHODS AND RESULTS: Growth of the parasite was inhibited by 50% in the presence of 4 mm NaNO2; immediate killing occurred at 10 mm . Mass spectrometric monitoring of gases showed that H2 and CO2 evolution were inhibited by NaNO2, and electron paramagnetic resonance spectrometry revealed a signal similar to that of a thiolate-iron-NO complex . Growth with sublethal concentrations of NaNO2 yielded organisms that produced ethanol rather than H2 . CONCLUSIONS: NaNO2 probably inactivates FeS protein(s) of hydrogenosomes so as to inhibit the conversion of pyruvate (derived from maltose in the growth medium) to H2 and acetate . SIGNIFICANCE AND IMPACT OF THE STUDY: The use of NaNO2 as a topical antitrichomonal agent in veterinary practice is a possibility . At present, slaughter of infected animals is the favoured method of control. Curr Genet, 2002 Jul, 41(4), 224 - 31 Epub 2002 Jun 15. Role of RNA surveillance proteins Upf1/CpaR, Upf2 and Upf3 in the translational regulation of yeast CPA1 gene; Messenguy F et al.; Gene CPA1, encoding one of the subunits of carbamoylphosphate synthetase (CPSase A) is subject to a translational control by arginine of which the essential element is a 25 amino acid peptide encoded by the CPA1 messenger . The peptide is the product of an open reading frame located upstream (uORF) of the coding phase of the gene, within a 250 nucleotide leader . In the past, a series of mutations impairing the repression of gene CPA1 by arginine had been selected in vivo . Most of the mutations were located in the CPA1 uORF, but mutations unlinked to the CPA1 gene were also isolated and mapped in a gene called CPAR . In this work, we show that the CPAR gene is identical to the UPF1 gene, encoding a protein responsible for the premature termination step of RNA surveillance by nonsense-mediated mRNA decay (NMD) . Deletion of UPF1, or deletion of UPF2 and UPF3, the other genes involved in the NMD pathway, enhances the synthesis of CPSase A, whether arginine is present or not in the growth medium . The regulatory effect of the NMD protein complex is only observed when the uORF is present in the CPA1 messenger, indicating that the arginine-peptide repression mechanism and the RNA surveillance pathway are complementary mechanisms . Our results indicate that the NMD destabilizes the 5' end of the CPA1 message and this decay is strongly enhanced when arginine is present in the growth medium. Parasitol Res, 2002 Sep, 88(9), 837 - 43 Epub 2002 Jun 04. Possible role of calcium ions, calcium channels and calmodulin in excystation and metacystic development of Entamoeba invadens; Makioka A et al.; The effect of calcium ions (Ca(2+)) and calmodulin (CaM) on the excystation and metacystic development of Entamoeba invadens was examined by transfer of cysts to a growth medium containing calcium antagonists and CaM inhibitors . Excystation, which was assessed by counting the number of metacystic amoebae after induction of excystation, was inhibited by the calcium chelators ethyleneglycol bis (beta-aminoethyl ether)- N,N'-tetraacetate (EGTA) and ethylene-diaminetetraacetate (EDTA), with EDTA being more potent than EGTA . The inhibitory effect of higher concentrations of these chelators on excystation was associated with reduced viability of cysts . Metacystic development, when determined by the number of nuclei in an amoeba, was delayed by EGTA, because the percentage of four-nucleate amoebae was higher than in controls at day 3 of incubation . EDTA made metacystic development unusual by producing a large number of metacystic amoebae with more than ten nuclei . The inhibition of excystation by these chelators was partially abrogated by their removal . A putative antagonist of intracellular calcium flux, 8-( N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8) also inhibited the excystation and metacystic development, but had little effect on cyst viability . The slow Na(+)-Ca(2+) channel blocker bepridil but not verapamil inhibited the excystation and metacystic development, associating with reduced cyst viability at higher concentrations . The inhibitory effect of bepridil on excystation was abrogated by removal of the drug . The CaM inhibitor trifIuoperazine (TFP) but not W-7 { N-(6-aminohexyl)-chloro-1-naphtalene sulphonamide} inhibited the excystation and metacystic development . The inhibitory effect of TFP on excystation was also abrogated by removal of the drug . These results indicate that extracellular calcium ions, amoebic intracellular calcium flux, calcium channels, and a CaM-dependent process contribute to the excystation and