Growth media

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, 2002 Sep, 84(3), 251 - 7
Phenolic removal in olive oil mill wastewater by strains of Pleurotus spp . in respect to their phenol oxidase (laccase) activity; Tsioulpas A et al.; The ability of several Pleurotus spp . strains to remove phenolic compounds from an olive oil mill wastewater (OMW) was studied . All strains tested in this work were able to grow in OMW without any addition of nutrients and any pre-treatment, except sterilization . High laccase activity was measured in the growth medium, while 69-76% of the initial phenolic compounds were removed . The black color of OMW became yellow-brown and brighter as the strains grew . The lowest phenolic concentrations were reached after 12/15 days . A decrease of the phytotoxicity, as described by the parameter Germination Index, was noticed in the OMW treated with some Pleurotus spp strains, although this decrease was not proportional to the phenolic removal . A new parameter, namely Phenol-toxicity Index, was considered in the present paper . Using this parameter it was found that the remaining phenolics and/or some of the oxidation products of the laccase reaction in the treated OMW were more toxic than the original phenolic compounds.

Plant Physiol, 2002 Jul, 129(3), 1359 - 67
Excess copper predisposes photosystem II to photoinhibition in vivo by outcompeting iron and causing decrease in leaf chlorophyll; Patsikka E et al.; Photoinhibition of photosystem II was studied in vivo with bean (Phaseolus vulgaris) plants grown in the presence of 0.3 (control), 4, or 15 microM Cu(2+) . Although photoinhibition, measured in the presence of lincomycin to block concurrent recovery, is faster in leaves of Cu(2+)-treated plants than in control leaves, thylakoids isolated from Cu-treated plants did not show high sensitivity to photoinhibition . Direct effects of excess Cu(2+) on chloroplast metabolism are actually unlikely, because the Cu concentration of chloroplasts of Cu-treated plants was lower than that of their leaves . Excess Cu in the growth medium did not cause severe oxidative stress, collapse of antioxidative defenses, or loss of photoprotection . Thus, these hypothetical effects can be eliminated as causes for Cu-enhanced photoinhibition in intact leaves . However, Cu treatment lowered the leaf chlorophyll (Chl) concentration and reduced the thylakoid membrane network . The loss of Chl and sensitivity to photoinhibition could be overcome by adding excess Fe together with excess Cu to the growth medium . The addition of Fe lowered the Cu(2+) concentration of the leaves, suggesting that Cu outcompetes Fe in Fe uptake . We suggest that the reduction of leaf Chl concentration, caused by the Cu-induced iron deficiency, causes the high photosensitivity of photosystem II in Cu(2+)-treated plants . A causal relationship between the susceptibility to photoinhibition and the leaf optical density was established in several plant species . Plant species adapted to high-light habitats apparently benefit from thick leaves because the rate of photoinhibition is directly proportional to light intensity, but photosynthesis becomes saturated by moderate light.

Am J Physiol Lung Cell Mol Physiol, 2002 Aug, 283(2), L256 - 64
Maintenance of the mouse type II cell phenotype in vitro; Rice WR et al.; The purpose of this study was to identify culture conditions for maintenance of isolated mouse type II cells with intact surfactant protein (SP) and phospholipid production . Type II cells were isolated from 6-wk-old mice and cultured on Matrigel matrix-rat tail collagen (70:30 vol/vol) in bronchial epithelial cell growth medium minus hydrocortisone plus 5% charcoal-stripped FBS and 10 ng/ml keratinocyte growth factor . Under these conditions, type II cells actively produced surfactant phospholipids and proteins for at least 7 days . Synthesis and secretion of surfactant phospholipids and SP-A, -B, -C, and -D declined on day 1 of culture but recovered by day 3, reaching levels comparable to or exceeding freshly isolated cells by day 5 . Abundant lamellar bodies were readily apparent in cells examined on days 5 and 7, and a surfactant pellet was recovered by centrifugation of media harvested on each day of culture . Secretion of SP-B, SP-C, and phosphatidylcholine was stimulated by phorbol 12-myristate 13-acetate and was inhibited by compound 48/80 . When tested with a bubble surfactometer, surfactant secreted by type II cells on day 5 of culture lowered surface tension to 5.2 +/- 2.3 mN/m . This is the first description of the synthesis and secretion of a functional surfactant complex by mouse type II cells after 7 days in primary culture.

J Cell Biochem, 2002, 86(2), 251 - 7
Effects of estrogen on collagen synthesis by cultured human osteoblasts depend on the rate of cellular differentiation; Ireland DC et al.; Estrogen is known to act on osteoblasts according to their stage of differentiation and estrogen receptor (ER) isoform expression . The aim of this study was to determine when type I collagen (COL1) synthesis by cultured low-passage, human bone-derived osteoblasts (hOBs) is upregulated in response to estrogen . Cell lines from female donors aged 1 and 66 years were cultured for 11 days on collagen in growth medium supplemented with human serum, hydrocortisone, and beta-glycerophosphate . Young-donor hOBs grew more quickly than old-donor hOBs and did not mineralize . Old-donor hOBs formed mineralized nodules 5 days after reaching confluence . Changes in mRNA levels with time for ERs, type I collagen, and alkaline phosphatase reflected the faster differentiation of the old-donor cells . The ERbeta/ERalpha ratio fell threefold in young-donor hOBs but rose 300-fold in old-donor hOBs . Increased ERbeta/ERalpha ratios prevented ligand-dependent downregulation of ERalpha transcription, resulting in reduced proliferation in old-donor hOBs . Upregulation of COL1 mRNA expression in response to estrogen was confined to intermediate stages of differentiation, resulting in significant increases in COL1 mRNA by estradiol only in young-donor cells . Since the young and old-donor hOBs were cultured under identical conditions, our results indicate that the response of hOBs to estrogen is largely dependent on intracellular mechanisms that control the timing of cellular differentiation .

J Neurosci Res, 2002 Jun 1, 68(5), 627 - 35
Mevastatin induces degeneration and decreases viability of cAMP-induced differentiated neuroblastoma cells in culture by inhibiting proteasome activity, and mevalonic acid lactone prevents these effects; Kumar B et al.; Statins with a closed-ring structure (mevastatin, lovastatin, and simvastatin) and with an open-ring structure (pravastatin and fluvastatin) are widely used in the human population to manage hypercholesterolemia . These statins may have neuroprotective or neurotoxic effects, but these effects remain controversial . We have utilized adenosine 3',5'-cyclic monophosphate-induced terminally differentiated murine neuroblastoma (NB) cells in culture as an experimental model to study the effect of statins . Results showed that mevastatin induced degenerative changes and reduced the viability of differentiated NB cells by inhibiting proteasome activity . Lactacystin, an established inhibitor of proteasome, also produced similar degenerative changes in these cells . In contrast, pravastatin neither affected the degeneration and viability of differentiated NB cells nor the proteasome activity . High-performance liquid chromatography (HPLC) analysis of the extract obtained from mevastatin-treated growth medium and differentiated cells revealed that about 50% of mevastatin is converted to an open-ring structure in the growth medium; however, differentiated cells did not convert any portion of mevastatin into an open-ring structure and accumulated only mevastatin with a closed-ring structure . Mevalonic acid lactone by itself did not affect the viability of differentiated NB cells or the proteasome activity, but it completely prevented mevastatin-induced degeneration and decreased viability by reducing the uptake of mevastatin and by blocking its action on proteasome activity . Mevalonic acid failed to prevent lactacystin-induced degeneration and inhibition of proteasome activity . Our results suggest that mevastatin could act as a neurotoxic agent or neuroprotective agent, depending upon the extent of its hydrolysis to an open-ring structure and the level of mevalonic acid .

Kidney Int, 2002 Aug, 62(2), 697 - 703
3,4-Dideoxyglucosone-3-ene (3,4-DGE): a cytotoxic glucose degradation product in fluids for peritoneal dialysis; Linden T et al.; BACKGROUND: Bioincompatible glucose degradation products (GDPs) in fluids for peritoneal dialysis (PD) develop during sterilization and storage . Their biological activity has successfully been monitored through the use of various in vitro methods but their molecular and chemical nature is less well understood . Many GDPs are highly reactive carbonyl compounds . Although some of the identified GDPs are extremely cytotoxic, none of them actually possess cytotoxicity at the concentrations found in PD fluids . Thus, the GDP responsible for the toxicity in PD fluids has not yet been identified . The intention of the present work was to investigate to what extent the unsaturated dicarbonyl compound, 3,4-dideoxyglucosone-3-ene (3,4-DGE) was present in PD fluids, and if it could be responsible for the in vitro effects on L-929 fibroblast cells . METHODS: A commercial preparation of 3,4-DGE and two different liquid chromatography methods were used for the chemical identification and quantification . In vitro bioincompatibility was determined as inhibition of cell growth using the L-929 fibroblast cell line . RESULTS: 3,4-DGE was present in conventionally manufactured PD fluids at a concentration of 9 to 22 micromol/L . In the newly developed PD fluid, Gambrosol trio, the concentrations were 0.3 to 0.7 micromol/L . When added as synthetic 3,4-DGE to cell growth media at the concentrations measured in conventional PD fluids, the inhibition of cell growth was significantly lower than for that seen with the conventional fluids . However, in the conventional PD fluids the total amount of 3,4-DGE available for toxic reactions most probably was higher than that measured, because 3,4-DGE was freshly recruited from a molecular pool when consumed . The speed of this recruitment was high enough to explain most of the growth inhibition seen for heat-sterilized PD fluids . CONCLUSION: 3,4-DGE is present in conventional PD fluids at a concentration between 9 and 22 micromol/L, and is the most biologically active of all GDPs identified to date . Thus, it is the main candidate to be held responsible for the clinical bioincompatibility caused by conventionally manufactured PD fluids.

Cent Eur J Public Health, 2002 Jun, 10(1-2), 66 - 71
The comparison of aluminium effects and its uptake by Escherichia coli in different media; Bojic A et al.; The investigation of the toxic effects and the uptake of aluminium by Escherichia coli in growth medium (GM) and in physiological solution (PS) have been studied . The toxicity was quantitatively determined according to the decrease of the colony forming units (CFU) in the physiological solution, that is its growth inhibition in the growth medium, vs . the aluminium concentration and incubation time, at pH 5.2, 6.2 and 7.2 . The uptake of aluminium was investigated by determining the intracellular aluminium in dry weights (DW), by graphite fumace atomic absorption spectrophotometry, considering that aluminium adsorbed to the cell surface was removed by washing with EDTA solution . The results show that toxicity and accumulation increase with the increase of the aluminium concentration and incubation time . However, the linearity of these functions was lost at higher values, which indicate dependence on time and concentration saturation . The effect of pH was specific, and correlated with the form of aluminium in solution . The increase in toxicity as the pH decreases, suggests that the Al(H2O)6(3+) ion is the major toxic form, among the remaining present ones aluminium in aqueous media . The results also show that the aluminium in a concentration range from 0.10 to 10.0 mg/l toxic to E . coli in PS, was significantly less toxic for bacteria in the GM, mainly because of living conditions and the accessibility of free Al.

Biochem Biophys Res Commun, 2002 Jul 5, 295(1), 182 - 6
Particulate methane monooxygenase from Methylosinus trichosporium is a copper-containing enzyme; Xin JY et al.; Particulate methane monooxygenase (pMMO) has been exfoliated and isolated from membranes of the Methylosinus trichosporium IMV 3011 . It appears that the stability of pMMO in the exfoliation process is increased with increasing copper concentration in the growth medium, but extensive intracytoplasmic membrane formed under higher copper concentration may inhibit the exfoliation of active pMMO from membrane . The highest total activity of purified pMMO is obtained with an initial concentration of 6 microM Cu in the growth medium . The purified MMO contains only copper and does not utilize NADH as electron donor . Treatment of purified pMMO with EDTA resulted in little change in copper level, suggesting that the copper in the pMMO is tightly bound with pMMO . (c) 2002 Elsevier Science (USA).

Physiol Plant, 2002 Jul, 115(3), 377 - 384
Effects of Pb-EDTA and EDTA on oxidative stress reactions and mineral uptake in Phaseolus vulgaris; Geebelen W et al.; Sequestration of Pb by synthetic chelates has been reported to increase bioavailability, uptake, and translocation of this metal in plants . In this work the potential phytotoxic effects of Pb-EDTA were investigated in Phaseolus vulgaris L . cv . Limburgse vroege plants grown on hydroponics . Addition of 50 microM Pb-EDTA to the nutrient solution caused a significant induction of syringaldazine peroxidase (SPOD; EC 1.11.1.7) in roots and primary leaves and guaiacol peroxidase (GPOD; EC 1.11.1.7) in leaves . Addition of 100 microM Pb-EDTA further exacerbated ascorbate peroxidase (APOD; EC 1.11.1.11), GPOD, dehydroascorbate reductase (DHAR; EC 1.8.5.1), glutathione reductase (GR; EC 1.6.4.2) and malic enzyme (ME; EC 1.1.1.40) in roots and APOD and ME in primary leaves . Addition of 200 microM Pb-EDTA also induced DHAR in leaves . This induction of peroxidases (SPOD, GPOD, APOD), enzymes of the ascorbate-glutathione cycle (DHAR, GR in roots) and of an NADP+ reducing enzyme in roots and primary leaves indicates that oxidative stress has been initiated . At 200 microM Pb-EDTA, chlorophyll a and b content in leaves was significantly reduced while visible effects on root morphology and shoot length were observed, while no significant morphological effects were found in the leaves, confirming the sensitive character of the measured enzymes as plant stress indicators . Elevation of the Pb-EDTA concentration in the growth medium significantly reduced the content of Ca, Fe, Mn and Zn taken up by plants, probably due to ion leakage as a result of observed toxicity . Addition of up to 200 microM EDTA increased chelation of divalent cations in nutrient solution resulting in reduced plant uptake of Zn, Cu, Fe and Mn . This did not result in phytotoxicity.

J Ind Microbiol Biotechnol, 2002 Feb, 28(2), 97 - 102
Expression of an alpha-galactosidase from Saccharomyces cerevisiae in Aspergillus awamori and Aspergillus oryzae; Murphy RA et al.; A gene encoding alpha-galactosidase activity was isolated by polymerase chain reaction (PCR) from Saccharomyces cerevisiae NCYC686 and separately placed under the control of transcriptional elements regulating alpha-amylase expression in Aspergillus oryzae and glucoamylase expression in A . awamori . Following transformation of both A . oryzae and A . awamori with their respective expression vectors, induction of heterologous alpha-galactosidase from positively selected clones was effected through the addition of soluble starch (10% wt/vol) to the growth medium . Upon induction in A . oryzae, a transcriptional instability resulted in degradation of mRNA encoding heterologous alpha-galactosidase, thus preventing expression of the active enzyme . The use of a gene fusion strategy in A . awamori overcame this instability and resulted in stable expression of S . cerevisiae alpha-galactosidase . Subsequent to initial (shake flask) experiments, a series of scale-up and optimisation studies led to heterologous expression of the recombinant enzyme in batch fermentation at 51 U mg(-1) total extracellular protein . This was higher than previously published works, which reported extracellular levels of heterologous alpha-galactosidase up to 38 U mg(-1) total protein . Analysis of crude extracts of the fermentation medium revealed significant differences between the activity parameters reported previously in the literature for this enzyme and those observed here . The recombinant enzyme exhibited thermostability properties not previously reported for S . cerevisiae alpha-galactosidase, a trait which would make it suitable for use in processes requiring high temperatures.

Eur J Haematol, 2002 Apr, 68(4), 189 - 93
Patients with idiopathic myelofibrosis show increased CD34+ cell concentrations in peripheral blood compared to patients with polycythaemia vera and essential thrombocythaemia; Andreasson B et al.; The aim of the present work was to compare the results for some haematological variables in 12 patients with idiopathic myelofibrosis (IMF) with those of 21 patients with polycythaemia vera (PV), 22 patients with essential thrombocythaemia (ET) and 10 healthy control subjects . In each patient and control subject peripheral blood was used for analysis of flow cytometric measurement of CD34-positive (CD34+) cells, in vitro colony growth and plasma erythropoietin (EPO) concentration . The mean concentration of CD34+ cells in the IMF group was 568 +/- 686 x 10(3) mL, which significantly (P<0.001 for each group) exceeded the means in PV, ET and control groups ((10.2 +/- 32.0) x 10(3) mL, 3.0 +/- 3.7 x 10(3) mL and 1.9 +/- 0.8 x 10(3) mL, respectively) . The mean number of EPO-independent erythroid colonies (EEC) was 110 +/- 215 colonies per 10(5) cells for the IMF patients . In comparison with the means for PV and ET patients (40 +/- 140 and 12 +/- 27 colonies per 10(5) cells, respectively) the difference did not reach statistical significance . The mean EEC for IMF patients was, however, significantly higher compared with the mean for the control subjects (P<0.05) . The means for total erythroid colony growth with EPO added to the growth medium as well as for granulocyte-macrophage colony-forming units were significantly higher in the IMF group compared with the means for PV, ET and control groups (P<0.001 for each group) . The mean plasma EPO was 204 +/- 290 IU/L in the patients with IMF compared with 6.6 +/- 7.9 IU/L in PV patients, 19.1 +/- 23.2 IU/L in ET and 10.3 +/- 7.8 IU/L in the control subjects . Due to considerable differences in haemoglobin concentrations no relevant conclusions could be drawn from the results for plasma EPO concentrations . Indeed, the majority of patients with IMF, PV and ET were on myelosuppressive treatment; additionally most PV patients received phlebotomy therapy . The results of the present study suggest that the circulating pool of stem cells and progenitor cells in peripheral blood is significantly increased in IMF patients compared with PV and ET patients as well as healthy control subjects . The most likely source for the elevated CD34+ cell concentration in peripheral blood is progenitor cells of extra-medullar origin.

Prikl Biokhim Mikrobiol, 2002 May-Jun, 38(3), 257 - 60
{Optimization of the medium and cultivation conditions of Penicillium roquefortii f39 producing the diketopiperazine alkaloid roquefortine}; Boichenko DM et al.; We optimized the medium for cultivation of Penicillium roquefortii f39, a producer of roquefortine . In this medium, the roquefortine yield increased 1.5-2-fold . The increase in roquefortine content was associated with high biomass yield, but not with an increase in biosynthetic activity of the mycelium . Direct correlation was found between extracellular roquefortine concentration and amount of the inoculum . The introduction of sucrose into the growth medium allowed us to increase the concentration of roquefortine during fermentation to 90 mg/l.