metacystic development of E . invadens. Anticancer Res, 2002 May-Jun, 22(3), 1615 - 27 Blockade of the epidermal growth factor receptor tyrosine kinase activity by quercetin and luteolin leads to growth inhibition and apoptosis of pancreatic tumor cells; Lee LT et al.; To glean insights into the mechanism of their action, we assessed the effects of two flavonoids, quercetin (Qu) and luteolin (Lu), on the growth and epidermal growth factor receptor (EGFR) tyrosine kinase activity of MiaPaCa-2 cancer cells . Exposure of these EGFR-expressing cells to 20 microM Qu or Lu resulted in concomitant decreases in cellular protein phosphorylation and growth . On the cellular level, Qu and Lu sensitivity correlated with EGFR levels and rapid cell proliferation, indicating the possibility of targeting those cells most prone to neoplastic progression . Cell treatment with the flavonoids markedly diminished the extent of cellular protein phosphorylation, by effectively modulating protein tyrosine kinase (PTK) activities, including that of EGFR . Immunocomplex kinase assay revealed that both Qu and Lu inhibited the PTK activities responsible for the autophosphorylation of EGFR as well as for the transphosphorylation of enolase . Treatment of the cells with Qu or Lu also reduced the phosphotyrosyl levels of 170-, 125-, 110-, 65-, 60-, 44-, 30- and 25-kDa proteins . We identified the 170-kDa phosphotyrosylprotein as EGFR . Qu and Lu exhibited a specific action in hampering the levels of phosphorylation of this and the aforementioned proteins, while having no discernible effect on their synthesis . A time-dependent attenuation of the phosphorylation of the above proteins was demonstrable . Treatment of the cells with Qu or Lu for 6 hours showed little inhibition, but prolonging the cell treatment for 24 hours caused the suppression of phosphorylation . Further continuation of the cell treatment culminated in the induction of apoptosis, characteristically exhibiting shrinkage of the cell morphology, DNA fragmentation and poly(ADP-ribose)polymerase (PARP) degradation . The onset of apoptosis and associated events occurred in a time-dependent fashion . The data clearly demonstrate that MiaPaCa-2 cells respond to Qu and Lu by a parallel reduction in cellular protein phosphorylation and cellular proliferation . The flavonoid-evoked attenuation of the phosphorylation of EFGR and of other proteins appeared to be transient, since removal of the flavonoid from the cell growth medium after 24 hours of incubation followed by exposure to 10 nm EGF, restored protein phosphorylation and cellular proliferation . Such an addition of EGF was also able to reverse Qu- or Lu-induced cell growth inhibition and diminish nuclear digestion evoked by 20 microM Qu or Lu . Both Qu and Lu were able to reverse the effect of EGF biochemically as well as functionally . Based on the evidence accrued, the above proteins could be implicated in growth signal transduction and the subtle changes in their phosphorylation, as effected by flavonoids, utilized as a reliable guide to predict growth response . The antiproliferative effect of flavonoids might result, at least in part, from the modulation of the EGF-mediated signaling pathway . The results indicate that the blockade of the EGFR-signaling pathway by the PTK inhibitors Qu and Lu significantly inhibits the growth of MiaPaCa-2 cells and induces apoptosis . The modulation of EGFR kinase appears to be a critically important, intrinsic component of Qu- and Lu-induced growth suppression, even though other mechanisms could also have contributed to the net effect. Int J Mol Med, 2002 Sep, 10(3), 257 - 61 Cultivation of fetal erythroid precursors from maternal blood: isolation and characterization by PCR and FISH; Hohmann H et al.; Cultivation of fetal progenitor cells from maternal blood offers the opportunity to produce sufficient fetal cells for prenatal molecular genetic and cytogenetic analysis . For in vitro cultivation, 10 ml of blood was collected from 22 women carrying a male fetus . After triple-density gradient centrifugation, the mononucleated cells were cultivated for 10 to 14 days in special hematopoietic growth medium . Red and white colored cell colonies were individually collected by micromanipulation . A representative sample from each colony was characterized by chromosome Y-specific polymerase chain reaction (PCR) systems . The remaining cells of the Y-positive colonies were used to perform chromosome preparations and fluorescence in situ hybridization (FISH) to detect XY-positive interphase nuclei and metaphases . Y-positive signals could be detected in 15 (68%) of the 22 analyzed blood samples . With SRY PCR 10.5% (40/379) of the collected red colonies were determined to be of fetal origin and 6.1% (32/522) of the colonies analyzed by amelogenin PCR were Y-positive . All collected white cell colonies were Y-negative . FISH analyses of PCR-positive colonies revealed that less than 30% of the cells within a colony are of fetal origin and reflect more precisely the actual situation within a single colony . Moreover the successful preparation of fetal metaphases in non-invasive prenatal diagnosis is presented. J Immunol Methods, 2002 Sep 15, 267(2), 165 - 71 A novel monoclonal antibody screening method using the Luminex-100 microsphere system; Seideman J et al.; We describe the development of a robust and sensitive assay system (detection limit <500 pg/ml biotin-IL-6, K(d)=75 ng/ml), using Luminex-100 microspheres, that could effectively screen for neutralizing antibody whenever a soluble form of the receptor for a target molecule is available . As an example, we coupled a recombinant human interleukin-6 soluble receptor to a Luminex carboxylated microsphere and used a biotin-labeled recombinant human interleukin-6 as a probe to assess binding competition . Three anti-human IL-6 monoclonal antibodies that bind distinct IL-6 epitopes were used as test articles to evince the stringency of the screen . Our assay was able to detect antibody concentration as low as 10 ng/ml without interference from hybridoma growth medium or cell supernatant . The time-saving benefits of this assay format make it ideal for high-throughput screening (HTS) applications for neutralizing monoclonal antibodies. J Cell Sci, 2002 Sep 1, 115(Pt 17), 3469 - 78 Paclitaxel-dependent mutants have severely reduced microtubule assembly and reduced tubulin synthesis; Barlow SB et al.; A subset of mutant cell lines selected for resistance to the antitumor drug paclitaxel are unable to progress normally through mitosis unless the drug is present in the growth medium . Without paclitaxel the cells form defective spindles, undergo aberrant mitoses, fail to complete cell division and eventually die . Analysis of these drug-dependent cells revealed a low amount of microtubule polymer and less tubulin production than wild-type cells . Ribonuclease protection experiments indicated that the decreased tubulin protein was due to decreased tubulin mRNA . Enhancing microtubule assembly by treating the cells with paclitaxel, restored tubulin to levels comparable with those of paclitaxel-treated wild-type cells, which demonstrated that the drug-dependent cells do not have a permanent impairment in their capacity to synthesize tubulin . Paclitaxel-resistant (but not dependent) cells have a smaller reduction in microtubule polymer with little or no decrease in tubulin production, whereas colcemidresistant cells have increased microtubule assembly but also exhibit little or no change in tubulin production . Finally, a mutant cell line producing an unstable beta-tubulin protein has normal growth as well as normal synthesis and polymerization of tubulin, despite an approximately 30% decrease in steady state tubulin content . These studies establish a lower limit of tubulin assembly needed for cell survival and indicate that tubulin assembly must fall below this point to trigger a significant decrease in tubulin synthesis. J Neurochem, 2002 Aug, 82(3), 495 - 503 CNTF inhibits high voltage activated Ca2+ currents in fetal mouse cortical neurones; Holm NR et al.; Neurotrophic factors yield neuroprotection by mechanisms that may be related to their effects as inhibitors of apoptosis as well as their effects on ion channels . The effect of ciliary neurotrophic factor (CNTF) on high-threshold voltage-activated Ca channels in cultured fetal mouse brain cortical neurones was investigated . Addition of CNTF into serum-free growth medium resulted in delayed reduction of the Ca2+ currents . The currents decreased to 50% after 4 h and stabilized at this level during incubation with CNTF for 48 h . Following removal of CNTF the inhibition was completely reversed after 18 h . CNTF reduced the current of all pharmacological subtypes of Ca channels as shown by use of selective blockers of L, N, and P/Q type Ca channels (nifedipine, omega-conotoxin MVIIA, omega-agatoxin IVA) . The Ca channel depression was mediated via the CNTF receptor, because enzymatic cleavage of the alpha-subunit glycerophosphatidylinositol anchor of the receptor eliminated the response . The CNTF effect was not elicited through pertussis toxin-sensitive