J Cell Physiol, 2002 May, 191(2), 162 - 72
Stimulation of the proliferation and differentiation of mouse pink-eyed dilution epidermal melanocytes by excess tyrosine in serum-free primary culture; Hirobe T et al.; The epidermal cell suspensions of the neonatal dorsal skin derived from wild type mouse at the pink-eyed dilution (p) locus (black, C57BL/10JHir-P/P) and their congenic mutant mouse (pink-eyed dilution, C57BL/10JHir-p/p) were cultured with a serum-free melanocyte growth medium supplemented with additional L-tyrosine (Tyr) from initiation of the primary culture . L-Tyr inhibited the proliferation of P/Pmelanocytes in a dose-dependent manner, whereas L-Tyr stimulated the proliferation of p/p melanoblasts and melanocytes regardless of dose . On the other hand, L-Tyr stimulated (P/P) or induced (p/p) the differentiation of epidermal melanocytes in a dose-dependent manner . In both P/P and p/p melanoblasts and melanocytes cultured with 2.0 mM L-Tyr for 14 days, slight increases in contents of eumelanin marker, pyrrole-2,3,5-tricarboxylic acid (PTCA) and pheomelanin marker, aminohydroxyphenylalanine (AHP) were observed . The average number of total melanosomes (stages I, II, III, and IV) per P/P melanocyte was not changed by L-Tyr treatment, but the proportion of stage IV melanosomes in the total melanosomes was increased . On the contrary, in p/p melanoblasts and melanocytes L-Tyr increased dramatically the number of stage II, III, and IV melanosomes as well as the proportion of stage III melanosomes . Contents of PTCA and eumelanin precursor, 5,6-dihydroxyindole-2-carboxylic acid (DHICA) of cultured media in p/p melanocytes were much more greatly increased than in P/P melanocytes . However, contents of AHP and pheomelanin precursor, 5-S-cysteinyldopa (5-S-CD) of cultured media in p/p melanocytes were increased in a similar tendency to P/Pmelanocytes . These results suggest that p/p melanocytes in the primary culture are induced to synthesize eumelanin by excess L-Tyr, but difficult to accumulate them in melanosomes.

Gene, 2002 May 15, 290(1-2), 1 - 18
Nitrogen regulation in Saccharomyces cerevisiae; Magasanik B et al.; Yeast cells can respond to growth on relatively poor nitrogen sources by increasing expression of the enzymes for the synthesis of glutamate and glutamine and by increasing the activities of permeases responsible for the uptake of amino acids for use as a source of nitrogen . These general responses to the quality of nitrogen source in the growth medium are collectively termed nitrogen regulation . In this review, we discuss the historical foundations of the study of nitrogen regulation as well as the current understanding of the regulatory networks that underlie nitrogen regulation . One focus of the review is the array of four GATA type transcription factors which are responsible for the regulation the expression of nitrogen-regulated genes . They are the activators Gln3p and Nil1p and their antagonists Nil2p and Dal80p . Our discussion includes consideration of the DNA elements which are the targets of the transcription factors and of the regulated translocation of Gln3p and Nil1p from the cytoplasm to the nucleus . A second focus of the review is the nitrogen regulation of the general amino acid permease, Gap1p, and the proline permease, Put4p, by ubiquitin mediated intracellular protein sorting in the secretory and endosomal pathways.

Int J Parasitol, 2002 Jun, 32(6), 655 - 75
Lipid metabolism in mucous-dwelling amitochondriate protozoa; Das S et al.; Entamoeba, Giardia, and trichomonads are the prominent members of a group known as 'mucosal parasites' . While Entamoeba and Giardia trophozoites colonise the small intestine, trichomonads inhabit the genitourinary tracts of humans and animals . These protozoa lack mitochondria, well-developed Golgi complexes, and other organelles typical of higher eukaryotes . Nonetheless, they have developed unique metabolic pathways that allow them to survive and multiply in the small intestine and reproductive tracts by scavenging nutrients from the host . Various investigators have shown that these protozoa are unable to synthesise the majority of their own lipids and cholesterol de novo; rather, they depend mostly on supplies from outside sources . Therefore, questions of how they transport and utilise exogenous lipids for metabolic purposes are extremely important . There is evidence suggesting that these parasites can take up the lipids and cholesterol they need from lipoprotein particles present in the host and/or in the growth medium . Studies also support the idea that individual lipid and fatty acid molecules can be transported without the help of lipoproteins . Exogenous phospholipids have been shown to undergo fatty acid remodelling (by deacylation/reacylation reactions), which allows these protozoa to alter lipids, bypassing the synthesis of entirely new phospholipid molecules . In addition, many of these amitochondriates are, however, capable of elongating/desaturating long-chain fatty acids, and assembling novel glycophospholipid molecules . In this review, progress in various aspects of lipid research on these organisms is discussed . Attempts are also made to identify steps of lipid metabolic pathways that can be used to develop chemotherapeutic agents against these and other mucosal parasites.

Acta Vet Hung, 2002, 50(1), 117 - 29
Equid herpesvirus 1 is neurotropic in mice, but latency from which infectious virus can be reactivated does not occur; Iqbal J et al.; Equid herpesvirus 1 (EHV-1) is the most common cause of virus-induced abortion in horses . After primary infection the virus becomes latent predominantly in the respiratory tract lymph nodes and the genome can also be detected in the peripheral nervous system . The role of mouse as a feasible model for the establishment of latency and reactivation of EHV-1 was investigated . Intracerebral and intranasal infections of 3- and 17-day-old mice were made and virus replication was confirmed by virus isolation and detected by indirect immunofluorescence (IIF) in brain . For reactivation studies, the mice were killed 8 weeks post infection and tissues were collected for cocultivation . In mice from both age groups, infectious virus was not detected by cocultivation . Following attempts to reactivate virus in vivo with corticosteroids, the viral antigen was detected at low levels by IIF and the expression of the gB gene by reverse transcription polymerase chain reaction (RT-PCR) in brain, trigeminal ganglia, olfactory lobe, lung and spleen . Virus was also detected by IIF following incubation of tissue explants in the growth medium containing pokeweed mitogen (PWM) . These results show the limitations of the mouse model for investigating EHV-1 latency and highlights the issue of 'ineffective reactivation' of virus.

Am J Physiol Lung Cell Mol Physiol, 2002 Jul, 283(1), L144 - 55
Inhibition of endogenous TRP1 decreases capacitative Ca2+ entry and attenuates pulmonary artery smooth muscle cell proliferation; Sweeney M et al.; Pulmonary vascular medial hypertrophy due to proliferation of pulmonary artery smooth muscle cells (PASMC) greatly contributes to the increased pulmonary vascular resistance in pulmonary hypertension patients . A rise in cytosolic free Ca2+ concentration ({Ca2+}cyt) is an important stimulus for cell growth in PASMC . Resting {Ca2+}cyt, intracellularly stored {Ca2+}, capacitative Ca2+ entry (CCE), and store-operated Ca2+ currents (I(SOC)) are greater in proliferating human PASMC than in growth-arrested cells . Expression of TRP1, a transient receptor potential gene proposed to encode the channels responsible for CCE and I(SOC), was also upregulated in proliferating PASMC . Our aim was to determine if inhibition of endogenous TRP1 gene expression affects I(SOC) and CCE and regulates cell proliferation in human PASMC . Cells were treated with an antisense oligonucleotide (AS, for 24 h) specifically designed to cleave TRP1 mRNA and then returned to normal growth medium for 40 h before the experiments . Then, mRNA and protein expression of TRP1 was downregulated, and amplitudes of I(SOC) and CCE elicited by passive depletion of Ca2+ from the sarcoplasmic reticulum using cyclopiazonic acid were significantly reduced in the AS-treated PASMC compared with control . Furthermore, the rate of cell growth was decreased by 50% in AS-treated PASMC . These results indicate that TRP1 may encode a store-operated Ca2+ channel that plays a critical role in PASMC proliferation by regulating CCE and intracellular {Ca2+}(cyt).

Bioresour Technol, 2002 Jun, 83(2), 71 - 9
Performance comparison of experimental constructed wetlands with different filter media and macrophytes treating industrial wastewater contaminated with lead and copper; Scholz M et al.; The aim of this study was to investigate the treatment efficiency of passive vertical-flow wetland filters containing different macrophytes (Phragmites and/or Typha) and granular media with different adsorption capacities . Gravel, sand, granular activated carbon, charcoal and Filtralite (light expanded clay) were used as filter media . Different concentrations of lead and copper sulfate were added to polluted urban stream inflow water to simulate pretreated mine wastewater . The relationships between growth media, microbial and plant communities as well as the reduction of predominantly lead, copper and five-day biochemical oxygen demand (BOD5) were investigated . An analysis of variance showed that concentration reductions (mg l(-1)) of lead, copper and BOD5 were significantly similar for the six experimental wetlands . Microbial diversity was low due to metal pollution and similar for all filters . There appears to be no additional benefit in using adsorption media and macrophytes to enhance biomass performance during the first 10 months of operation.

Phytochemistry, 2002 Jul, 60(5), 475 - 81
Aromadendrane transformations by Curvularia lunata ATCC 12017; Collins DO et al.; The naturally occurring sesquiterpene squamulosone (1), isolated from Hyptis verticillata (Labiatae), was synthetically reduced to five analogues that were identified as (1S,10S)-9alpha-hydroxy-allo-aromadendrane (2), (1R,10R)-9beta-hydroxyaromadendrane (3), (1S,10S)-allo-aromadendran-9-one (4), (1R,10R)-aromadendran-9-one (5) and aromadendra-1,9-diene (6) . Each congener was incubated with the fungus Curvularia lunata ATCC 12017 in two different growth media . All the substrates except the deoxy compound 6 underwent a simple redox reaction . Ketone 5 additionally experienced remote hydroxylation while analogue 6, possessing a conjugated diene system, was most extensively metabolised . The substrates and products presented here, but one, are all novel.

Biotechnol Prog, 2002 May-Jun, 18(3), 418 - 23
Improved paclitaxel and baccatin III production in suspension cultures of Taxus media; Cusido RM et al.; A cell suspension culture of Taxus media was established from a stable callus line of this species . The growth rate and production of paclitaxel and baccatin III of this cell suspension were significantly increased during the shake flask culture in its respective optimum media for cell growth and product formation, which were selected after assaying 24 different culture media . The highest yields of paclitaxel (2.09 mg L(-1)) and baccatin III (2.56 mg L(-1)) in the production medium rose (factors of 7.0 and 3.0, respectively) in the presence of methyljasmonate (220 microg g(-1) FW) . When the elicitor was added together with mevalonate (0.38 mM) and N-benzoylglycine (0.2 mM), the increase in the yields of paclitaxel and baccatin III was even higher (factors of 8.3 and 4.0, respectively) . Thereafter, a two-stage culture for cell suspension was carried out using a 5-l stirred bioreactor running for 36 days, the first stage being in the cell growth medium until cells entered their stationary growth phase (12 days) and the second stage being in the production medium supplemented with the elicitor and two putative precursors in the concentrations indicated above . Under these conditions, 21.12 mg L(-1) of paclitaxel and 56.03 mg L(-1) of baccatin III were obtained after 8 days of culture in the production medium.

Arch Biochem Biophys, 2002 Jun 1, 402(1), 104 - 9
Manganese supplementation relieves the phenotypic deficits seen in superoxide-dismutase-null Escherichia coli; Al-Maghrebi M et al.; Escherichia coli, lacking cytoplasmic superoxide dismutases, exhibits a variety of oxygen-dependent phenotypic deficits . Enrichment of the growth medium with Mn(II) relieved those deficits . Extracts of cells grown on Mn(II)-rich medium exhibited superoxide dismutase-like activity that was due partially to low-molecular-weight and partially to high-molecular-weight complexes . The high-molecular-weight activity was sensitive to proteolysis . Hence this activity is likely associated with low-affinity binding of Mn to proteins.

Toxicology, 2002 Jun 14, 175(1-3), 201 - 13
Impact of protein binding on the availability and cytotoxic potency of organochlorine pesticides and chlorophenols in vitro; Gulden M et al.; In vitro toxicity data are generally based on nominal concentrations and thus depend on both activity and availability of a compound . The aims of the present study were to examine the influence of protein binding on the cytotoxicity of selected organochlorine pesticides and chlorophenols in Balb/c 3T3 cell cultures and to determine parameters of protein binding which can be used to estimate protein bound fractions and to model distribution in vitro . EC(50)-values derived from concentration-effect relationships determined in the presence of various concentrations of bovine serum albumin (BSA) were linearly correlated to BSA concentration . Increasing the BSA concentration from about 1.2 to 40 mg/ml increased the EC(50)-values by factors between 3.4 and 34.4 . Molar ratios of substance bound to albumin ranged from 0.11 to 2.42 . Calculated fractions bound to albumin in the normal growth medium were 0.075-0.17 (p,p'-DDT, p,p'-DDE, dieldrin, lindane), 0.09-0.1 (4-mono- and 2,4-dichlorophenol), 0.68 (2,4,5-trichlorphenol) and almost 1.0 (pentachlorophenol) . At 40 mg/ml BSA any compound was largely bound to albumin (fractions bound > or = 0.74) . Distribution modelling revealed that the availability of the highly hydrophobic organochlorines additionally was significantly reduced by partitioning into lipids . The results clearly demonstrate that nominal and relative toxic potencies of organochlorine pesticides and chlorophenols determined in vitro are substantially influenced by effects of protein binding on availability.

Clin Exp Med, 2002 May, 2(1), 45 - 52
Detection and quantification of the soluble form of the human erythropoietin receptor (sEpoR) in the growth medium of tumor cell lines and in the plasma of blood samples; Westphal G et al.; The erythropoietin receptor (EpoR) belongs to the cytokine superfamily and is a type I transmembrane protein . Like other members of this family, EpoR is also synthesized as a soluble form, and is subsequently secreted by the cell . To investigate whether soluble EpoR (sEpoR) is expressed in human tumor cell lines, we developed a sensitive quantitative ELISA, using an anti-human EpoR antibody and recombinant sEpoR as standard control . With this ELISA, sEpoR could be detected in the supernatant of human tumor cell lines . Analysis of blood samples showed that sEpoR was coprecipitated during coagulation . Therefore only plasma was suitable for analysis and first measurements of plasma samples were investigated . In conclusion, an ELISA to quantify the sEpoR was established and the expression of sEpoR in human tumor cell lines was demonstrated for the first time.

J Environ Sci (China), 2002 Apr, 14(2), 216 - 20
Joint enhancement of lead accumulation in Brassica plants by EDTA and ammonium sulfate in sand culture; Xiong ZT et al.; When EDTA was added alone in the Pb-contaminated sand, the plant biomass and the total Pb amount in Plant decreased in both species, Brassica pekinensis and B . juncea var . multiceps, though the shoot Pb amount increased . In contrast, when (NH4)2SO4 was added alone in the Pb-contaminated sand, little effect was observed on the shoot Pb amount, though the root Pb amount was significantly increased in B . juncea var . multiceps . When amending EDTA and (NH4)2SO4 in combination, however, the shoot Pb amount in both species substantially increased, being, on an average, 2 times and 9 times higher than that in EDTA alone or (NH4)2SO4 alone amended treatment, respectively . The two amendments showed antagonism for plant growth, but synergism for Pb bioaccumulation . B . pekinensis showed its highest level of shoot and total Pb amount in the treatment amended with EDTA and (NH4)2SO4 only a half as much as in the other treatments . It is inferred that the mechanisms responsible for the joint-enhanced Pb accumulation might be concerned with the acidification of the growth medium, cation exchange reaction and relieving EDTA induced toxicity as results by amending ammonium sulfate.

J Vasc Surg, 2002 Jun, 35(6), 1226 - 32
Inhibition of vascular smooth muscle cell proliferation with red wine and red wine polyphenols; Araim O et al.; OBJECTIVE: The potential beneficial effects of red wine consumption on the development of atherosclerotic disease have been previously suggested in the literature . Vascular smooth muscle cell (SMC) proliferation is an important component of atherogenesis . Inhibition of vascular SMC proliferation may have a beneficial effect in retarding the development of atherosclerotic disease . The goal of this study was to determine the effect of red wine, red wine polyphenol extract, and resveratrol, a polyphenol commonly found in red wine, on the proliferation of vascular SMC in culture . METHODS: Bovine aortic SMCs were used for all experiments . SMCs were treated with growth media supplemented with dealcoholized red wine, red wine polyphenol extract, or resveratrol at various concentrations for as long as 48 hours . SMC proliferation was assessed with (3)H-thymidine DNA incorporation assay . SMC viability was assessed with trypan blue exclusion studies and a colorimetric lactic dehydrogenase cytotoxicity assay . RESULTS: Our results show that red wine and red wine polyphenol extract inhibit SMC proliferation in a dose-dependent fashion . Resveratrol also inhibits vascular SMC proliferation . SMC viability studies show that this inhibition of SMC proliferation is not the result of a cytotoxic effect . CONCLUSION: Our findings show that red wine and red wine polyphenols have an inhibitory effect on the proliferation of vascular SMCs in culture . These results suggest that the observed beneficial effects of red wine may be the result, in part, of the inhibition of vascular SMC proliferation . Furthermore, the antiproliferative properties of red wine may be caused by its component polyphenols.

Br J Plast Surg, 2002 Apr, 55(3), 219 - 27
The co-application of sprayed cultured autologous keratinocytes and autologous fibrin sealant in a porcine wound model; Grant I et al.; We have explored the potential for cultured autologous keratinocytes to form an epidermis when delivered as a spray intermixed with autologous fibrin sealant . Twelve full-thickness wounds in Large White pigs (six wounds in each of two pigs) were isolated from the surrounding skin by 4 cm diameter polytetrafluoroethylene chambers, and grafted with Integra artificial skin (Ethicon) . Autologous fibrin sealant was produced 10 days later, using an automated processor unit (Vivostat System, ConvaTec, Bristol Myers Squibb), from 120 ml of autologous citrated blood taken 30 min before keratinocyte application . Nine wounds were sprayed, using a Vivostat System automated applicator unit, with a mixture of the sealant preparation and freshly trypsinised cultured autologous keratinocytes in growth medium, at a density of 1-3 x 10(5) cm(-2) . Three control wounds were sprayed with the same mixture without cells . The sealant-cell mixture polymerised and adhered to the wound surface immediately . Histological analysis of biopsies taken following sealant-cell application showed that isolated spherical keratinocytes were distributed throughout the sealant at between 3.1 x 10(4) cm(-2) and 7.6 x 10(4) cm(-2) . After 4 days discreet colonies of keratinocytes were observed on the wound bed . At 14 days a multi-layered undulating epidermis was formed, punctuated by sporadic epidermal cysts; the mean area of epithelium was 50.1% (s.d . = 19.7%, n = 9) . There was no epithelium in the controls (s.d . = 0, n = 3) . The difference was statistically significant (P=0.016) . This study suggests that co-sprayed cultured keratinocytes and autologous fibrin sealant may be an effective means of delivering epithelial cells to assist wound healing .

Mycopathologia, 2002, 154(1), 51 - 4
Studies on physiology, zoospore morphology and entomopathogenic potential of the aquatic oomycete: Lagenidium giganteum; Sur B et al.; The oomycete Lagenidium giganteum, a facultative parasite of mosquito larvae requires exogenous sterols for the genesis of zoospores when grown saprobically . Growth media prepared from oil rich materials such as soy or sunflower seed were very effective inducers of virulent zoospores . The external morphology of zoospores of L . giganteum was studied with the aid of philips scanning electron microscope 515 . Zoospores were ovoid, bluntly pointed with the groove parallel to the long axis and 0.7 x 1.4 microm . Insect cell walls are known to contain lipid and chitin . L . giganteum was tested for chitinase activity and found to possess 0.76 +/- SD0.14 chitinase activity . Use of oil seed for growth of the organism confirms phospholipase activity . Phospholipase production was studied further by egg-yolk plate method . Presence of these two key enzymes that can initiate host cell damage suggests the entomopathogenic potential of L . giganteum . L . giganteum failed to grow at 37 degrees C limiting its effectiveness in warmer climates . Introduction of this organism to variety of habitats with various mosquito species will demonstrate the efficacy of the organism as a bioinsecticide.

Int J Hyg Environ Health, 2002 Apr, 205(3), 235 - 8
Evaluation of serum-free culture conditions for primary human nasal epithelial cells; Mattinger C et al.; Defined culture conditions are essential for the interpretation of effects caused by volatile substances on human nasal epithelial cells (HUNEC) cultured in vitro . Conventionally, serum-containing media are used . However, the results of these experiments are of restricted value as serum contains many unknown and undefined substances . Not all serum-free media on the market are suitable for culturing primary HUNEC . Therefore, serum-free defined cell culture media were compared to evaluate optimal conditions for HUNEC and their cell lines . HUNEC were generated by trypsin digestion of mucosal tissue of the inferior turbinate . Cells were cultured on uncoated polystyrene dishes adding pre-warmed medium . Viability was controlled by trypan blue dye exclusion; colony forming units and cell morphology were controlled microscopically . The expression of different cytokeratins was studied immunocytochemically . Dulbecco's modified Eagle's medium was not suitable to grow HUNEC and passage them . HUNEC cultured in bronchial epithelial growth medium presented a more homogeneous cell morphology compared to other media and had a doubling time of 1.2 days . The maximum number of cell passages was 11 with bronchial epithelial growth medium.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(5), 537 - 541
An Expression Release System for Heterogeneous Proteins in E.coli Mediated by the kil Gene; Tong Q et al.; An expression release system for heterogeneous proteins in Escherichia coli mediated by the colicin release gene(kil gene) was constructed . The system is based on the ability of the Kil protein to release periplasmic proteins into the growth medium . beta-lactamase, an E.coli periplasmic protein, and prolyl endopeptidase (PEP), a periplasmic protein of Aeromonas punctata subsp . punctata were used as report proteins . Commonly, these two proteins are seldom released into the growth medium . The results indicated that the released amounts of the beta-lactamase and of the prolyl endopeptidase were nearly 5 fold and 4 fold than control, respectively.

J Biol Chem, 2002 Aug 9, 277(32), 28981 - 6 Epub 2002 May 30.
A two-component signal transduction pathway regulates manganese homeostasis in Synechocystis 6803, a photosynthetic organism; Ogawa T et al.; Elemental manganese is essential for the production of molecular oxygen by cyanobacteria, plants, and algae . In the cyanobacterium Synechocystis sp . PCC 6803, transcription of the mntCAB operon, encoding a high affinity Mn transporter, occurs under Mn starvation (nm Mn) conditions but not in Mn-sufficient (microm Mn) growth medium . Using a strain in which the promoter of this operon directs the transcription of the luxAB reporter genes, we determined that inactivation of the slr0640 gene, which encodes a histidine kinase sensor protein component of a two-component signal transduction system, resulted in constitutive high levels of lux luminescence . Systematic targeted inactivation mutagenesis also identified slr1837 as the gene encoding the corresponding response regulator protein . We have named these two genes manS (manganese-sensor) and manR (manganese-regulator), respectively . A polyhistidine-tagged form of the ManS protein was localized in the Synechocystis 6803 cell membrane . Directed replacement of the conserved catalytic His-205 residue of this protein by Leu abolished its activity, although the mutated protein was present in cyanobacterial membrane . This mutant also showed suboptimal rates of Mn uptake under either Mn-starved or Mn-sufficient growth condition . These data suggest that the ManS/ManR two-component system plays a central role in the homeostasis of manganese in Synechocystis 6803 cells.

Gene, 2002 Feb 20, 285(1-2), 49 - 57
Regulated expression of proteins in yeast using the MAL61-62 promoter and a mating scheme to increase dynamic range; Finley RL Jr et al.; The ability to express heterologous genes in yeast has become indispensable for many biological research techniques . Expression systems that can be regulated are particularly useful because they allow an experimenter to control the timing and levels of gene expression . Despite their many advantages, however, surprisingly few conditional expression systems are available for yeast . Moreover, of those that have been described, many are not ideal either because they have high background expression levels, low induced levels, or because they require restrictive growth conditions . Here we describe a conditional expression system that takes advantage of the yeast MAL62 promoter (MAL62p), which can be controlled by adding maltose or glucose to the growth medium to induce or repress transcription, respectively . In addition, we use a mating scheme to dramatically increase the dynamic range of expression levels possible . We show that MAL62p background activity can be effectively eliminated by maintaining expression constructs in a mal(-) yeast strain . High-level expression can be induced in diploids formed by mating the mal(-) strain with a MAL(+) strain . A similar mating scheme may be useful for other conditional expression systems as well . Among other uses, this approach should aid high throughput yeast two-hybrid assays, which rely on maintaining large libraries of expression strains, which are eventually mated to conduct assays for protein interactions . We demonstrate a two-hybrid system in which MAL62p is used in conjunction with the yeast GAL1 promoter to independently regulate expression of both hybrid proteins, and to allow detection of interactions involving toxic proteins.

Microb Ecol, 2002 Apr, 43(3), 353 - 66 Epub 2002 Mar 05.
Can phosphorus limitation inhibit dissolved organic carbon consumption in aquatic microbial food webs? A study of three food web structures in microcosms; Olsen LM et al.; Microcosms with three different food web structures and phosphorus (P) limited growth medium were used to study the interactions between P and organic carbon (C) fractions in pelagic food webs . The cultures were run with low dilution to allow the biological processes to determine the outcome . A double isotope technique was used to follow the C and P compartments . In all systems the primary production was P limited . The measured P:C ratios and the observed accumulation of degradable dissolved organic carbon (DOC) indicated that the growth of heterotrophic bacteria was also P limited . The presence of neither algal grazers nor flagellates feeding on bacteria altered the limitation pattern . A net loss of P from the bacterial fraction was observed after the bloom . Different strategies for nutrient aquisition and growth are proposed as mechanisms enabling simultaneous P limitation of algae and bacteria, and a concomitant accumulation of degradable DOC . The ability of the algae to grow with low P:C ratio keeps the regeneration of P through grazers low enough to cause sustained P limitation of both algae and bacteria . The grazers were important producers of DOC when present . This implies that the usual assumption of carbon limited bacterial growth may lead to wrong conclusions regarding the dynamics of plankton communities and the DOC pool.

Life Sci Space Res, 1965, 3, 155 - 64
A technique and some results of meteorite microbiological investigations; Abyzov SS et al.; The possibility of the introduction of living organisms on earth with meteorites is a question of interest for scientists . It is quite possible that bacterial spores can remain in an anabiotic state for a long time, thus there is no reason to refuse the possibility of microorganisms present in meteorite . A special box was constructed in order to take samples from meteorites under sterile conditions because penetration of a soil microflora inside meteorites upon their fall on the earth surface is quite possible . The sterilized rock pieces were put into soil, samples were removed at intervals and subjected to microbiological assay . In all cases different soil microorganisms were found in the central parts of the samples tested . In another study a canal was drilled in the sterile rock and meteorite samples and a bacterial culture was poured into this canal, the sample being submerged into a liquid growth medium . Cell penetration through the rock took place and bacterial growth was found in the liquid growth medium . Therefore meteorites extracted from soil not at once after their fall are hardly suitable for microbiological assay.

Anal Chem, 2002 May 1, 74(9), 2064 - 71
HPLC/ESI-quadrupole ion trap mass spectrometry for characterization and direct quantification of amphoteric and nonionic surfactants in aqueous samples; Levine LH et al.; An amphoteric (cocamidopropylbetaine, CAPB) and a nonionic (alcohol polyethoxylate, AE) surfactant were characterized by electrospray ionization quadrupole ion trap mass spectrometry (ESI-MS) as to their homologue distribution and ionization/fragmentation chemistry . Quantitative methods involving reversed-phase gradient HPLC and (+)ESI-MSn were developed to directly determine these surfactants in hydroponic plant growth medium that received simulated graywater . The predominant homologues, 12 C alkyl CAPB and 9 EO AE, were monitored to represent the total amount of the respective surfactants . The methods demonstrated dynamic linear ranges of 0.5-250 ng (r2 > 0.996) for CAPB and 8-560 ng (r2 > 0.998) for AE homologue mixture, corresponding to minimum quantification limits of 25 ppb CAPB and 0.4 ppm AE with 20-microL injections . This translated into an even lower limit for individual components due to the polydispersive nature of the surfactants . The procedure was successfully employed for the assessment of CAPB and AE biodegradation in a hydroponic plant growth system used as a graywater bioreactor.

Trans ASAE, 2001 Jul-Aug, 44(4), 989 - 96
Design and development of an automated and non-contact sensing system for continuous monitoring of plant health and growth; Kacira M et al.; An automated system was designed and built to continuously monitor plant health and growth in a controlled environment using a distributed system approach for operational control and data collection . The computer-controlled system consisted of a motorized turntable to present the plants to the stationary sensors and reduce microclimate variability among the plants . Major sensing capabilities of the system included machine vision, infrared thermometry, time domain reflectometry, and micro-lysimeters . The system also maintained precise growth-medium moisture levels through a computer-controlled drip irrigation system . The system was capable of collecting required data continuously to monitor and to evaluate the plant health and growth.

Appl Biochem Biotechnol, 2002 Spring, 98-100, 833 - 40
Cadmium recovery by a sulfate-reducing magnetotactic bacterium, Desulfovibrio magneticus RS-1, using magnetic separation; Arakaki A et al.; Cadmium recovery by a sulfate-reducing magnetotactic bacterium, Desulfovibrio magneticus strain RS-1, was investigated . D . magneticus precipitated >95% of cadmium at an initial concentration of 1.3 ppm in the growth medium . Electron microscopic analysis revealed that D . magneticus formed electron-dense particles on its surface when cultivated in the presence of cadmium ions (Cd2+) . Sulfide was also found in the precipitate, and the composition ratio of sulfide/cadmium was 0.7 . Sixty percent of viable RS-1 cells was recovered by a simple magnetic separation revealing the removal of 58% cadmium from the culture medium.

Appl Biochem Biotechnol, 2002 Spring, 98-100, 243 - 55
Presence and physiologic regulation of alcohol oxidase activity in an indigenous fungus isolated from petroleum-contaminated soils; Alvarado-Caudillo Y et al.; A soluble alcohol oxidase (AO) activity was detected in the mycelium of a filamentous fungus strain named YR-1, isolated from petroleum-contaminated soils . AO activity from aerobically grown mycelium was detected in growth media containing the hydrocarbons decane or hexadecane; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde . Zymogram analysis conducted with purified fractions from aerobic mycelium of YR-1 strain extracts indicated the existence of two AO enzymes (AO-1 and AO-2) . Purified samples of both enzymes analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis indicated the presence of three protein bands with molecular sizes 20,38, and 46 kDa that could be part of the native enzyme . In samples of both enzymes, the 46-kDa protein gave a positive reaction in immunodetection experiments with antibodies directed against AO from Hansenula polymorpha . The purified AO-2 enzyme oxidized different alcohols, although higher activity was displayed with hexadecanol . Km values obtained for methanol and hexa-decanol indicated a higher affinity for the latter . Analysis of the aminoter-minal sequence of the 46-kDa protein of AO-2 enzyme indicated significant similarity to enzymes involved in the metabolism of biphenyl polychloride compounds.

Appl Microbiol Biotechnol, 2002 May, 58(6), 743 - 50 Epub 2002 Mar 20.
Production of canthaxanthin by Haloferax alexandrinus under non-aseptic conditions and a simple, rapid method for its extraction; Asker D et al.; The production of carotenoids from Haloferax alexandrinus strain TM(T) was investigated at various concentrations of NaCl (10-25%) in culture media under non-aseptic conditions . PCR and dot blot hybridization assays were employed to monitor the growth of Hfx . alexandrinus in the culture under aseptic and non-aseptic conditions . The amplified PCR products of 16S rDNA from Hfx . alexandrinus grown under aseptic conditions were used as specific probes, which bound with amplified PCR products of 16S rDNA dots from both aseptic and non-aseptic conditions (20-25% NaCl) . The results indicated that contamination of the culture was precluded at high NaCl concentrations (20-25%) . Therefore, it is not necessary to perform asepsis during the biotechnological processes of carotenoid production by Hfx . alexandrinus . A 1-l-scale cultivation of the cells in flask cultures under non-aseptic conditions produced 3.12+/-0.5 g dry weight, 6.34+/-2.5 mg total carotenoids and 2,156.67+/-0.1 microg canthaxanthin . Further experiments in a batch fermenter, under non-aseptic conditions, also demonstrated increases in the biomass concentration and carotenoid production . When grown in a standard growth medium at 25% NaCl, the cells of Hfx . alexandrinus lysed spontaneously in fresh water and hence carotenoids could be extracted directly from the cells without any mechanical disintegration . These results demonstrate the feasibility and simplicity of commercial production of carotenoids using Hfx . alexandrinus.

Infect Immun, 2002 Jun, 70(6), 2846 - 52
NikR mediates nickel-responsive transcriptional induction of urease expression in Helicobacter pylori; van Vliet AH et al.; The important human pathogen Helicobacter pylori requires the abundant expression and activity of its urease enzyme for colonization of the gastric mucosa . The transcription, expression, and activity of H . pylori urease were previously demonstrated to be induced by nickel supplementation of growth media . Here it is demonstrated that the HP1338 protein, an ortholog of the Escherichia coli nickel regulatory protein NikR, mediates nickel-responsive induction of urease expression in H . pylori . Mutation of the HP1338 gene (nikR) of H . pylori strain 26695 resulted in significant growth inhibition of the nikR mutant in the presence of supplementation with NiCl(2) at > or =100 microM, whereas the wild-type strain tolerated more than 10-fold-higher levels of NiCl(2) . Mutation of nikR did not affect urease subunit expression or urease enzyme activity in unsupplemented growth media . However, the nickel-induced increase in urease subunit expression and urease enzyme activity observed in wild-type H . pylori was absent in the H . pylori nikR mutant . A similar lack of nickel responsiveness was observed upon removal of a 19-bp palindromic sequence in the ureA promoter, as demonstrated by using a genomic ureA::lacZ reporter gene fusion . In conclusion, the H . pylori NikR protein and a 19-bp operator sequence in the ureA promoter are both essential for nickel-responsive induction of urease expression in H . pylori.

Plant J, 2002 Apr, 30(2), 203 - 12
Synchronous Arabidopsis suspension cultures for analysis of cell-cycle gene activity; Menges M et al.; Synchronized suspension cultures are powerful tools in plant cell-cycle studies . However, few Arabidopsis cell cultures are available, and synchrony extending over several sequential phases of the cell cycle has not been reported . Here we describe the first useful synchrony in Arabidopsis, achieved by selecting the rapidly dividing Arabidopsis cell suspensions MM1 and MM2d . Synchrony may be achieved either by removing and re-supplying sucrose to the growth media or by applying an aphidicolin block/release . Synchronization with aphidicolin produced up to 80% S-phase cells and up to 92% G2 cells, together with clear separation of different cell-cycle phases . These synchronization procedures can be used for analysis of gene expression and protein activity . We show that representatives of three CDK gene classes of Arabidopsis (CDKA, CDKB1 and CDKB2) show differential expression timing, and that three CDK inhibitor genes show strikingly different expression patterns during cell-cycle re-entry . We propose that ICK2 (KRP2) may have a specific role in this process.

Br J Biomed Sci, 2002, 59(1), 11 - 4
Image analysis of Transwell assays in the assessment of invasion by malignant cell lines; Connolly L et al.; This study aims to determine if layering of extracellular matrix (ECM) can achieve a physiological basement membrane thickness of 8 microm and to assess the use of paraffin wax-embedded Transwell plates coupled with digital image analysis as a means of determining invasion by malignant cell lines . Layers of Matrigel, a sarcoma-derived ECM was built to a concentration of 7.4 microg/mm2 in the upper chamber of a Transwell plate invasion assay . Two cell lines from extrahepatic bile duct adenocarcinoma were tested in serum-free growth medium . Conditioned medium was added to the lower chamber to act as a chemoattractant . Following attachment, cells were incubated for 48 h and the Matrigel-coated insert cut from its holder and fixed in 10% unbuffered formalin saline . Each insert was bisected and processed to paraffin wax . Serial levels were stained by haematoxylin and eosin . A Kontron image analysis system was used to measure the mean thickness of Matrigel for each cell line and the degree of invasion was assessed by measuring the depth to which cells had degraded the Matrigel . A mean thickness of 8 microm was achieved using 5.0 microg/mm2 for the OCUCh-LM1 cell line and 7.4 microg/mm2 for the SKChA-1 cell line . No significant difference was seen in the ability of either cell line to degrade Matrigel . Immunocytochemistry for laminin and cytokeratin helped to identify ECM components and cells, respectively . In conclusion, digital image analysis of paraffin wax-embedded inserts can be used to determine the invasive capacity of various cell lines; immunocytochemistry may help to identify ECM components and cells; and the assay used to assess different cell lines and their ability to degrade Matrigel.

Biosci Biotechnol Biochem, 2002 Feb, 66(2), 344 - 50
Production of brain-derived neurotrophic factor in Escherichia coli by coexpression of Dsb proteins; Hoshino K et al.; When brain-derived neurotrophic factor (BDNF) is produced in the Escherichia coli periplasm, insoluble BDNF proteins with low biological activity and having mismatched disulfide linkages are formed . The coexpression of cysteine oxidoreductases (DsbA and DsbC) and membrane-bound enzymes (DsbB and DsbD), which play an important role in the formation of disulfide bonds in the periplasm, was investigated to improve the production of soluble and biologically active BDNF . The expression levels of Dsb proteins changed when the growth medium and the Dsb expression plasmids were changed, and the production rate of soluble BDNF was almost proportional to the expression level of DsbC protein with disulfide isomerase activity in the case of a low expression level of BDNF . The rate of soluble BDNF production with coexpression of DsbABCD was as high as 35% . These results show that coexpression of BDNF and Dsb proteins can effectively increase the production of soluble and biologically active BDNF.