G proteins . Other neurotrophic factors like neurotrophin-3 and insulin-like growth factor-I had no effect on the Ca2+ currents . These results may have important implications for the possible functions of CNTF in the nervous system, such as altered synaptic activity, neuronal excitability and susceptibility to brain ischaemia. Am J Respir Cell Mol Biol, 2002 Aug, 27(2), 179 - 85 Asthmatic bronchial epithelium is more susceptible to oxidant-induced apoptosis; Bucchieri F et al.; Abnormal apoptotic mechanisms are associated with disease pathogenesis . Because the asthmatic bronchial epithelium is characteristically damaged with loss of columnar epithelial cells, we postulated that this is due to unscheduled apoptosis . Using an antibody directed toward the caspase cleavage product of poly(ADP-ribose) polymerase, immunohistochemistry applied to endobronchial biopsies showed higher levels of staining in the bronchial epithelium of subjects with asthma as compared with normal control subjects (% epithelial staining {median (range) = 10.5 (1.4-24.5) versus 0.4 (0.0-9.7)}; P < 0.001) . Because we were unable to determine whether this difference was due to ongoing inflammation in vivo, cultures of normal and asthmatic bronchial epithelial cells were used to study apoptosis in vitro . In complete growth medium, these cells showed no difference in their rate of proliferation or viability . However, cells from subjects with asthma were more susceptible to the apoptotic effects of H2O2 than cells from normal control subjects (% apoptotic cells = 32.2 {8.8-54.9} versus 14.3 {6.4-24.7}; P < 0.05), even though both were similarly affected by treatment with actinomycin D . These data indicate that the susceptibility of asthmatic bronchial epithelium to oxidants is greater than normal . This susceptibility may contribute to the rising trends in asthma associated with air pollution and diets low in antioxidants. Sci Total Environ, 2002 May 27, 291(1-3), 1 - 32 Soils: their implications to human health; Abrahams PW; This paper reviews how the health of humans is affected by the world's soils, an association that to date has been under appreciated and under reported . Soils significantly influence a variety of functions (e.g . as a plant growth medium; its importance on the cycling of water; as a foundation for buildings) that sustains the human population . Through ingestion (either deliberate or involuntary), inhalation and dermal absorption, the mineral, chemical and biological components of soils can either be directly beneficial or detrimental to human health . Specific examples include: geohelminth infection and the supply of mineral nutrients and potentially harmful elements (PHEs) via soil ingestion; cancers caused by the inhalation of fibrous minerals or Rn gas derived from the radioactive decay of U and Th in soil minerals; and tetanus, hookworm disease and podoconiosis caused by skin contact and dermal absorption of appropriate soil constituents . Human health can also be influenced in more indirect ways as soils interact with the atmosphere, biosphere and hydrosphere . Examples include: the volatilisation of persistent organic pollutants (POPs) from soils and their subsequent global redistribution that has health implications to the Aboriginal people of the Arctic; the frequent detrimental chemical and biological quality of drinking and recreational waters that are influenced by processes of soil erosion, surface runoff, interflow and leaching; and the transfer of mineral nutrients and PHEs from soils into the plants and animals that constitute the human food chain . The scale and magnitude of soil/health interactions are variable, but at times a considerable number of people can be affected as demonstrated by the extent of hookworm infection or the number of people at risk because they live in an I-deficient environment . Nevertheless, it can often be difficult to establish definite links between soils and human health . This, together with the emergence of new risks, knowledge, or discoveries, means that there is considerable scope for research in the future . Such investigations should involve a multidisciplinary approach that both acquires knowledge and ensures its dissemination to people in an understandable way . This requires an infrastructure and finance that governments need to be responsive to. Appl Environ Microbiol, 2002 Aug, 68(8), 3978 - 87 Cyclic AMP and acyl homoserine lactones increase the cultivation efficiency of heterotrophic bacteria from the central Baltic Sea; Bruns A et al.; The effect of signal molecules on the