Appl Biochem Biotechnol, 2002 Mar, 97(3), 147 - 63
Anaerobic reduction of a sulfonated azo dye, Congo Red, by sulfate-reducing bacteria; Diniz PE et al.; The capacity for anaerobic decolorization of a sulfonated azo dye, Congo Red, by a strain of a sulfate-reducing bacterium was evaluated . After optimizing the growth rate of the bacteria on a simple carbon source and terminal electron acceptor pair, lactate and sulfate, respectively, the effect of the dye concentration on their growth rate was analyzed . The decolorization rate was affected by the dye concentration in the growth medium . The azo-bond cleavage mechanism of reductive decolorization with the formation of benzidine was consistent with the results, as this metabolite was identified by high-performance liquid chromatography . Several fractions of the culture medium, including lysed cell extracts, were examined for the capacity to reduce the azo dye . This reduction capacity was found in the culture medium in which the cells had previously grown . The results showed that the mechanism of reductive decolorization of this sulfonated azo dye was extracellular and nonenzymatic, consistent with the production of sulfide anion by the microorganisms while growing on lactate and sulfate . The sulfide anions were the cause of the reduction leading to the disappearance of color in the medium . To increase the rate of decolorization, the presence of ferrous ion was also necessary together with the lactate and sulfate substrates.

J Ind Microbiol Biotechnol, 2002 Apr, 28(4), 207 - 12
Solubilization of low-rank coal by Trichoderma atroviride: evidence for the involvement of hydrolytic and oxidative enzymes by using 14C-labelled lignite; Holker U et al.; The deuteromycete Trichoderma atroviride is able to solubilize lignite in dependence on a given carbon source for growth . When cultivated on media containing glutamate, this mold excreted a set of different enzymes with hydrolytic activity . Addition of lignite to the growth media induced the synthesis of extracellular lignite-specific esterase activity but no evidence has been provided for its direct involvement in the process of lignite solubilization . Hence, the basic capability of T . atroviride enzymes to degrade a variety of ester and ether bonds at the surface or within the bulky lignite structure was tested using coal following its direct labelling with 14C-alkyl iodide . The participation of hydrolytic and oxidative enzymes in lignite degradation was assessed by measuring the release of 14C radioactivity from selectively alkylated carboxylic and phenolic OH groups . T . atroviride cleaved both carboxylic esters using esterases and the phenolic ether bonds by using oxidative enzymes, most likely laccases.

J Hematother Stem Cell Res, 2002 Apr, 11(2), 337 - 47
Comparison of phenotypic and functional dendritic cells derived from human umbilical cord blood and peripheral blood mononuclear cells; Joshi SS et al.; Dendritic cells (DC) are important accessory cells that are capable of initiating an immune response . Generation of functional DC has potential clinical use in treating diseases such as cancer . In this report, we have demonstrated the generation of functional DC from mononuclear cells isolated from human umbilical cord blood cells (UCBC) and peripheral blood cells (PBC) using a defined medium Prime Complete Growth Medium (PCGM) (GenePrime LLC, Gaithersburg, MD) . DC generated using PCGM showed the typical phenotype of DC as determined by flow cytometry and electron microscopy . Further analysis of the DC using confocal microscopy showed localization of the antigen and major histocompatibility complex (MHC) molecules in the cytoplasm 3-5 days following tumor antigen loading into DC . Subsequently, the tumor antigen-MHC complex was localized on the surface of DC . DC generated from UCBC or PBC also increased (p < 0.001) the allogeneic mixed lymphocyte reaction, confirming their immune accessory functions compared to a control mixed lymphocyte reaction (MLR) without DC added . Interestingly, DC generated using PCGM medium also significantly enhanced the hematopoietic colony (CFU-C)-forming ability . Furthermore, addition of 5% DC derived from cord blood loaded with tumor antigen also significantly (p < 0.001) increased peripheral and cord blood-derived antigen-specific cytotoxic T lymphocyte (CTL)-mediated killing of human leukemic cells (K562) and breast cancer cells (MDA-231) . Thus, these results show that functional DC generated from cord blood using a defined medium are a useful source of accessory cells for augmenting CTL-mediated cytotoxicity and have potential use in cellular therapy for human leukemia and breast cancer.

Hepatology, 2002 May, 35(5), 1237 - 46
Cellular response to conditional expression of hepatitis C virus core protein in Huh7 cultured human hepatoma cells; Li K et al.; Data suggesting that the hepatitis C virus (HCV) core protein influences normal cellular processes remain controversial . To determine the effects of core on cellular gene expression in hepatocytes, we developed a human hepatoma (Huh7)-derived cell line with tightly regulated core expression under the control of a tetracycline-regulated promoter . Cells expressing core did not have impaired proliferative abilities . Changes in gene expression profiles in response to core expression were determined using commercial oligonucleotide microarrays (Affymetrix GeneChip) . Significant increases were observed in the abundance of mRNA-encoding members of the metallothionein (MT) family, as well as nicotinamide N-methyltransferase (NNMT) and glutathione peroxidase-like protein (GPLP) . These changes did not result from removal of tetracycline from growth media, and were confirmed in reverse-transcription polymerase chain reaction (RT-PCR) assays . They suggest that core protein expression leads to intracellular oxidative stress, and that vital cellular functions are, in turn, protected by up-regulation of cellular antioxidant defense mechanisms . In conclusion, these findings can explain many potentially conflicting prior observations concerning the effects of core on cellular physiology, and are of relevance to the role of core protein in the pathogenesis of HCV-related fibrosis and hepatocellular carcinoma.

Life Sci Space Res, 1967, 5, 250 - 60
Exobiology and the effect of physical factors on micro-organisms; Imshenetsky AA et al.; A study of the action of different physical factors on micro-organisms is necessary for a further development of exobiology . The action of temperature on crystalline preparations of catalase and peroxidase was studied by means of oscillographic polarography . A determination of the height of polarographic waves at the decrease of temperature from 20 degrees C to 0 degrees C has shown that structural elements of the peroxidase molecule connected with the enzymatic activity are more stable with the decrease of temperature cf . catalase . A relative resistance of the dehydrogenase activity in Az . vinelandii cells to high vacuum was found . Incubation of azotobacter cells under vacuum of 10(-9) mm Hg during 72 hr did not decrease the activity of alcohol and succinic dehydrogenase . Bac . cereus spores can be preserved from bactericidal UV action by thin films of chrome . The thickness of chrome film being 200-670 angstroms, spores are killed by a dose of 7.8 x 10(7) erg/cm2 at 253.7 microns wave length . Spores covered by chrome film thicker than 800 angstroms remain alive after this treatment . Investigations carried out with an 'Artificial Mars' camera led to the following results . The growth of Bac . megaterium on liquid growth media in this camera ceases as a result of UV rays killing all cells after 3 weeks . Untreated bacteria grow in the camera for a long time . Spore-forming bacteria isolated from the sand of the Kara-Kum Desert grow in ground limonite (with the addition of 2% garden soil) having maximum hygroscopic humidity (3.8%) . Freezing and thawing (from -60 degrees C to +25 degrees C) corresponding to day temperature deviations on Mars, low pressure (P=10 mm Hg) and the composition of the atmosphere (CO2-50%, N2-40%, Ar-10%) do not influence the growth of xerophylic bacteria under study . Humidity is the main factor limiting the growth of micro-organisms under 'Artificial Mars' conditions . According to the further development of the microbiological meteorite analysis methods, samples of rocks and stone meteorites were sterilized, incubated in the desert or on a snow surface in the Arctic and after different times (from 100 days to 7 months), investigated . In all cases, microbes were found only on the sample surfaces, whereas 1 cm from the surface and in the central parts micro-organism were completely absent . Hence, microbiological analysis of central parts of meteorites fallen in the Arctic or during dry periods of the year in the desert can give reliable results.

Life Sci Space Res, 1967, 5, 187 - 203
Ecological patterns of micro-organisms in desert soils; Opfell JB et al.; The Atacama Desert's hardy aerobic microbial populations have been found to occupy ecological niches substantially different from those occupied by aerobic micro-organisms in the California and Valley of 10 000 Smokes deserts . Two specialized groups of microflora, the actinomycetes and micro-aerophiles, appear to be abundant in some portions of the Atacama Desert, though many of the bacterial and mold types and species common in the California deserts are absent . The factors defining the ecological niches and relationships of microbial populations in these soils are largely unknown . In several respects, moreover, micro-environments in these soils are similar to some of those expected on Mars . For the purpose of establishing the engineering constraints on the design of life detection instruments and their sampling and processing components, the nature and diversity of species of micro-organisms prevalent in the soils of extremely dry climates of the earth have not been adequately investigated . Useful ecological information about these soils will include an estimate of the likely lower limit in numbers or mass of viable aggregates in the autochthonous populations and the survivability of these populations under mechanical processing and isolated storage . Knowledge of the artificial niches, growth media for example, which members of these populations accept, will be useful in designing life detection instruments themselves . The range of practical designs for soil sampling devices will be determined by the nature of the particles and surfaces, and their depths, on which various physiological groups or largest populations of micro-organisms reside in the extremely dry deserts of the earth . The results of several exploratory experiments are used to provide some understanding of the environment and the ecological patterns of micro-organisms in soils of the earth's driest deserts.

Sheng Li Xue Bao, 2000 Feb, 52(1), 34 - 8
{Cardiocyte hypertrophy induced by cultured neonatal rat ventricular fibroblast conditioned growth medium}; Gong SZ et al.; The present work demonstrated that cultured neonatal rat ventricular fibroblast conditioned growth medium (FCGM) could significantly increase cell surface area and protein content and promote (3)H -Leucine incorporation on neonatal rat cardiomyocyte . The above effect was strongest on the third day, and was dose-dependent . BQ(123), an ET-A receptor antagonist, significantly blocked the effect, while CV11974, an Ang II I-type receptor antagonist, and regitin, an alpha-adrenergic receptor antagonist, did not . These results suggest that there are some substances promoting hypertrophy of cardiomyocytes in FCGM, which may be ET-1 . The FCGM-induced increases in cardiomyocyte protein synthesis and cell surface area were inhibited partially by pertusis toxin (PTX) and PKC inhibitor staurosporine (ST), suggesting that the hypertrophic effect is related with PTX sensitive G protein and PKC.

Biofizika, 2002 Mar-Apr, 47(2), 304 - 8
{Stimulation and inhibition of Escherichia coli cell growth during cultivation in the catholyte and anolyte of culture medium}; Muroshnikov AI; The effect of pretreatment of growth medium M-9 with direct electric current in the cathode and the anode compartments of a diaphragm electrolyzer on the growth of Escherichia coli cells was studied . The cells were cultured separately in the catholyte and the anolyte of the growth medium . The cell growth was registered as a change in optical density of the culture suspension by the method of turbidimetry . It was found that cells grown in the catholyte at a temperature of 37 degrees C yielded a 20-30% increase in amount as compared to the control . No cell growth was observed in the anolyte, and a part of the initial cells were lysed . Possible mechanisms of stimulation and inhibition of cell growth and the reasons of discrepancies in the earlier published data are discussed.

J Cell Biochem, 2002, 85(4), 775 - 84
PKC-MEK-MAPK-dependent signal transduction pathway mediates the stimulation of lysyl oxidase expression by serum and PDGF in rat aortic smooth muscle cells; Smith-Mungo L et al.; Lysyl oxidase (LO) plays a critical role in the stabilization and insolubilization of fibrous structural proteins of the extracellular matrix and has been implicated in the suppression of Ras-induced tumorigenesis . Several prior reports demonstrate that the expression of this catalyst is strongly influenced by a variety of effectors of cell function and is responsive to the growth state of fibrogenic cells . Using specific inhibitors of components of signal transduction pathways, the present study reveals that a PKC-MEK-MAPK-dependent pathway is critical to the enhanced expression of the LO gene in response to variations in the levels of the serum component of the growth medium and in response to platelet-derived growth factor (PDGF) . PDGF is shown to be the major component of fetal bovine serum, which stimulates the activity of a LO promoter construct .

Appl Biochem Biotechnol, 2001 Spring, 91-93, 185 - 93
Utilization of cyanobacteria in photobioreactors for orthophosphate removal from water; Gaffney AM et al.; The effectiveness of photosynthetic free-living and polyurethane foam (PU) immobilized Anabaena variabilis cells for removal of orthophosphate (P) from water in batch cultures and in a photobioreactor was studied . Immobilization in PU foams was found to have a positive effect on P uptake by cyanobacteria in batch cultures . The efficiency of P uptake by immobilized cells was higher than by free-living cells . A laboratory scale photobioreactor was constructed for removal of P from water by the immobilized cyanobacteria . The photobioreactor was designed so that the growth medium (water) from a reservoir was pumped through a photobioreactor column with immobilized cyanobacteria and back to the reservoir . This created a closed system in which it was possible to measure P uptake . No leakage of cells into the photobioreactor medium reservoir was observed during the operation . The immobilized cells incorporated into a photobioreactor column removed P continuously for about 15 d . No measurable uptake was demonstrated after this period . Orthophosphate uptake efficiency of 88-92% was achieved by the photobioreactor.

Eur J Biochem, 2002 Apr, 269(7), 1835 - 43
Residues near the N-terminus of protein B control autocatalytic proteolysis and the activity of soluble methane mono-oxygenase; Callaghan AJ et al.; Soluble methane mono-oxygenase (sMMO) of Methylococcus capsulatus (Bath) catalyses the O2-dependent and NAD(P)H-dependent oxygenation of methane and numerous other substrates . During purification, the sMMO enzyme complex, which comprises three components and has a molecular mass in excess of 300 kDa, becomes inactivated because of cleavage of just 12 amino acids from the N-terminus of protein B, which is the smallest component of sMMO and the only one without prosthetic groups . Here we have shown that cleavage of protein B, to form the inactive truncated protein B', continued to occur when intact protein B was repeatedly separated from protein B' and all detectable contaminants, giving compelling evidence that the protein was cleaved autocatalytically . The rate of autocatalytic cleavage decreased when the residues flanking the cleavage site were mutated, but the position of cleavage was unaltered . Analysis of a series of incremental truncates showed that residue(s) essential for the activity of sMMO, and important in determining the stability of protein B, lay in the region Ser4-Tyr7 . Protein B was shown to possess intrinsic nucleophilic activity, which we propose initiates the cleavage reaction via a novel mechanism . Proteins B and B' were detected in approximately equal amounts in the cell, showing that truncation of protein B is biologically relevant . Increasing the growth-medium copper concentration, which inactivates sMMO, did not alter the extent of in vivo cleavage, therefore the conditions under which cleavage of protein B may fulfil its proposed role as a regulator of sMMO remain to be identified.

Mol Cell Biochem, 2002 Feb, 231(1-2), 179 - 85
Influence of zinc ions on protein secretion in a heavy metal tolerant strain of the ericoid mycorrhizal fungus Oidiodendron maius; Martino E et al.; A heavy metal tolerant strain of the ericoid mycorrhizal species Oidiodendron maius, isolated from soil heavily contaminated with zinc, was previously shown to tolerate high concentrations of zinc and cadmium ions in the growth medium . We have investigated some of the specific molecular responses of this fungal strain to the presence of increasing concentrations of zinc ions in the growth medium . In particular, we show that zinc ions induce a general change in the array of secreted proteins, with a shift towards the production of more basic, low molecular weight polypeptides . Some of these proteins were microsequenced and identified through homology search in databases . Among them are hydrolytic enzymes (nuclease, proteinase, lysozyme) and two superoxide dismutase isoforms . The latter are antioxidant enzymes known to play a role in heavy metal response in plants, animals and microorganisms.

FEBS Lett, 1969 Nov 29, 5(4), 253 - 256
Evolution de la composition lipidique de Nocardia asteroides au cours de la croissance; Bordet C et al.; By addition of 1-(14)C-sodium acedate to the growth medium of Nocardia asteroides, it can be shown that the lipid content increases during the exponential phase, but does not vary during the stationary phase of the growth . Nocardic acid biosynthesis from the medium molecular weight fatty acids occurs chiefly during te stationary phase . As these compounds are localised in the cell walls, it becomes evident that the lipid envelope of the walls is still increasing when the cell growth and division have stopped.

Mol Ther, 2002 Apr, 5(4), 473 - 8
Highly efficient retroviral gene transfer based on centrifugation-mediated vector preloading of tissue culture vessels; Kuhlcke K et al.; Efficient retroviral gene transfer into primary cells is a prerequisite for various gene therapeutic strategies . We have developed a transduction protocol based on the preloading of tissue culture vessels with retroviral particles by low-speed (1000g) centrifugation . We show that vector-preloaded tissue culture vessels allow highly efficient gene transfer into various target cells . We obtained transduction rates of up to 85% for primary T lymphocytes after just a single round of transduction . Under clinically relevant conditions using a vector developed for suicide gene therapy and produced under good manufacturing practice (GMP) conditions, the described method allowed generation of large numbers (>2x10(9)) of gene-modified T cells . The preloading concept ensures transduction of target cells in their optimal growth medium regardless of the medium used for vector production . This facilitated highly efficient gene transfer into quite different target cells such as CD34(+) and AC133(+) bone marrow progenitor as well as mesenchymal stem cells . The presented method combines high gene-transfer rates with a great potential for standardization in accordance with GMP guidelines and is consequently well suited for both research and clinical applications . (c)2002 Elsevier Science (USA).

Mol Cell Biol, 2002 May, 22(9), 2893 - 905
The MyoD-inducible p204 protein overcomes the inhibition of myoblast differentiation by Id proteins; Liu CJ et al.; The murine p204 protein level is highest in heart and skeletal muscle . During the fusion of cultured myoblasts to myotubes, the p204 level increases due to transcription dependent on the muscle-specific MyoD protein, and p204 is phosphorylated and translocated from the nucleus to the cytoplasm . p204 overexpression accelerates myoblast fusion in differentiation medium and triggers this process even in growth medium . Here we report that p204 is required for the differentiation of C2C12 myoblasts . We propose that it enables the differentiation, at least in part, by overcoming the inhibition of the activities of the MyoD and E47 proteins by the Id proteins: Id1, Id2, and Id3 . These are known to inhibit skeletal muscle differentiation by binding and blocking the activity of MyoD, E12/E47, and other myogenic basic helix-loop-helix (bHLH) proteins . Our hypothesis is based on the following findings . (i) A decrease in the p204 level in C2C12 myoblasts by antisense RNA (a) increased the level of the Id2; (b) inhibited the MyoD-, E12/E47-, and other bHLH protein-dependent accumulation of the muscle-specific myosin heavy-chain protein; and (c) inhibited the fusion of myoblasts to myotubes in differentiation medium . (ii) p204 bound to the Id proteins in vitro and in vivo . (iii) In the binding of p204 to Id2, the b segment of p204 and the HLH segment of Id2 were involved . (iv) Addition of p204 overcame the inhibition by the Id proteins of the binding of MyoD and E47 to DNA in vitro . (v) Overexpression of p204 in myoblasts (a) decreased the level of the Id proteins, even in a culture in growth medium, and (b) overcame the inhibition by the Id proteins of MyoD- and E47 dependent transcription and also overcame the inhibition by Id2 of the fusion of myoblasts to myotubes.

Lett Appl Microbiol, 2002, 34(4), 300 - 3
A liquid synchronized-growth culture assay for the identification of true positive and negative yeast three-hybrid transformants; Srivastava R et al.; AIMS: To develop a simple and easy-to-use assay for detection of truely positive transformants from a yeast three-hybrid assay . METHODS AND RESULTS: The yeast three-hybrid system is a new powerful system for studying RNA-protein interactions in vivo . There are, however, many reports from investigators about the difficulty in distinguishing a positive from a negative result due to the hard-to-detect differences between truely positive transformants and the negative ones . A liquid synchronous-growth culture approach has been described for all positive and negative transformants and their growth densities have been compared to each other at fixed intervals of incubation times . We have designed a simple yet effective procedure to assay for positive and negative RNA-protein interactions based on liquid culture analysis of synchronously growing yeast cells . Results obtained from this new procedure clearly show differences in positive and negative transformants after a 24-h incubation of synchronously growing transformants in liquid culture . CONCLUSIONS: The procedure mentioned in this report shows clearly the differences between positive and negative results from a three-hybrid system . SIGNIFICANCE AND IMPACT OF THE STUDY: The method proposed here is a clear advantage over existing methods based on measuring growth on restrictive growth medium plates by the naked eye . This method will have substantial usage with investigators using the yeast three-hybrid studies for RNA-protein interactions.

Cell Mol Biol (Noisy-le-grand), 2001, 47 Online Pub, OL7 - 14
The infrared spectroscopic Gram stain; Lang PL et al.; The attenuated total reflectance (ATR) spectra of bacterial colonies were obtained in situ, without their removal from the agar growth media using the infrared microscope . Principal components regression (PCR) analysis was used to obtain a measure of the differentiating potential of the ATR spectra obtained on two standard sets consisting of 31 and 44 species, respectively, grown on trypticase soy agar . A quantitative response value of +1 was assigned to Gram positive species and -1 was assigned to Gram negative species . In a cross validation experiment, the Gram stain property correlated with the factor loadings with a multiple correlation of 0.9502 and 0.9520 for each of the standard sets, respectively . Also, it was found that ATR spectra can be obtained from bacteria grown on blood agar and still have good differentiating capabilities for Gram stain . In addition, ATR spectra provide significantly better differentiating spectral information and predicting capabilities than specular reflectance spectra obtained in situ . Finally, the numerical value obtained for the Gram stain can be used as a measure of Gram variability . Results indicate that the Gram positive values shift to Gram negative values as the bacterial cell wall deteriorates with time.

Appl Microbiol Biotechnol, 2002 Mar, 58(3), 303 - 7 Epub 2002 Jan 10.
Production of gamma-linolenic acid by Cunninghamella echinulata cultivated on glucose and orange peel; Gema H et al.; A newly isolated strain of Cunninghamella echinulata grown on glucose produced significant quantities of biomass and cellular lipids in media with high C/N ratio . The oil yield from glucose consumed increased after nitrogen exhaustion in the growth medium, but gamma-linolenic acid (GLA) content in cellular oil systematically decreased during the lipid accumulation process . When lipid accumulation was completed, GLA concentration in the cellular lipids progressively increased . The highest GLA production (720 mg/l) was achieved in medium with a C/N ratio equal to 163 . C . echinulata was also able to grow on orange peel . The C/N ratio in the orange peel decreased from 50 to 26 during solid-state fermentation . Maximum oxygen uptake was observed during assimilation of reducing sugars, whereas a polygalacturonase activity was detected after reducing sugars had been exhausted . The maximum GLA production was 1.2-1.5 mg/g of fermented peel, calculated on a dry weight basis . After enrichment of the pulp with inorganic nitrogen and glucose, an increase in the production of oil and GLA was observed.

Biochem J, 2002 Apr 15, 363(Pt 2), 321 - 7
Copper supplementation restores cytochrome c oxidase activity in cultured cells from patients with SCO2 mutations; Salviati L et al.; Human SCO2 is a nuclear-encoded Cu-binding protein, presumed to be responsible for the insertion of Cu into the mitochondrial cytochrome c oxidase (COX) holoenzyme . Mutations in SCO2 are associated with cardioencephalomyopathy and COX deficiency . Studies in yeast and bacteria have shown that Cu supplementation can restore COX activity in cells harbouring mutations in genes involving Cu transport . Therefore we investigated whether Cu supplementation could restore COX activity in cultured cells from patients with SCO2 mutations . Our data demonstrate that the COX deficiency observed in fibroblasts, myoblasts and myotubes from patients with SCO2 mutations can be restored to almost normal levels by the addition of CuCl(2) to the growth medium.

Proc Natl Acad Sci U S A, 2002 Apr 2, 99(7), 4319 - 24
FKBP12-rapamycin-associated protein associates with mitochondria and senses osmotic stress via mitochondrial dysfunction; Desai BN et al.; FKBP12-rapamycin associated protein (FRAP, also known as mTOR or RAFT) is the founding member of the phosphatidylinositol kinase-related kinase family and functions as a sensor of physiological signals that regulate cell growth . Signals integrated by FRAP include nutrients, cAMP levels, and osmotic stress, and cellular processes affected by FRAP include transcription, translation, and autophagy . The mechanisms underlying the integration of such diverse signals by FRAP are largely unknown . Recently, FRAP has been reported to be regulated by mitochondrial dysfunction and depletion of ATP levels . Here we show that exposure of cells to hyperosmotic conditions (and to glucose-deficient growth medium) results in rapid and reversible dissipation of the mitochondrial proton gradient . These results suggest that the ability of FRAP to mediate osmotic stress response (and glucose deprivation response) is by means of an intermediate mitochondrial dysfunction . We also show that in addition to cytosolic FRAP a large portion of FRAP associates with the mitochondrial outer membrane . The results support the existence of a stress-sensing module consisting of mitochondria and mitochondrial outer membrane-associated FRAP . This module allows the cell to integrate a variety of stress signals that affect mitochondrial function and regulate a growth checkpoint involving p70 S6 kinase.

Proc Natl Acad Sci U S A, 2002 Apr 16, 99(8), 5632 - 7 Epub 2002 Apr 02.
Genetic analysis of the archaeon Methanosarcina barkeri Fusaro reveals a central role for Ech hydrogenase and ferredoxin in methanogenesis and carbon fixation; Meuer J et al.; Ech hydrogenase (Ech) from the methanogenic archaeon Methanosarcina barkeri catalyzes the reversible reduction of ferredoxin by H(2) and is a member of a distinct group of membrane-bound {NiFe} hydrogenases with sequence similarity to energy-conserving NADH:quinone oxidoreductase (complex I) . To elucidate the physiological role(s) of Ech a mutant lacking this enzyme was constructed . The mutant was unable to grow on methanol/H(2)/CO(2), H(2)/CO(2), or acetate as carbon and energy sources but showed wild-type growth rates with methanol as sole substrate . Addition of pyruvate to the growth medium restored growth on methanol/H(2)/CO(2) but not on H(2)/CO(2) or acetate . Results obtained from growth experiments, cell suspension experiments, and enzyme activity measurements in cell extracts provide compelling evidence for essential functions of Ech and a 2{4Fe-4S} ferredoxin in the metabolism of M . barkeri . The following conclusions were made . (i) In acetoclastic methanogenesis, Ech catalyzes H(2) formation from reduced ferredoxin, generated by the oxidation of the carbonyl group of acetate to CO(2) . (ii) Under autotrophic growth conditions, the enzyme catalyzes the energetically unfavorable reduction of ferredoxin by H(2), most probably driven by reversed electron transport, and the reduced ferredoxin thus generated functions as low potential electron donor for the synthesis of pyruvate in an anabolic pathway . (iii) Reduced ferredoxin in addition provides the reducing equivalents for the first step of methanogenesis from H(2)/CO(2), the reduction of CO(2) to formylmethanofuran . Thus, in vivo genetic analysis has led to the identification of the electron donor of this key initial step of methanogenesis.

Protoplasma, 2002 Feb, 219(1-2), 89 - 98
Arabinogalactan proteins, pollen tube growth, and the reversible effects of Yariv phenylglycoside; Mollet JC et al.; Arabinogalactan proteins (AGPs) are abundant complex macromolecules involved in both reproductive and vegetative plant growth . They are secreted at pollen tube tips in Lilium longiflorum . Here, we report the effect of the (beta-D-glucosyl)3 Yariv phenylglycoside, known to interact with AGPs, on pollen tube extension in several plant species . In Annona cherimola the Yariv reagent clearly inhibited pollen tube extension within 1-2 h of treatment, as demonstrated previously for L . longiflorum, but had no effect on Lycopersicon pimpinellifolium, Aquilegia eximia, and Nicotiana tabacum . With the monoclonal antibody JIM13 we also examined these same species for evidence that they secreted AGPs at their pollen tube tips . Only A . cherimola showed evidence of AGPs at the pollen tube tip as does lily . The Yariv reagent causes arrest of tube growth in both A . cherimola and lily, but its removal from the medium allows regeneration of new tip growth in both species . We show that the site of the new emerging tip in lily can be predicted by localization of AGP secretion . Labeling with JIM13 appeared on the flanks of the arrested tip 1 h after removal of the Yariv reagent from the growth medium . After 4 h, many of the Yariv reagent-treated pollen tubes had regenerated new pollen tubes with the tips brightly labeled by JIM13 and with a collar of AGPs left at the emergence site . During this recovery, esterified pectins colocalized with AGPs . Secretion at the site of the new tip may be important in the initial polarization event that occurs on the flanks of the arrested tube tip and results in a new pollen tube.

Anal Chem, 2002 Mar 15, 74(6), 1302 - 6
Bioimprinting of polymers and sol-gel phases . Selective detection of yeasts with imprinted polymers; Dickert FL et al.; Coated quartz crystal microbalances were modified with a surface-imprinting process using whole yeast cells . These molded polymer and sol-gel surfaces show honeycomb-like structures as shown by atomic force microscopy . Reinclusion of cells allows a selective on-line monitoring of these microorganism concentrations in water over 5 orders of magnitude . The sensitivity to cells holds up in growth media up to 21 g/L . Even cell fragments can be detected in flowing conditions . The highly robust polymers on the sensor devices are suitable for biotechnological applications.

Alcohol Clin Exp Res, 2002 Mar, 26(3), 295 - 302
Ethanol decreases the efficiency of phosphorylation of thymidine kinase in a human T-lymphocytic cell line; Prakash O et al.; BACKGROUND: Thymidine kinase (TK) and thymidylate kinase (TMPK) are the two rate-limiting enzymes in the cascade of activation of the anti-human immunodeficiency virus (HIV) drug 3'-azido-3'-deoxythymidine (AZT) to its active triphosphate form . We examined the effect of ethanol and a combination of ethanol and AZT on TK and TMPK activities in human Jurkat T cells . METHODS: Jurkat T cells were exposed to 0.2 and 0.5% (v/v) ethanol concentrations alone or in combination with 5 or 10 microM AZT for 48 hr in growth medium . TK and TMPK activities were determined by measuring the conversion of {3H} substrates (thymidine or AZT for TK and thymidine monophosphate for TMPK) to their respective monophosphate or diphosphate forms . The effect of ethanol on the transcriptional activity of TK was determined by reverse transcription-polymerase chain reaction and on the growth of Jurkat cells by {3H}-thymidine incorporation and cell-cycle analysis . RESULTS: Treatment of Jurkat cells with 0.2 and 0.5% ethanol concentrations resulted in 25 and 50% decreases (p < or = 0.05) in TK activity, respectively . No significant changes were observed in the TMPK activities . However, ethanol decreased the formation of thymidine diphosphate from thymidine in coupled TK/TMPK reactions, suggesting that decreases in TK activity could result in an overall decrease in the phosphorylation of AZT . The effect of ethanol on TK was independent of its transcript level . AZT in combination with ethanol decreased the inhibitory effect of ethanol on TK activity . However, it did not block the ethanol effect even at higher concentrations . Ethanol significantly decreased the proliferative capacity and cell cycle progression of the Jurkat cells . CONCLUSIONS: Our in vitro study in human Jurkat T cells indicates that at physiologically achievable concentrations in humans, ethanol can decrease TK activity through decreases in cell proliferation, and it suggests that ethanol ingestion in HIV-1-infected individuals could compromise activation of AZT and related drugs through decreased TK activity.

Appl Environ Microbiol, 2002 Apr, 68(4), 1872 - 81
Effect of growth conditions and staining procedure upon the subsurface transport and attachment behaviors of a groundwater protist; Harvey RW et al.; The transport and attachment behaviors of Spumella guttula (Kent), a nanoflagellate (protist) found in contaminated and uncontaminated aquifer sediments in Cape Cod, Mass., were assessed in flowthrough and static columns and in a field injection-and-recovery transport experiment involving an array of multilevel samplers . Transport of S . guttula harvested from low-nutrient (10 mg of dissolved organic carbon per liter), slightly acidic, granular (porous) growth media was compared to earlier observations involving nanoflagellates grown in a traditional high-nutrient liquid broth . In contrast to the highly retarded (retardation factor of approximately 3) subsurface transport previously reported for S . guttula, the peak concentration of porous-medium-grown S . guttula traveled concomitantly with that of a conservative (bromide) tracer . About one-third of the porous-medium-grown nanoflagellates added to the aquifer were transported at least 2.8 m downgradient, compared to only approximately 2% of the broth-grown nanoflagellates . Flowthrough column studies revealed that a vital (hydroethidine {HE}) staining procedure resulted in considerably less attachment (more transport) of S . guttula in aquifer sediments than did a staining-and-fixation procedure involving 4',6'-diamidino-2-phenylindole (DAPI) and glutaraldehyde . The calculated collision efficiency (approximately 10(-2) for porous-medium-grown, DAPI-stained nanoflagellates) was comparable to that observed earlier for the indigenous community of unattached groundwater bacteria that serve as prey . The attachment of HE-labeled S . guttula onto aquifer sediment grains was independent of pH (over the range from pH 3 to 9) suggesting a primary attachment mechanism that may be fundamentally different from that of their prey bacteria, which exhibit sharp decreases in fractional attachment with increasing pH . The high degree of mobility of S . guttula in the aquifer sediments has important ecological implications for the protistan community within the temporally changing plume of organic contaminants in the Cape Cod aquifer.

Appl Environ Microbiol, 2002 Apr, 68(4), 1647 - 51
Proline reverses the abnormal phenotypes of Colletotrichum trifolii associated with expression of endogenous constitutively active Ras; Memmott SD et al.; Colletotrichum trifolii is the causative organism of alfalfa anthracnose . We previously cloned and characterized the small prototypical G protein, Ras, of C . trifolii, which is involved in the signaling pathways that mediate interaction between the pathogen and its host . Transformants expressing constitutively active forms of Ras have growth medium-dependent phenotypes . In nutrient-rich media (e.g., yeast extract and peptone), the phenotype of the transformants was indistinguishable from that of the wild type . However, during nutrient starvation, the transformants lose polarity, have distended hyphae, and fail to sporulate and produce appressoria . Since peptone caused the phenotype to revert, amino acids were tested singly and in combination to identify the responsible amino acid(s) . We found that 1.6 mM proline in the medium reverses the constitutively active Ras phenotype.

Plant Cell, 2002 Mar, 14(3), 575 - 88
The Arabidopsis salt overly sensitive 4 mutants uncover a critical role for vitamin B6 in plant salt tolerance; Shi H et al.; Salt stress is a major environmental factor influencing plant growth and development . To identify salt tolerance determinants, a genetic screen for salt overly sensitive (sos) mutants was performed in Arabidopsis . We present here the characterization of sos4 mutants and the positional cloning of the SOS4 gene . sos4 mutant plants are hypersensitive to Na(+), K(+), and Li(+) ions . Under NaCl stress, sos4 plants accumulate more Na(+) and retain less K(+) compared with wild-type plants . SOS4 encodes a pyridoxal kinase that is involved in the biosynthesis of pyridoxal-5-phosphate, an active form of vitamin B6 . The expression of SOS4 cDNAs complements an Escherichia coli mutant defective in pyridoxal kinase . Supplementation of pyridoxine but not pyridoxal in the growth medium can partially rescue the sos4 defect in salt tolerance . SOS4 is expressed ubiquitously in all plant tissues . As a result of alternative splicing, two transcripts are derived from the SOS4 gene, the relative abundance of which is modulated by development and environmental stresses . Besides being essential cofactors for numerous enzymes, as shown by pharmacological studies in animal cells, pyridoxal-5-phosphate and its derivatives are also ligands for P2X receptor ion channels . Our results demonstrate that pyridoxal kinase is a novel salt tolerance determinant important for the regulation of Na(+) and K(+) homeostasis in plants . We propose that pyridoxal-5-phosphate regulates Na(+) and K(+) homeostasis by modulating the activities of ion transporters.

Genetics, 2002 Mar, 160(3), 877 - 89
Telomeric and rDNA silencing in Saccharomyces cerevisiae are dependent on a nuclear NAD(+) salvage pathway; Sandmeier JJ et al.; The Sir2 protein is an NAD(+)-dependent protein deacetylase that is required for silencing at the silent mating-type loci, telomeres, and the ribosomal DNA (rDNA) . Mutations in the NAD(+) salvage gene NPT1 weaken all three forms of silencing and also cause a reduction in the intracellular NAD(+) level . We now show that mutation of a highly conserved histidine residue in Npt1p results in a silencing defect, indicating that Npt1p enzymatic activity is required for silencing . Deletion of another NAD(+) salvage pathway gene called PNC1 caused a less severe silencing defect and did not significantly reduce the intracellular NAD(+) concentration . However, silencing in the absence of PNC1 was completely dependent on the import of nicotinic acid from the growth medium . Deletion of a gene in the de novo NAD(+) synthesis pathway BNA1 resulted in a significant rDNA silencing defect only on medium deficient in nicotinic acid, an NAD(+) precursor . By immunofluorescence microscopy, Myc-tagged Bna1p was localized throughout the whole cell in an asynchronously growing population . In contrast, Myc-tagged Npt1p was highly concentrated in the nucleus in approximately 40% of the cells, indicating that NAD(+) salvage occurs in the nucleus in a significant fraction of cells . We propose a model in which two components of the NAD(+) salvage pathway, Pnc1p and Npt1p, function together in recycling the nuclear nicotinamide generated by Sir2p deacetylase activity back into NAD(+).