cultivation efficiency of bacteria from the Gotland Deep in the central Baltic Sea was investigated . Numbers of cultivated cells were determined by the most-probable-number (MPN) technique . Artificial brackish water supplemented with different carbon substrates at low concentrations (200 microM each) was employed as the growth medium . Compared to the results of previous studies, this approach yielded significantly higher cultivation efficiencies (up to 11% in fluid media) . A further and pronounced increase in cultivation success was accomplished by the addition of cyclic AMP (cAMP), N-butyryl homoserine lactone, or N-oxohexanoyl-DL-homoserine lactone at a low concentration of 10 microM . The most effective inducer was cAMP, which led to cultivation efficiencies of up to 100% of total bacterial counts . From the highest positive dilutions of these latter MPN series, several strains were isolated in pure culture and one strain (G100) was used to study the physiological effect of cAMP . Dot blot hybridization revealed, however, that strain G100 represented only a small fraction of the total bacterial community . This points towards an inherent limitation of the MPN approach, which does not necessarily recover abundant species from highly diverse communities . Bacterial cells of strain G100 that were starved for 6 weeks attained a higher growth rate and a higher biomass yield when resuscitated in the presence of cAMP instead of AMP. Bioresour Technol, 2002 Oct, 85(1), 99 - 101 Characterization of carex peat using extinction values of humic acids; Baran A; Recent developments in technology and soil science have drawn particular attention to peatlands and their behaviour, properties and utilisation . Further, characteristics of peat are very important in evaluating it as a plant growth medium . The objective of this study was to characterise a number of samples collected from different depths of five different profiles of Yenicaga peat (carex) by measuring the organic carbon, total nitrogen, and extinction values (Er) of their humic acid fractions (HAs) in comparison with decomposition degrees . Peat samples having a high decomposition degree were near to the Er45 axis in the ordinate and had high Er67 values, whereas peats with low decomposition degree were far from the Er45 axis and had low Er67 values. Mol Med, 2002 Mar, 8(3), 149 - 57 Insulin-like growth factor family and combined antisense approach in therapy of lung carcinoma; Pavelic J et al.; BACKGROUND: Perturbation in a level of any peptide from insulin-like growth factor (IGF) family (ligands, receptors, and binding proteins) seems to be implicated in lung cancer formation; IGF ligands and IGF-I receptor through their mitogenic and anti-apoptotic action, and the mannose 6-phosphate/insulin-like growth factor II receptor (M6-P/IGF-IIR) possibly as a tumor suppressor . MATERIALS AND METHODS: To determine the identity, role, and mutual relationship of IGFs in lung cancer growth and maintenance, we examined IGF's gene (by RT-PCR) and protein (by immunohistochemistry) expression in 69 human lung carcinoma tissues . We also examined IGF-I receptor numbers (Scatchard analysis) and IGF-II production and release (by Western blot) in IGF-II/IGF-IR mRNA positive and negative lung carcinomas . Finally, the potential role of IGF-IR and IGF-II as growth promoting factors in lung cancer was studied using antisense oligodeoxynucleotides that specifically inhibit IGF-IR and IGF-II mRNA . RESULTS: Thirty-two tumors were positive for IGF-I, 39 for IGF-II, 48 for IGF-IR, and 35 for IGFBP-4 mRNA . Seventeen tumors were concomitantly positive for all four IGFs, whereas 34 were positive for IGF-II, IGF-IR, and IGFBP-4 mRNA . An elevated amount of IGF-II peptide was secreted into the growth medium of cell cultures established from five different IGF-II/IGF-IR mRNA positive lung cancer tissues . The cells also expressed elevated numbers of IGF-IR . Nine IGF-II-negative and 19 IGF-II-positive lung cancers of different stages were selected, and M6-P/ IGF-II receptor was determined immunohistochemically . Most of the IGF-II-negative tumors were strongly positive for M6-P/IGF-IIR . IGF-II-positive tumors were mostly negative for M6-P/IGF-II receptors . Antisense oligodeoxynucleotides to IGF-II significantly inhibited, by 25-60%, the in vitro growth of all six lung cancer cell lines . However, the best results (growth inhibition of up to 80%) were achieved with concomitant antisense treatment (to IGF-IR and IGF-II) . CONCLUSION: Our data suggest that lung cancer cells produce IGF-IR and IGF-II, which in turn