Folia Microbiol (Praha), 2001, 46(5), 407 - 12
Effect of osmotic stress on Aspergillus chevalieri respiratory system; Hefnawy MA; The osmotolerant fungus Aspergillus chevalieri tolerates up to 80% sucrose concentration in the growth medium . At 50% sucrose the growth rate is 1.5-fold higher than in control (3% sucrose), at 80% sucrose it drops to 30% of the control level . Total proteins and lipids in the mitochondrial fractions obtained from the mycelium rise with increasing sucrose concentration during growth (2.6 and 2.1 times at 80% sucrose) . The rate of respiration by whole cells and mitochondrial fractions increases with increased sucrose level in the growth medium . The activity of respiratory enzymes, such as succinate dehydrogenase, cytochrome oxidase, NADH oxidase and succinate oxidase, were also higher in cells grown in the presence of elevated sucrose concentrations . The largest increase was observed with NADH dehydrogenase . A . chevalieri cells grown at high osmotic stress exhibited enlarged mitochondria . The mean mitochondrial diameter at 50 and 80% sucrose was approximately 2.9- and 2.6-fold larger than in the control, respectively . Agarose gel electrophoresis of nucleic acids revealed the presence of high-density bands of RNA in mitochondrial fractions from cells grown at elevated sucrose levels.

Am J Physiol Regul Integr Comp Physiol, 2002 Apr, 282(4), R1200 - 9
Electrophysiological properties of rainbow trout cardiac myocytes in serum-free primary culture; Nurmi A et al.; A low-density primary culture of trout ventricular myocytes in serum-free growth medium was established and maintained for up to 10 days at 17 degrees C . The myocytes retained their normal rod shaped morphology, capacitive surface area of the sarcolemma (SL), and contractile quiescence . However, sarcolemmal cation currents changed significantly, some permanently, some transiently, after 8-10 days of culture . TTX-sensitive sodium current (I(Na)) and Ba(2+)-sensitive background inward rectifier potassium current (I(K1)) were permanently depressed to 24-28% of their control density measured in freshly isolated myocytes . In contrast, L-type calcium current (I(Ca)) was only transiently downregulated; after 2-3 days in culture, the density of the current was 32% of the control and recovered to the control value after 8-10 days in culture . The changes in membrane currents were reflected in the shape of the action potential (AP) . After 2-3 days in culture, maximal overshoot potential and resting potential were significantly reduced, and the durations of the AP at 50 and 90% repolarization were significantly increased . These changes became significantly more pronounced after 8-10 days of culture, with the exception of AP duration at 50% repolarization level . The shortening of the early plateau phase may reflect an additional change to an outward current, presumably the rapid component of the delayed rectifier (I(Kr)) . Although the present findings indicate that fish cardiac myocytes can be maintained in serum-free primary culture for at least 10 days at 17 degrees C, some but not all of the electrophysiological characteristics of the myocytes change markedly during culture . The changes in ion currents were not due to loss of sarcolemmal membrane and therefore are likely to represent altered expression of cation currents as an adaptive response to culture conditions.

Int J Med Microbiol, 2002 Feb, 291(6-7), 545 - 50
The neutrophil-activating protein of Helicobacter pylori; Dundon WG et al.; Infection of the stomach mucosa by the gastric pathogen Helicobacter pylori is accompanied by a large infiltration of neutrophils and monocytes which are believed to contribute substantially to H . pylori-induced gastritis . A protein was identified (HP-NAP for neutrophil-activating protein from H . pylori) that was capable of increasing the adhesion of neutrophils to endothelial cells . We have demonstrated that HP-NAP is a dodecamer composed of identical 17-kDa subunits that induces the production of reactive oxygen radicals (ROIs) by neutrophils via a cascade of intracellular activation events . HP-NAP has also been shown to be chemotactic for neutrophils and monocytes, and a majority of H . pylori-infected patients have been found to produce antibodies specific for HP-NAP making it a strong vaccine candidate . More recently it has been shown that HP-NAP can stimulate tissue factor and plasminogen activator inhibitor-2 production by human monocytes . While structurally similar to the Escherichia coli DNA-binding protein Dps, HP-NAP has characteristics that are more similar to bacterioferritins being capable of binding up to 500 atoms of iron in vitro . Further study, however, has revealed that synthesis of HP-NAP in H . pylori is not altered by the addition or subtraction of metal ions from its growth medium suggesting that the primary role of the protein in vivo is not as a metal-binding protein . A number of other reports have proposed that HP-NAP acts as an adhesin being capable of binding several different compounds in vitro . Sequence analysis of the genomes of several other bacteria reveal that many possess Dps/HP-NAP-like proteins . The preliminary characterisation of some of these proteins will be discussed.

Can J Microbiol, 2002 Jan, 48(1), 1 - 6
Simplified media for spiroplasmas associated with tabanid flies; Moulder RW et al.; Traditionally, isolation, maintenance, and testing of Spiroplasma species (Mollicutes: Entomoplasmatales) from horse flies (Tabanus spp.) and deer flies (Chrysops spp.) (Diptera: Tabanidae) have been accomplished in the complex M1D medium . A relatively inexpensive, simplified medium for tabanid spiroplasmas could expedite procedures that require large quantities of growth medium . Nine strains of spiroplasmas, eight from tabanids and one from mosquitoes, were cultured in three simplified broth media, R2, R8-1, and C-3G, and in M1D . There was no significant difference in the rate of spiroplasma growth in M1D and the three simplified media . R2 medium supported the growth of tabanid spiroplasmas more consistently and with better morphology through 10 subcultures than did the other simplified media . Primary isolations were made in R2 medium from tabanids collected (i) in Georgia, U.S.A., with 10 isolations from 10 flies and (ii) in coastal Costa Rica, with isolation rates of 70% (28/40) and 73% (27/37), respectively, for R2 and M1D . Of the seven group VIII field isolates from Costa Rica, four were capable of sustained growth in R2, and three were triply cloned in this simplified medium . These results suggest that the simplified medium R2 is suitable for many procedures with tabanid spiroplasmas.

Tsitologiia, 2001, 43(12), 1168 - 73
{Radioadaptive enhancement of the repair of UV-induced postreplication gaps in Escherichia coli DNA}; Zhestianikov VD et al.; Postreplication DNA repair (PRR) in UV-irradiated Escherichia coli WP2 uvrA (tryptophan-dependent strain) and K12 AB1886 uvrA6 pre-irradiated by gamma-rays in low doses (radioadaptation, the first stress effect) has been investigated . PRR was found to be more effective after incubation in the growth medium (for 45-60 min) than in non-radioadapted cells: the repair of postreplication gaps increased by 6-15% . If cells of WP2 uvrA strain were incubated after UV-irradiation in media lacking tryptophan or casamin acids (the second stress effect), PRR was seen to increase as early as within 15 min of incubation and it is more effective than at the first stress . After a 30-60 min incubation the double stress effect leads to an increase in postreplication gap repair by 23-45% . In this case almost all the gaps prove to be repaired . The second stress alone exerts no influence on PPR efficiency . It is supposed that a preliminary radioadaptation may stimulate synthesis of a protein (proteins) of the SOS-response (presumably DNA polymerase V) . The second stress effect apparently induces synthesis of an unknown factor (or depreesses synthesis of a MmrA-like protein), and this in cooperation with a protein newly synthesized during radioadaptation significantly increases the efficiency of PPR.

J Agric Food Chem, 2002 Mar 13, 50(6), 1393 - 9
Eutypa dieback in grapevines: differential production of acetylenic phenol metabolites by strains of Eutypa lata; Molyneux RJ et al.; The production of acetylenic phenol metabolites in vitro by three strains of the ascomycete Eutypa lata, the causative agent of dying-arm disease in grapevines, has been investigated . Metabolite composition and yields differed significantly between strains and with growth medium but usually reached a maximum after 24-30 days of fungal growth . A general method for the analysis and identification of metabolites by gas chromatography/mass spectrometry of their trimethylsilyl ether derivatives was developed and individual compounds were quantitated by analytical high-performance liquid chromatography (HPLC) and separated by preparative HPLC . The phenolic aldehyde, eutypine (1), reported to be the grape phytotoxin, occurred in only one of the strains examined whereas the primary metabolite was the corresponding alcohol, eutypinol (2), the presumptive detoxification product . A novel metabolite was isolated as a major constituent, together with a minor component, and their structures were established by spectroscopic methods as a methoxyquinol, named eulatinol (4), and a chromene analog (9) of 2, respectively . The evidence suggests that 1 is not solely responsible for phytotoxicity in grapevines but that dying-arm disease may result from a suite of compounds elaborated by the fungus, with the composition dependent on fungal strain and nutritional source.

J Pediatr Surg, 2002 Mar, 37(3), 472 - 6
Insulin-like growth factor-I protects neuroblastoma against starvation-induced apoptosis and is associated with increased Bcl-2 expression; Beierle EA et al.; BACKGROUND/PURPOSE: Aggressive neuroblastomas avoid apoptosis and have increased expression of the antiapoptotic protein, Bcl-2 . Insulin-like growth factor-I (IGF-I) is mitogenic and may promote tumor survival by inhibiting apoptosis . The authors hypothesize that IGF-I may protect neuroblastoma cells from apoptosis by upregulating their Bcl-2 expression . METHODS: Human neuroblastoma cells (IMR-32) are cultured, and 3 experimental groups are established: 1 group with cells cultured in standard growth media (control), 1 with cells grown in serum-depleted media (starvation), and 1 with neuroblastoma cells cultured in starvation media plus IGF-I . The cells are harvested at 14 and 24 hours, and cytospin slides are made . Bcl-2 expression is measured by immunohistochemistry . Apoptosis is detected with the TUNEL method . RESULTS: Bcl-2 expression is decreased 90% in the serum starved neuroblastoma cells . In addition, apoptosis is 150 times higher in the starved neuroblastoma cells . These changes are abrogated by the addition of IGF-I, where apoptosis is decreased 50% and Bcl-2 is 14-fold higher in the IGF-I-treated group . These changes are most apparent at 24 hours . CONCLUSIONS: IGF-I protects neuroblastoma cells from apoptosis and increases Bcl-2 expression . Growth factors may have a direct role in promoting tumorigenesis by inducing the expression of antiapoptotic proteins by the tumor .

J Cell Sci, 2002 Mar 1, 115(Pt 5), 1041 - 8
Substrate proteolysis is inhibited by dominant-negative Nedd4 and Rsp5 mutants harboring alterations in WW domain 1; Shcherbik N et al.; Mammalian Nedd4 and its budding yeast orthologue Rsp5 are members of a large family of HECT-domain-containing ubiquitin ligases . Besides possessing a Ca(2+)/lipid-binding domain, both ligases have multiple protein-interacting modules termed WW domains . The C-terminal WW domains mediate interactions with substrates, but the function of the first WW domain remains unclear . We found that expression of a WW domain 1 Nedd4 mutant inhibits the growth of budding yeast by affecting the rsp5-ole1 pathway . The WW domain 1 mutant-induced phenotype is suppressed by ole1 cDNA overexpression or oleic acid supplementation of growth media and ole1 RNA levels are reduced in cells expressing this Nedd4 mutant . Also, the WW domain 1 Nedd4 mutant associates via WW domains 2 and 3 with Spt23, a Rsp5 target and ole1 transactivator . The dominant-negative activity of this mutant is associated with promoting accumulation of unprocessed Spt23 and inhibiting generation of processed and presumably active protein . Also, Spt23 processing is inhibited by a Nedd4 mutant that lacks ubiquitin ligase activity and Spt23-binding-competent Rsp5 mutants harboring WW domain 1 or ligase domain mutations . Interestingly, in mammalian cells, wild-type Nedd4 promotes proteasome-mediated degradation of the precursor form of Spt23 . WW domain 1 and ligase domain Nedd4 mutants block its degradation . These results indicate that WW domain 1 of these ligases interacts with cofactors that are required for ubiquitin/proteasome-dependent proteolysis of bound substrates.

Plant Cell Physiol, 2002 Feb, 43(2), 177 - 85
A gene encoding multidrug resistance (MDR)-like protein is induced by aluminum and inhibitors of calcium flux in wheat; Sasaki T et al.; A cDNA clone exclusively induced by aluminum (Al) was isolated from root apices of wheat (Triticum aestivum L.) by the differential display method . The predicted amino acid sequence exhibited homology to the multidrug resistance (MDR) proteins that is known as a member of the ATP-binding cassette (ABC) protein superfamily . Thus this gene was named TaMDR1 (Triticum aestivum MDR) . TaMDR1 was induced as a function of Al concentration in the range from 5 to 50 microM, which is in the range of Al content in natural acid soil environment . The concentration required for the induction was lower in the Al-sensitive cultivar than in the Al-tolerant cultivar, indicating that the accumulation of TaMDR1 mRNA was associated with the events caused by Al toxicity rather than Al tolerance . TaMDR1 was significantly induced by the exposure to lanthanum, gadolinium and ruthenium red, which are known as inhibitors of calcium channels . Furthermore, decreasing of calcium ion in growth medium caused stimulation of the gene expression . These results suggested that the induction of TaMDR1 is caused by the breaking of calcium homeostasis which occurred at early stage of Al toxicity.

J Biol Chem, 2002 May 10, 277(19), 16567 - 75 Epub 2002 Feb 26.
Analysis of tissue transglutaminase function in the migration of Swiss 3T3 fibroblasts: the active-state conformation of the enzyme does not affect cell motility but is important for its secretion; Balklava Z et al.; Increasing evidence suggests that tissue transglutaminase (tTGase; type II) is externalized from cells, where it may play a key role in cell attachment and spreading and in the stabilization of the extracellular matrix (ECM) through protein cross-linking . However, the relationship between these different functions and the enzyme's mechanism of secretion is not fully understood . We have investigated the role of tTGase in cell migration using two stably transfected fibroblast cell lines in which expression of tTGase in its active and inactive (C277S mutant) states is inducible through the tetracycline-regulated system . Cells overexpressing both forms of tTGase showed increased cell attachment and decreased cell migration on fibronectin . Both forms of the enzyme could be detected on the cell surface, but only the clone overexpressing catalytically active tTGase deposited the enzyme into the ECM and cell growth medium . Cells overexpressing the inactive form of tTGase did not deposit the enzyme into the ECM or secrete it into the cell culture medium . Similar results were obtained when cells were transfected with tTGase mutated at Tyr(274) (Y274A), the proposed site for the cis,trans peptide bond, suggesting that tTGase activity and/or its tertiary conformation dependent on this bond may be essential for its externalization mechanism . These results indicate that tTGase regulates cell motility as a novel cell-surface adhesion protein rather than as a matrix-cross-linking enzyme . They also provide further important insights into the mechanism of externalization of the enzyme into the extracellular matrix.

Clin Microbiol Infect, 1997 Aug, 3(4), 468 - 473
Multicenter evaluation of mycobacteria growth indicator tube (MGIT) compared with the BACTEC radiometric method, BBL biphasic growth medium and Löwenstein---Jensen medium; Tortoli E et al.; OBJECTIVE: To evaluate the new BBL mycobacteria growth indicator tube (MGIT) in comparison with other media . METHODS: MGIT was evaluated in 10 Italian centers on 433 clinical samples, mainly of respiratory origin and mainly smear positive, in comparison with Lowenstein---Jensen and with one or more other methods represented, according to participating centers, by the BACTEC radiometric method or by the biphasic BBL Septi-Chek AFB system . While MGIT and Lowenstein---Jensen were used for all the samples, 285 of them were also inoculated in BACTEC vials and 274 in biphasic bottles . Of these samples, 132 were investigated with all the four methods . RESULTS: Although less rapid and sensitive than the radiometric method, the results of MGIT were equal when compared with the other two media with respect to overall isolation yield; furthermore, it allowed the detection of growth in significantly shorter times . CONCLUSIONS: The results of this study indicate the value of MGIT for the detection of mycobacteria and, thanks to its extreme simplicity of use, its suitability for small and large laboratories . Its combined use with a solid medium can substantially improve the diagnosis of mycobacterial infection.

Mol Genet Genomics, 2002 Feb, 266(6), 1004 - 11 Epub 2002 Jan 12.
A novel cis-acting cysteine-responsive regulatory element of the gene for the high-affinity glutathione transporter of Saccharomyces cerevisiae; Miyake T et al.; We cloned a DNA fragment from Saccharomyces cerevisiae that complemented the deficiency in high-affinity glutathione transport activity conferred by a gsh11 mutation, and found that the ORF responsible was YJL212c, which had already been designated as OPT1 and HGT1 by others . Northern analysis clearly demonstrated that this ORF, now referred to as OPT1/ HGT1/ GSH11, was induced by sulfur starvation and repressed by adding cysteine to the growth medium . Reporter gene assays showed that a segment spanning the region between positions -371 and -355 was essential for the regulation of this gene . A sequence of 9 nt, CCGCCACAC (from -364 to -356), in this region was shown to be required for protein binding, using an electrophoretic mobility shift assay . Based on these results, we propose that CCGCCACAC comprises the core of a cis-acting element involved in cysteine-responsive gene regulation in S . cerevisiae.

Genetics, 2002 Feb, 160(2), 417 - 27
Genetic control of extracellular protease synthesis in the yeast Yarrowia lipolytica; Gonzalez-Lopez CI et al.; Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes an acidic protease or an alkaline protease, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins . Previous results have indicated that the alkaline protease response to pH was dependent on YlRim101p, YlRim8p/YlPalF, and YlRim21p/YlPalH, three components of a conserved pH signaling pathway initially described in Aspergillus nidulans . To identify other partners of this response pathway, as well as pH-independent regulators of proteases, we searched for mutants that affect the expression of either or both acidic and alkaline proteases, using a YlmTn1-transposed genomic library . Four mutations affected only alkaline protease expression and identified the homolog of Saccharomyces cerevisiae SIN3 . Eighty-nine mutations affected the expression of both proteases and identified 10 genes . Five of them define a conserved Rim pathway, which acts, as in other ascomycetes, by activating alkaline genes and repressing acidic genes at alkaline pH . Our results further suggest that in Y . lipolytica this pathway is active at acidic pH and is required for the expression of the acidic AXP1 gene . The five other genes are homologous to S . cerevisiae OPT1, SSY5, VPS28, NUP85, and MED4 . YlOPT1 and YlSSY5 are not involved in pH sensing but define at least a second protease regulatory pathway.

J Dermatol Sci, 2002 Feb, 28(2), 152 - 8
Sustained ability for fibroblast outgrowth from stored neonatal foreskin: a model for studying mechanisms of fibroblast outgrowth; Nahm WK et al.; Fibroblast cultures derived from neonatal foreskin explants have been an important model for understanding the basic mechanisms of fibroblast function and activity . Neonatal foreskin has been routinely used for establishing such fibroblast cultures in vitro . In general, tissue explants and fibroblast cultures are established from freshly harvested neonatal foreskin tissue . It is unknown whether prolonged storage of tissue, even in optimal growth media and conditions would still result in successful explant outgrowth . Specifically, no studies have properly examined the maximal duration of foreskin storage that can produce viable fibroblasts with normal growth and synthetic characteristics . We, therefore, set out to isolate and propagate fibroblast cultures from neonatal foreskin tissue that had been kept in complete culture media at 4 degrees C for various periods of time . We found that outgrowth and propagation of viable fibroblasts in vitro occurred with foreskin samples obtained within 24 days after surgical harvesting . The morphology, cellular proliferative capacity (measured by {3H}-thymidine uptake), steady state levels of alpha1(I) procollagen mRNA, and collagenous protein synthesis were comparable among fibroblast cultures derived from foreskin explants established 3 days (control) and up to 24 days (but not 38 days) after tissue harvesting . These studies demonstrate that viable and functional fibroblasts, with apparently similar in vitro characteristics, can be isolated and propagated from explants of neonatal foreskin tissue that have undergone prolonged storage . Moreover, these findings may be useful in understanding the lack of fibroblast growth in such conditions as found in delayed wound healing and aging.

Toxicol Appl Pharmacol, 2002 Feb 1, 178(3), 144 - 54
The surface charge of visible particulate matter predicts biological activation in human bronchial epithelial cells; Veronesi B et al.; The physicochemical complexity of airborne particulate matter (PM) has hampered identifying a specific mechanism(s) for its toxicity . In this study, selected physicochemical characteristics (i.e., size, particle number, acidity, and surface charge) were measured on various field PM, derived from urban ambient (St . Louis, Ottawa, Canada), residential (Woodstove), volcanic dust from Mt . St . Helen (MSH), and industrial {oil fly ash (OFA) coal fly ash (CFA)} sources . Morphometric analysis of visible (< or = 2.0 to >10 microm) field particles indicated that the industrial PM (OFA, CFA) had the smallest diameter and lowest total number of particles per weight while Woodstove and Ottawa had the largest diameter and highest number of particles . All PM lowered the pH of an unbuffered 10 mM NaCl solution from pH 7.4 to pH 4.7-6.8 but did not change the neutral pH of the cell culture medium, keratinocyte growth media (KGM) . The surface charge (i.e., zeta potential) of microscopically visible (> or = 2.0 microm) field particles, suspended in either a Hepes-buffered KCl solution or in KGM, was measured by microelectrophoresis . In KCl solution, the mean zeta potential of all tested PM ranged from -36 +/- 2 (Woodstove) to -27 +/- 4.3 mV (MSH) . When measured in KGM medium, the mean zeta potential value of each PM was significantly less (p > 0.001) than those measured in KCl solution, with values ranging from -17 +/- 0.3 mV (St . Louis) to -9 +/- 0.6 mV (MSH) . Suspensions of field PM, its soluble and washed particulate fractions, were next prepared from each PM . The biological effects (i.e., increases in intracellular calcium ({Ca2+}i), cytokine release) of their exposure were measured in human, immortalized, tracheal-bronchial epithelial cells (BEAS-2B) . Exposure of BEAS-2B cells to each fraction produced an immediate, but differential increase in {Ca2+}i and the subsequent release of the inflammatory cytokine IL-6, 4 and 16 h later . Increases in {Ca2+}i by field PM significantly correlated with the IL-6 released by each fraction (r2 > or = 0.76) after both 4 and 16 h exposures . The biological effects of each PM were compared with their physicochemical characteristics . No correlation was found between increases in {Ca2+}i or cytokine release and a PM's acidity or the number or size of its visible (> or = 2.0 microm) particles . However, the surface charge of PM field particles, when measured in the KGM exposure medium, showed a high correlation (r2 > or = 0.94) with the IL-6 release by field PM after both 4 and 16 h exposure . Increases in {Ca2+}i also correlated (r2 = 0.85) with the surface charge of PM field particles when measured in KGM . These data indicate that the surface charge (i.e., zeta potential) carried on PM's visible field particles predicts their differential release of the inflammatory cytokine IL-6 in cultures of human respiratory epithelial cells.

J Exp Zool, 2002 Mar 1, 292(4), 351 - 66
Effects of genic substitution at the pink-eyed dilution locus on the proliferation and differentiation of mouse epidermal melanocytes in vivo and in vitro; Hirobe T et al.; Cells positive to the dopa reaction (melanocytes) as well as to the combined dopa-premelanin reaction (melanoblasts and melanocytes) in the epidermis of C57BL/10JHir-p/p (pink-eyed dilution) mice were fewer and less reactive than in C57BL/10JHir (black, P/P) mice, suggesting that the proliferation and differentiation of p/p melanocytes are inhibited . To confirm the inhibitory effects of p gene on the proliferation and differentiation of epidermal melanocytes, we cultured epidermal cell suspensions of neonatal skins from P/P and p/p in a serum-free medium . The proliferation and differentiation of p/p melanoblasts/melanocytes in primary culture were greatly inhibited as compared to P/P melanoblasts/melanocytes . The morphology of p/p melanoblasts/melanocytes cultured in melanocyte growth medium, though non-pigmented, was similar to P/P melanocytes; namely, dendritic, polygonal, or epithelioid . About 8% of p/p cells cultured in melanocyte growth medium were positive to the dopa reaction, and about 25% were reactive to the combined dopa-premelanin reaction . Eumelanin content in p/p was extremely reduced compared to P/P . The immunocytochemical staining of p/p melanoblasts/melanocytes revealed that they are negative to tyrosinase, but reactive to tyrosinase-related protein (TRP)-1, TRP-2, and c-kit . However, the reactivities in p/p were lower than in P/P . Although the differentiation of p/p melanoblasts was not induced by endothelin (ET)-1, ET-2, and ET-3, the proliferation of p/p melanoblasts was stimulated by them . These results suggest for the first time that p gene exerts its influence on the proliferative activities of mouse epidermal melanoblasts by affecting the regulatory mechanisms dependent on the function of ETs .

Phytochemistry, 2002 Mar, 59(5), 489 - 92
Biotransformation of cadina-4,10(15)-dien-3-one and 3alpha-hydroxycadina-4,10(15)-diene by Curvularia lunata ATCC 12017; Collins DO et al.; Cadina-4,10(15)-dien-3-one (1) was metabolised by Curvularia lunata ATCC 12017 in two different growth media to give three metabolites, one of which, 12-hydroxycadina-4,10(15)-dien-3-one (4), was new . Incubation of 3alpha-hydroxycadina-4,10(15)-diene (2) with the fungus produced three new analogues, namely, (4S)-1alpha,3alpha-dihydroxycadin-10(15)-ene (5), 3alpha,14-dihydroxycadina-4,10(15)-diene (6) and 3alpha,12-dihydroxycadina-4,10(15)-diene (7).

Oncogene, 2002 Jan 24, 21(5), 761 - 8
Estrogen-like effects of thyroid hormone on the regulation of tumor suppressor proteins, p53 and retinoblastoma, in breast cancer cells; Dinda S et al.; T47D cells represent an estrogen-responsive human ductal carcinoma cell line which expresses detectable levels of estrogen receptor (ER) . We have previously shown that estradiol (E(2)) treatment of T47D cells causes an increase in the level of p53 and a concomitant phosphorylation of retinoblastoma protein (pRb) . In the present study, we have analysed the expression of p53 and phosphorylation state of pRb and compared the effects of E(2) and triiodothyronine (T(3)) on these phenomena . Cells were grown in a medium containing charcoal-treated serum to deplete the levels of endogenous steroids . Upon confluency, the cells were treated with T(3) (10(-12) to 10(-7) M) for 24 h and the presence of p53 and pRb was detected by Western analysis . E(2) treatment of cells caused a 2-3-fold increase in the level of p53 . Presence of T(3) in the medium caused a gradual increase in the level of p53 in a concentration-dependent manner . Under the above conditions, pRb was phosphorylated (detected as an upshift during SDS-PAGE) in the presence of E(2) and T(3) . Supplementation of growth medium with T(3) (1 microM) caused an increase in the rate of proliferation of T47D cells and induced hyperphosphorylation of pRb within 4 h; this effect was maintained for up to 12 h . When ICI 164 384 (ICI) (1 microM), an ER antagonist, was combined with E(2) (1 nM) or T(3) (1 microM), effects of hormones on cell proliferation and hyperphosphorylation of pRb were blocked . Western analysis of p53 was supplemented with its cytolocalization by immuno-labeling using laser scanning confocal fluorescence microscopy, which revealed an ICI-sensitive increase in the abundance of p53 in hormone-treated cells . Steroid binding analysis revealed lack of competition by T(3) for the {(3)H}E(2) binding . These results indicate that T(3) regulates T47D cell cycle progression and proliferation raising the p53 level and causing hyperphosphorylation of pRb by a common mechanism involving ER and T(3) receptor (T(3)R)-mediated pathways.

J Appl Microbiol, 2002, 92(2), 315 - 21
Reduction of ferric iron by acidophilic heterotrophic bacteria: evidence for constitutive and inducible enzyme systems in Acidiphilium spp; Johnson DB et al.; AIMS: To compare the abilities of two obligately acidophilic heterotrophic bacteria, Acidiphilium acidophilum and Acidiphilium SJH, to reduce ferric iron to ferrous when grown under different culture conditions . METHODS AND RESULTS: Bacteria were grown in batch culture, under different aeration status, and in the presence of either ferrous or ferric iron . The specific rates of ferric iron reduction by fermenter-grown Acidiphilium SJH were unaffected by dissolved oxygen (DO) concentrations, while iron reduction by A . acidophilum was highly dependent on DO concentrations in the growth media . The ionic form of iron present (ferrous or ferric) had a minimal effect on the abilities of harvested cells to reduce ferric iron . Whole cell protein profiles of Acidiphilium SJH were very similar, regardless of the DO status of the growth medium, while additional proteins were present in A . acidophilum grown microaerobically compared with aerobically-grown cells . CONCLUSIONS: The dissimilatory reduction of ferric iron is constitutive in Acidiphilium SJH while it is inducible in A . acidophilum . SIGNIFICANCE AND IMPACT OF THE STUDY: Ferric iron reduction by Acidiphilium spp . may occur in oxygen-containing as well as anoxic acidic environments . This will detract from the effectiveness of bioremediation systems where removal of iron from polluted waters is mediated via oxidation and precipitation of the metal.

Cryobiology, 2001 Sep, 43(2), 106 - 13
Overexpression of trehalose synthase and accumulation of intracellular trehalose in 293H and 293FTetR:Hyg cells; Lao G et al.; A humanized clone containing the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase (otsA/B) has been constructed . Using the Gateway Cloning System (Invitrogen, Inc.), the otsA/B genes have been placed under the control of the CMV promoter (pEXPcmv-otsA/B) or the CMV promoter and the tet operator (pEXP cmv TetO-otsA/B) . The pEXPcmv-otsA/B clone has been introduced into 293H cells using LIPOFECTAMINE 2000 and the intracellular concentration of trehalose has been evaluated . The 293H cells accumulate 4-5 microg trehalose/mg dry weight and this concentration increases to 7-10 microg trehalose/mg dry weight if trehalose is included in the growth medium . The pEXPcmv TetO-otsA/B clone has been transfected into 293FTetR:Hyg cells which contain the tet repressor integrated into the genome . When these transfected cells are grown in the absence of tetracycline, no intracellular trehalose is detected . Inclusion of 0.3 microg/ml tetracycline in the growth medium results in the accumulation of 11-14 microg trehalose/mg dry weight, a value which increases to 19-20 microg trehalose/mg dry weight if trehalose is included in the growth medium . The data for the 293FTetR:Hyg cells indicate that intracellular trehalose accumulates in response to the addition of tetracycline . This system will allow us to manipulate the intracellular concentration of trehalose and to evaluate the desiccation tolerance of these cells as a function of intracellular trehalose concentration .

Int J Prosthodont, 2001 Jan-Feb, 14(1), 48 - 52
Growth of Candida species on commercial denture adhesives in vitro; Makihira S et al.; PURPOSE: Although denture adhesives are widely used by the elderly, it is unknown whether denture adhesives support microbial growth . Therefore, we investigated the growth of Candida species on six commercial denture adhesives . MATERIALS AND METHODS: The growth of a single isolate of C albicans and C tropicalis on six commercial denture adhesives was investigated by monitoring pH changes in growth media . RESULTS: In the preliminary study, the effect of each product on the pH value of the medium was examined; a single product itself significantly reduced the pH of medium below 5.0 . When the yeast was grown on the materials, the pH changes in the media varied depending on the adhesive materials and, to a greater or lesser extent, on whether they showed antifungal activity . Two products (Cushion Collect and Collect Soft A) significantly suppressed C albicans growth (P < 0.01), and one product (Collect Soft A) effectively reduced C tropicalis growth (P < 0.01) . CONCLUSION: Our results suggest that denture adhesives possess antifungal activity to a greater or lesser degree . However, one product caused the reduction of pH below 5.0 . Thus, in the daily use of denture adhesives, attention should be paid to both the materials and their microbiologic properties.

Plant Physiol, 2002 Feb, 128(2), 472 - 81
Arabidopsis seedling growth, storage lipid mobilization, and photosynthetic gene expression are regulated by carbon:nitrogen availability; Martin T et al.; The objective of the current work was to establish the degree to which the effects of carbon and nitrogen availability on Arabidopsis seedling growth and development are due to these nutrients acting independently or together . Growth of seedlings on low (0.1 mM) nitrogen results in a significant reduction of seedling and cotyledon size, fresh weight, chlorophyll, and anthocyanin content but a slight increase in endogenous sugars . The addition of 100 mM sucrose (Suc) to the nitrogen-depleted growth media results in a further reduction in cotyledon size and chlorophyll content and an overall increase in anthocyanins and endogenous sugars . Storage lipid breakdown is almost completely blocked in seedlings grown on low nitrogen and 100 mM Suc and is significantly inhibited when seedlings are grown on either low nitrogen or high Suc . Carbohydrate repression of photosynthetic gene expression can only be observed under low nitrogen conditions . Low (0.1 mM) nitrogen in the absence of exogenous carbohydrate results in a significant decrease in chlorophyll a/b-binding protein and ribulose bisphosphate carboxylase small subunit gene transcript levels . Thus, carbon to nitrogen ratio rather than carbohydrate status alone appears to play the predominant role in regulating various aspects of seedling growth including storage reserve mobilization and photosynthetic gene expression.

World J Gastroenterol, 2002 Feb, 8(1), 31 - 5
Morphological and functional changes of mitochondria in apoptotic esophageal carcinoma cells induced by arsenic trioxide; Shen ZY et al.; AIM: To demonstrate that mitochondrial morphological and functional changes are an important intermediate link in the course of apoptosis in esophageal carcinoma cells induced by As2O3 . METHODS: The esophageal carcinoma cell line SHEEC1, established in our laboratory, was cultured in 199 growth medium, supplemented with 100mL x L(-1) calf serum and 3 mol x L(-1)As2O3 (the same below) . After 2, 4, 6, 12, 24 h of drug adding, the SHEEC1 cells were collected for light-and electron-microscopic examination . The mitochondria were labeled by Rhodamine fluorescence probe and the fluorescence intensity of the mitochondria was measured by flow cytometer and cytofluorimetric analysis . Further,the mitochondrial transmembrane potential (MTP, psim) change was also calculated . RESULTS: The mitochondrial morphological change after adding As2O3 could be divided into three stages . In the early-stage (2-6h) after adding As2O3, an adaptive proliferation of mitochondria appeared; in the mid-stage (6-12 h) a degenerative change was observed; and in the late-stage (12-24 h) the mitochondria swelled with outer membrane broken down and then cells death with apoptotic changes of nucleus . The functional change of the mitochondria indicated by fluorescent intensity, which reflected the MTP status of mitochondria, was in accordance with morphological change of the mitochondria . The fluorescent intensity increased at early-stage, declined in mid-stage and decreased to the lowest in the late-stage . 24 h after As2O3 adding, the cell nucleus showed typical apoptotic changes . CONCLUSION: Under the inducement of As2O3, the early apoptotic changes of SHEEC1 cells were the apparent morphological and functional changes of mitochondria, afterwards the nucleus changes followed . It is considered that changes of mitochondria are an important intermediate link in the course of apoptosis of esophageal carcinoma cells induced by As2O3.

Microbiology, 2002 Feb, 148(Pt 2), 381 - 90
Osmotic regulation of the Streptomyces lividans thiostrepton-inducible promoter, ptipA; Ali N et al.; Transcriptional activation of the thiostrepton-inducible promoter, ptipA, in Streptomyces lividans is mediated by TipAL . This transcriptional activator belongs to the MerR/SoxR family that characteristically binds an operator sequence located between the -10 and -35 hexamers normally occupied by RNA polymerase . As for the Escherichia coli merT promoter, the ptipA hexamers are separated by a long 19 bp spacer and hence a topological transition of the DNA is likely to be a requisite for alignment with RNA polymerase . Growth conditions that could facilitate this conformational change were investigated using transcriptional fusions of ptipA with reporter genes . Adjustment of growth medium osmolarity led to increased and prolonged TipAL-dependent expression, both with and without the inducer, thiostrepton . These effects correlated with increases in negative DNA supercoiling . Moreover, an inability to induce the promoter with thiostrepton in strain TK64 was corrected by increasing the concentration of osmolyte, compensating for an apparent reduced level of negative DNA supercoiling in the strain . Prolonging the time of activation of tipA in the wild-type by manipulating growth conditions revealed that mycelial autolysis could be induced by thiostrepton in 4-d-old cultures.

Am J Physiol Renal Physiol, 2002 Mar, 282(3), F465 - 71
Mechanism and regulation of vitamin B(6) uptake by renal tubular epithelia: studies with cultured OK cells; Said HM et al.; The kidneys play an important role in regulating vitamin B(6) body homeostasis, but limited information exists regarding the mechanism of pyridoxine uptake by renal epithelial cells, and no study exists on its regulation . To address these issues, we used the renal opossum-derived tubular epithelial (opossum kidney; OK) cells and found pyridoxine uptake to 1) be temperature and energy dependent, 2) be pH dependent, with a higher uptake at alkaline or neutral buffer pH compared with acidic pH, 3) be Na(+) independent, 4) involve a saturable component (apparent Michaelis- Menten constant of 2.40 +/- 0.23 microM), 5) be inhibited by structural analogs, and 6) be amiloride sensitive . Maintaining OK cells in a vitamin B(6)-deficient growth medium (for 48 h) led to a significant upregulation of pyridoxine uptake . This upregulation was found to be specific for pyridoxine, inhibited by cyclohexamide and actinomycin D, reversible, and mediated via an increase in maximal velocity . Pretreating OK cells with modulates of a Ca(2+)/calmodulin-mediated pathway led to a significant downregulation in pyridoxine uptake via inhibition of maximal velocity . These results demonstrate that pyridoxine uptake by renal tubular epithelial OK cells is via a specialized pH-sensitive carrier-mediated mechanism . This mechanism appears to be regulated by extracellular vitamin B(6) levels and an intracellular Ca(2+)/calmodulin-mediated pathway.

Blood Cells Mol Dis, 2001 Nov-Dec, 27(6), 1013 - 9
Deformin, a substance found in Bartonella bacilliformis culture supernatants, is a small, hydrophobic molecule with an affinity for albumin; Derrick SC et al.; Culture supernatants of Bartonella bacilliformis were previously shown to contain a factor, called deforming factor or deformin, which causes deformation and invagination of red cell membranes and formation of intracellular vacuoles . This factor is here shown to be a small water-soluble molecule, approximately 1400 Da as estimated by gel-filtration chromatography . Deforming factor binds tightly to albumin, especially albumin dimers and multimers, present in the growth medium . It can be released from albumin with 50% ethanol and has been partially purified by filtration and HPLC .