stimulates their proliferation by autocrine mechanism . Cancer cell proliferation can be abrogated or alleviated by blocking the mRNA activity of these genes indicating that an antisense approach may represent an effective and practical cancer gene therapy strategy. J Bacteriol, 2002 Aug, 184(16), 4640 - 3 The NorR protein of Escherichia coli activates expression of the flavorubredoxin gene norV in response to reactive nitrogen species; Hutchings MI et al.; The Escherichia coli norVW genes encode a flavorubredoxin and NADH:(flavo)rubredoxin reductase, respectively, which are involved in nitric oxide detoxification under anaerobic growth conditions . Here it is shown that the norVW genes also have a role in protection against reactive nitrogen intermediates generated from nitroprusside . Transcription from the norV promoter is activated by the presence of nitroprusside in the growth medium; activation requires the product of a divergently transcribed regulatory gene, norR. Mol Cell Biol, 2002 Aug, 22(16), 5879 - 86 RIP2, a checkpoint in myogenic differentiation; Munz B et al.; Using a subtractive cDNA library hybridization approach, we found that receptor interacting protein 2 (RIP2), a tumor necrosis factor receptor 1 (TNFR-1)-associated factor, is a novel early-acting gene that decreases markedly in expression during myogenic differentiation . RIP2 consists of three domains: an amino-terminal kinase domain, an intermediate domain, and a carboxy-terminal caspase activation and recruitment domain (CARD) . In some cell types, RIP2 has been shown to be a potent inducer of apoptosis and an activator of NF-kappa B . To analyze the function of RIP2 during differentiation, we transduced C2C12 myoblasts with retroviral vectors to constitutively produce RIP2 at high levels . When cultured in growth medium, these cells did not show an enhanced rate of proliferation compared to controls . When switched to differentiation medium, however, they continued to proliferate, whereas control cells withdrew from the cell cycle, showed increased expression of differentiation markers such as myogenin, and began to differentiate into multinucleated myotubes . The complete RIP2 protein appeared to be necessary to inhibit myogenic differentiation, since two different deletion mutants lacking either the amino-terminal kinase domain or the carboxy-terminal CARD had no effect . A mutant deficient in kinase activity, however, had effects similar to wild-type RIP2, indicating that phosphorylation was not essential to the function of RIP2 . Furthermore, RIP proteins appeared to be important during myogenic differentiation in vivo, as we detected a marked decrease in expression of the RIP2 homolog RIP in several muscle tissues of the dystrophic mdx mouse, a model for continuous muscle degeneration and regeneration . We conclude that RIP proteins can act independently of TNFR-1 stimulation by ligand to modulate downstream signaling pathways, such as activation of NF-kappa B . These results implicate RIP2 in a previously unrecognized role: a checkpoint for myogenic proliferation and differentiation. J Agric Food Chem, 2002 Jul 31, 50(16), 4567 - 71 Biochemical basis for wheat seedling allelopathy on the suppression of annual ryegrass (Lolium rigidum); Wu H et al.; The chemical basis for wheat seedling allelopathy on the growth of annual ryegrass was investigated by the identification and quantification of multiple allelochemicals from wheat seedlings . Results indicated that 58 wheat accessions differed significantly in seedling allelopathy and inhibited the root growth of ryegrass from 10 to 91%, depending on accession . Analysis of allelochemicals by GC/MS/MS indicated that allelopathy was significantly correlated with the levels of measured allelochemicals in the shoots and roots of young wheat seedlings . Ryegrass root growth was also negatively correlated with the levels of p-hydroxybenzoic, vanillic, and trans-ferulic acids in root exudates . Wheat allelopathic potential was negatively correlated with the levels of the eight known allelochemicals quantified in the shoots, roots, and water-agar medium, with multiple regression coefficients (r) of -0.61, -0.71, and -0.71, respectively . In comparison with weakly allelopathic accessions, strongly allelopathic accessions produced significantly higher amounts of allelochemicals in the shoots and roots of the wheat seedlings and also exuded larger quantities of allelochemicals into the growth medium . Wheat accessions with strong seedling allelopathy might be useful for management of weeds during the establishment stage, thereby reducing the need for commercial herbicides in early-season application. Bioresour Technol, 2002 Aug, 84(1), 7 - 14 The influence of humic acids derived from earthworm-processed organic wastes on plant growth; Atiyeh RM et al.; Some effects of humic acids, formed during the breakdown of organic wastes by earthworms (vermicomposting), on plant growth were evaluated . In the first experiment, humic acids were extracted from pig manure vermicompost using the classic alkali/acid fractionation procedure and mixed with a soilless container medium (Metro-Mix 360), to provide a range of 0, 50, 100, 150, 200, 250, 500, 1,000, 2,000, and 4,000 mg of humate per kg of dry weight of container medium, and tomato seedlings were grown in the mixtures . In the second experiment, humates extracted from pig manure and food wastes vermicomposts were mixed with vermiculite to provide a range of 0, 50, 125, 250, 500, 1,000, and 4,000 mg of humate per kg of dry weight of the container medium, and cucumber seedlings were grown in the mixtures . Both tomato and cucumber seedlings were watered daily with a solution containing all nutrients required to ensure that any differences in growth responses were not nutrient-mediated . The incorporation of both types of vermicompost-derived humic acids, into either type of soilless plant growth media, increased the growth of tomato and cucumber plants significantly, in terms of plant heights, leaf areas, shoot and root dry weights . Plant growth increased with increasing concentrations of humic acids incorporated into the medium up to a certain proportion, but this differed according to the plant species, the source of the vermicompost, and the nature of the container medium . Plant growth tended to be increased by treatments of the plants with 50-500 mg/kg humic acids, but often decreased significantly when the concentrations of humic acids derived in the container medium exceeded 500-1,000 mg/kg . These growth responses were most probably due to hormone-like activity of humic acids from the vermicomposts or could have been due to plant growth hormones adsorbed onto the humates. Arch Biochem Biophys, 2002 Aug 1, 404(1), 48 - 54 Kinetic and thermodynamic characterization of adenylyl cyclase from Euglena gracilis; Jasso-Chavez R et al.; Some kinetic and thermodynamic properties of the plasma membrane adenylyl cyclase (AC) from the protist Euglena gracilis were examined . The AC kinetics for Mg-ATP was hyperbolic with a K(m) value of 0.33-0.43 mM, whereas the inhibition exerted by 2('),5(')-dideoxyadenosine was of the mixed type with a K(i) of 80-147 microM . The V(m) value (0.9 or 1.8 nmol(mg protein)(-1)min(-1)) changed, depending upon the carbon source in the growth medium (lactic acid or glutamate plus malate) . Lactic acid membrane AC was slightly more thermolabile (from 28 to 40 degrees C) and showed higher activation energy (range 15-25 degrees C) . With lactate, the total and saturated fatty acid percentage content in the plasma membrane was significantly greater than with glutamate plus malate, whereas the percentage content of polyunsaturated (n-3) fatty acids was lower . The data suggest that the fatty acid composition, as changed by the carbon source in the growth medium, may modulate the AC activity in Euglena. Phytomedicine, 2002 May, 9(4), 319 - 24 The fruiting body and its caterpillar host of Cordyceps sinensis show close resemblance in main constituents and anti-oxidation activity; Li SP et al.; Cordyceps (summer-grass, winter-worm), one of the most valued traditional Chinese medicines, is used commonly for the replenishment of body health . It consists of the dried fungus Cordyceps sinensis growing on caterpillar larvae . For medication, the fruiting body (fungus) and the worm (caterpillar) are used together . However, the pharmacological efficiency and the main constituents of the individual parts have not been determined . In the present study the water extracts from the fruiting body and worm of natural Cordyceps were analyzed for their content of nucleosides and polysaccharides; the results showed that the worm had chemical composition similar to the fruiting body . In addition, both the fruiting body and worm of Cordyceps showed similar potency in their anti-oxidation activities in the xanthine oxidase assay, the induction of hemolysis assay and the lipid-peroxidation assay . These results suggest that the function of the worm in Cordyceps is to provide a growth medium for the fruiting body, and that eventually, the worm is totally invaded by C . sinensis mycelia. Bioresour Technol,