J Anim Sci, 2002 Jan, 80(1), 202 - 7
Circulating cortisol, tumor necrosis factor-alpha interleukin-1beta, and interferon-gamma in pigs infected with Actinobacillus pleuropneumoniae; Balaji R et al.; This study evaluated the time course of systemic cytokine concentrations in an acute model of pneumonia in pigs challenged intranasally with Actinobacillus pleuropneumoniae . Feed intake and serum cortisol were measured as overt clinical and systemic markers of disease onset, respectively, and serum tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma as representative systemic inflammatory markers . Crossbred barrows (n = 15), approximately 5 wk of age, were used in the study . Pigs were housed in an environmentally controlled facility at 25 degrees C and under continuous illumination in pens measuring approximately 1.5 m2 . Pigs had free access to water and an unmedicated diet . Approximately 1 wk prior to disease challenge, pigs were fitted nonsurgically with venous catheters . At challenge, pigs were given 5 x 10(8) CFU Actinobacillus pleuropneumoniae intranasally (n = 8) or a similar volume of sterile growth media intranasally (Control; n = 7) . Feed intake was estimated by the change in feeder weight at 12-h intervals from -12 to 72 h relative to the time of disease challenge . Blood sampling began 12 h prior to challenge and continued until 72 h after challenge . Pigs were sampled at -12, -6, and 0 h, then at 90-min intervals until 12-h post-challenge, continuing at 3-h intervals until 24-h post-challenge, then again at 6-h intervals until 72 h after challenge . Serum was harvested and frozen until assayed for cortisol, tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma . Feed intake was reduced in Actinobacillus pleuropneumoniae pigs during the intervals 0 to 12 h (P < 0.001), 24 to 36 h (P < 0.001), 48 to 60 h (P <0.05), and 60 to 72 h (P < 0.05) . TheActnobacillus pleuropneumoniae-challenged pigs had elevated serum cortisol from 180-min to 18-h post-challenge (P < 0.001) and also at 36 (P < 0.05), 42 (P < 0.001), and 60 (P < 0.05) h following infection . Circulating cytokines were not affected by disease challenge . Thus, in this experimental model of pneumonia, weaned pigs demonstrated expected behavioral and endocrine characteristics of disease in the absence of significant changes in circulating inflammatory cytokines.

Appl Environ Microbiol, 2002 Feb, 68(2), 799 - 806
Optimization of reverse transcriptase PCR to detect viable Shiga-toxin-producing Escherichia coli; McIngvale SC et al.; The ability of reverse transcriptase PCR (RT-PCR) to detect viable Shiga-toxin-producing Escherichia coli (STEC) was investigated . Four primer sets, each targeting a specific region in the slt-II operon, were evaluated for their stringency and specificity for slt-II mRNA . STEC were evaluated for toxin expression under various conditions, including cell growth phase, growth medium, incubation temperature, and aeration . Following primer optimization, STEC were inoculated into Trypticase soy broth and cooked ground beef enrichments . Cells were harvested and RNA or DNA was extracted at 4, 8, 12, and 24 h . RT-PCR or PCR was conducted, and the products were visualized by gel electrophoresis and by Southern blots . mRNA targets were detected in 12-h cooked ground meat enrichments with an initial inoculum of 1 CFU/g . These results indicate that RT-PCR of E . coli slt-II mRNA is useful for detection of viable STEC in ground beef.

World J Gastroenterol, 2001 Jun, 7(3), 370 - 5
Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction; Cheng JL et al.; AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro . METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL) . Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month . Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR . RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year . The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently . Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly . The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm . The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion . CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.

FEMS Microbiol Lett, 2002 Jan 10, 206(2), 151 - 6
The nitrogen assimilation control (Nac) protein represses asnC and asnA transcription in Escherichia coli; Poggio S et al.; In this work, we show that the expression of the asnA and asnC genes is regulated by the availability of ammonium in the growth medium . Our results suggest that, under nitrogen-limiting growth conditions, the nitrogen assimilation control (Nac) protein is involved in the repression of the asnC gene, whose product is required to activate the transcription of asnA . We also show that asparagine negatively affects the expression of asnA, independently of the presence of Nac . These results allow us to conclude that asnA transcription is regulated by two different mechanisms that respond to different effectors: nitrogen and asparagine availability.

Toxicol In Vitro, 2002 Feb, 16(1), 11 - 21
In vitro response of human gingival epithelioid S-G cells to minocycline; Babich H et al.; Minocycline, a broad-spectrum antibiotic used in the treatment of acne and periodontal disease and to control inflammatory diseases such as rheumatoid arthritis, has recently been shown to induce a spectrum of adverse health effects . In the light of these contradictory data, this research was directed to provide basic information on the toxicology of minocycline, using in vitro cell culture models, and to evaluate its efficacy in periodontal therapies, particularly for wound healing . The human gingival epithelioid S-G cell line was used as the bioindicator . The greater toxicity of minocycline over doxycycline and tetracycline, related antimicrobial agents, probably correlated with its higher lipophilicity . The cytotoxicity of minocycline was unaffected by an S9 hepatic microsomal fraction, indicating that it is a direct-acting, rather than a metabolism-mediated, cytotoxicant . In comparative toxicity studies, much variation in the degree of sensitivity to minocycline was noted for different cell types . No correlation in the extent of sensitivity to minocycline and the physiologic state of the bioindicator cell (normal, transformed or malignant) was noted . The toxicity of minocycline to the S-G cells was dependent on its concentration and length of exposure . For a continuous 3-day exposure of the S-G cells to minocycline, the midpoint cytotoxicity (or, NR(50)) value, as quantified in the neutral red (NR) assay, was 204 microg/ml on day 1, 84 microg/ml on day 2, and 59 microg/ml on day 3 . For a 1-h exposure of the S-G cells in phosphate buffered saline (PBS), the NR(50) value was 780 microg/ml minocycline . Although a 1-h exposure in PBS to 200 microg/ml minocycline exerted some toxicity, the S-G cells recovered on exposure to growth medium; irreversible, progressive damage occurred at 400 microg/ml minocycline and greater . Minocycline, at 50 microg/ml, enhanced attachment of the S-G cells to a gelatin-coated surface and cell migration towards an immobilized fibronectin gradient, both biologic parameters important in periodontal wound healing . Minocycline generally had little or no effect on production of the pro-inflammatory cytokines, interleukin-6 (IL-6) and interleukin-8 (IL-8), by non-activated S-G cells, the exception being stimulation of IL-6 at 48 h . IL-1beta, however, greatly stimulated IL-6 and IL-8 production, which was further increased by concurrent exposure to minocycline . This suggested that minocycline may enhance the ability of gingival epithelial cells to participate in the early, inflammatory phase of periodontal wound healing . The limitation of minocycline efficacy to a rather narrow window of concentration, centering about 50 microg/ml, and primarily for short-term exposures may possibly explain, in part, the contradictory clinical data on the health effects of this drug.

Protein Expr Purif, 2002 Feb, 24(1), 43 - 50
Bacterial expression and purification of recombinant bovine Fab fragments; O'Brien PM et al.; We have previously described a recombinant phagemid expression vector, pComBov, designed for the production of native sequence bovine monoclonal antibodies (mAb) generated by antibody phage display . Bovine mAb Fab fragments isolated from libraries constructed using pComBov in Escherichia coli strain XL1-Blue, which is routinely used for antibodies expressed on the surface of phage, were expressed at very low yields . Therefore, a study was undertaken to determine optimal growth conditions for maximal expression of bovine Fab fragments in E . coli . By varying the E . coli strain, and the temperature and length of the culture growth, we were able to substantially increase the yield of soluble Fab fragments . A high yield of Fab fragments was found in the culture growth medium, which enabled us to devise a rapid and simple single-step method for the purification of native (nondenatured) Fabs based on immobilized metal affinity chromatography against a six-histidine amino acid carboxyl-terminal extension of the heavy-chain constant region . Using these methods we were able to express and purify antigen-specific bovine Fab fragments from E . coli .

Anal Biochem, 2002 Feb 1, 301(1), 8 - 13
Silver electrode as a sensor for determination of zinc in cell cultivation medium; Kizek R et al.; Use of the silver electrode as a sensor for the monitoring of zinc in cell growth medium is described . Zinc at silver electrodes provides specific voltammetric signal, which is affected by solution components . Signals of zinc ions in phosphate buffer solutions with and without cell growth medium were compared . Common DMEM cell culture medium was used for the cultivation of a cell line of v-myb-transformed chicken monoblasts and its variants expressing v-jun and c-jun in a zinc-dependent manner . Electrochemical results showed zinc concentrations in the medium coincide very well with the jun expression . With respect to the low toxicity of silver for eukaryotic cells, silver electrodes represent promising tools for the determination of zinc concentrations in vivo without the potential risk of a cell culture damage.

Phytother Res, 2002 Feb, 16(1), 43 - 7
Effect of a lipopolysaccharide from E . coli on the proliferation of fibroblasts and keratinocytes in vitro; Yang H et al.; Studies previously conducted in our laboratory have shown that an extract from the leaves of Chromo-laena odorata is mitogenic for human skin fibroblasts and keratinocytes . However, lipopolysaccharides, sometimes present in plant extracts, can also play a role in cell growth and might have been responsible for or contributed to the mitogenic activity observed . The present study aimed to investigate whether a lipopolysaccharide would have any effect on the proliferation of human fibroblasts and keratinocytes . Cells were seeded in 96-well plates and concentrations from 0.0 to 5.0 microg/mL of lipopolysaccharide in basal or growth medium were added . Cell growth was determined over a period of 10 days using a colorimetric assay . Lipopolysaccharide at concentrations between 0.05 microg/mL and 0.5 microg/mL in the growth medium significantly stimulated fibroblast proliferation after incubation for more than 6 days . In basal medium, more than 8 days of incubation was needed for significant stimulation of growth . Lipopolysaccharide stimulation of keratinocytes was evident at 0.5 microg/mL by day 3 in basal medium and by day 5 in growth medium . Although the lipopolysaccharide did stimulate cell growth it did so only at higher concentrations than were present in our plant extracts and to a lesser degree .

Planta, 2001 Dec, 214(2), 189 - 95
Phenols and phenol oxidases are involved in cadmium accumulation in the water plants Nymphoides peltata (Menyanthaceae) and Nymphaeae (Nymphaeaceae); Lavid N et al.; This comparative study investigates the mechanism of cadmium accumulation in the semiaquatic plant Nymphoides peltata (Menyanthaceae) and the aquatic plant Nymphaea (Nymphaeaceae) . It was conducted as part of an ongoing study of the use of water plants for phytoremediation . Epidermal structures, known as hydropotes, are located on the abaxial epidermis of the leaf laminae of Nymphoides peltata and are shown to contain phenols, peroxidase and polyphenol oxidase activities . When plants are subjected to 50 mg/l of cadmium in the growth medium, these hydropotes accumulate cadmium . Cadmium-induced increases in phenols, peroxidase and polyphenol oxidase activities were determined in plant extracts . Cadmium binding by polymerized phenols was demonstrated in vivo . In comparison with Nymphaeae epidermal glands, N . peltata hydropotes are larger, open, and create bigger crystal, the latter principally composed of calcium and, proportionally, less cadmium . Although both plants showed similar levels of cadmium accumulation, N . peltata was sensitive while Nymphaeae was resistant to this cadmium level . It is suggested that in these water plants the main mechanism for cadmium accumulation is based on the trapping of cadmium crystals by polymerized phenols in specialized epidermal structures and this is due to peroxidase and polyphenol oxidase activities . Nymphaeae, with greater peroxidase activity and more polyphenols, is more resistant to this heavy metal than N . peltata.

Ann N Y Acad Sci, 2001 Nov, 944, 135 - 43
Mass production of embryoid bodies in microbeads; Magyar JP et al.; Embryonic stem cells (ESC) are totipotent cells that can differentiate into a large number of different cell types . Stem cell-derived, differentiated cells are of increasing importance as a potential source for non-proliferating cells (e.g., cardiomyocytes or neurons) for future tissue engineering applications . Differentiation of ESC is initiated by the formation of embryoid bodies (EB) . Current protocols for the generation of EB are either of limited productivity or deliver EB with a large variation in size and differentiation state . To establish an efficient and robust EB production process, we encapsulated mouse ESC into alginate microbeads using various microencapsulation technologies . Microencapsulation and culturing of ESC in 1.1% alginate microbeads gives rise to discoid colonies, which further differentiate within the beads to cystic EB and later to EB containing spontaneously beating areas . However, if ESC are encapsulated into 1.6% alginate microbeads, differentiation is inhibited at the morula-like stage, so that no cystic EB can be formed within the beads . ESC colonies, which are released from 1.6% alginate microbeads, can further differentiate to cystic EB with beating cardiomyocytes . Extended supplementation of the growth medium with retinoic acid promotes differentiation to smooth muscle cells.

Appl Environ Microbiol, 1996 Sep, 62(9), 3107 - 11
Nisin resistance distinguishes Mycoplasma spp . from Acholeplasma spp . and provides a basis for selective growth media; Abu-Amero KK et al.; The sensitivity of 11 Mycoplasma and 5 Acholeplasma species to the bacteriocin nisin was determined . When applied on filter paper discs to lawns of acholeplasma cells, nisin (20 nmol per disc) gave 3.5- to 7.0-mm zones of growth inhibition . The inclusion of 0.2 mM nisin in agar medium reduced the number of Acholeplasma laidlawii colonies by a factor of more than 10(6), and in a salts solution, 75 microM nisin killed more than 99.9% of cells within 1 min . Under similar conditions, nisin had no significant effect upon the growth or survival of Mycoplasma species . At low concentrations (1 to 3 microM), nisin stimulated glucose oxidation by A . laidlawii and Acholeplasma oculi . However, in comparison with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a recognized protonophore and uncoupler of respiration, the maximum extent of stimulation was low, < or = 20%, compared with up to 180% for CCCP . Also, in contrast to results obtained with CCCP, at concentrations only slightly above those causing stimulation of acholeplasma oxygen uptake, nisin strongly inhibited respiration . Inhibition of oxygen uptake was greater for A . laidlawii cells grown in the absence of cholesterol, and on agar medium, growth inhibition by nisin decreased with increasing concentrations of cholesterol . Nisin resistance may be a valuable characteristic in the selection and identification of Mycoplasma spp.

Int J Cancer, 2002 Jan 10, 97(2), 135 - 41
Inhibition of spontaneous metastases formation by amifostine; Grdina DJ et al.; Amifostine was investigated for its ability to inhibit spontaneous metastases formation using the well-characterized murine sarcoma, Sa-NH . Amifostine was administered intraperitoneally at a dose of 50 mg/kg every other day for 6 days to C3Hf/Kam mice until tumors reached an average size of 8-8.5 mm in diameter . Amifostine was again administered immediately after surgical removal of the tumor-bearing limbs by amputation, and then once more 2 days later . Twenty-one days later, animals were evaluated for the presence of spontaneously developed pulmonary metastases . Nontumor-bearing control animals were sham treated using the same dosing and surgery schedules . Treatment with amifostine appeared to slightly delay tumor growth, that is, 13 vs . 12 days for tumors to reach an average diameter of 8 mm . Amifostine reduced both the incidence of pulmonary metastases formed in experimental animals from 77% to 57% (p < 0.05), and their average number per animal from 12.8 +/- 5.4 (SEM) to 2.9 +/- 1.1 (SEM) . The effect of amifostine exposure on serum levels of the angiogenesis inhibitor angiostatin was also determined using Western blot analysis . Consistent with the antimetastatic effect, exposure of animals to 50 mg/kg of amifostine resulted in a 4-fold enhanced serum level of angiostatin above control levels . This phenomenon occurred in tumor-bearing and nontumor-bearing animals . The effects of amifostine on matrix metalloproteinase (MMP) enzymatic activity was also determined using gelatin zymography . Conditioned growth medium collected from Sa-NH cells grown to confluency was exposed to various concentrations of SH, i.e., 2-{(aminopropyl)amino}ethane-thiol (WR-1065), the active thiol form of amifostine, for either 30 min or 18 hr . WR-1065, as a function of increasing dose and time, inhibited the enzymatic activities of MMP-2 and MMP-9 . At a concentration and time of exposure likely to be achieved in vivo, that is, 40 microM and 30 min, MMP-2 and MMP-9 activities were reduced to between 30% and 40% of control values . Consistent with these affects, WR-1065 was also found to be effective in inhibiting the ability of Sa-NH cells to migrate through Matrigel membranes . After an 18-hr exposure under in vitro conditions, WR-1065 at concentrations of 4, 40 and 400 microM, and 4 mM, inhibited Sa-NH migration to 11%, 44%, 81% and 97% of control values, respectively . The abilities of amifostine and its active thiol WR-1065 to stimulate angiostatin production in mice, and to inhibit the MMP enzymatic activities and invasion ability of Sa-NH cells under in vitro conditions, are consistent with the observed antimetastatic effects exhibited against Sa-NH tumors growing in vivo .

Appl Environ Microbiol, 2002 Jan, 68(1), 31 - 6
Novel alkylsulfatases required for biodegradation of the branched primary alkyl sulfate surfactant 2-butyloctyl sulfate; Ellis AJ et al.; Recent reports show that contrary to common perception, branched alkyl sulfate surfactants are readily biodegradable in standard biodegradability tests . We report here the isolation of bacteria capable of biodegrading 2-butyloctyl sulfate and the identification of novel enzymes that initiate the process . Enrichment culturing from activated sewage sludge yielded several strains capable of growth on 2-butyloctyl sulfate . Of these, two were selected for further study and identified as members of the genus PSEUDOMONAS: Strain AE-A was able to utilize either sodium dodecyl sulfate (SDS) or 2-butyloctyl sulfate as a carbon and energy source for growth, but strain AE-D utilized only the latter . Depending on growth conditions, strain AE-A produced up to three alkylsulfatases, as shown by polyacrylamide gel electrophoresis zymography . Growth on either SDS or 2-butyloctyl sulfate or in nutrient broth produced an apparently constitutive, nonspecific primary alkylsulfatase, AP1, weakly active on SDS and on 2-butyloctyl sulfate . Growth on 2-butyloctyl sulfate produced a second enzyme, AP2, active on 2-butyloctyl sulfate but not on SDS, and growth on SDS produced a third enzyme, AP3, active on SDS but not on 2-butyloctyl sulfate . In contrast, strain AE-D, when grown on 2-butyloctyl sulfate (no growth on SDS), produced a single enzyme, DP1, active on 2-butyloctyl sulfate but not on SDS . DP1 was not produced in broth cultures . DP1 was induced when residual 2-butyloctyl sulfate was present in the growth medium, but the enzyme disappeared when the substrate was exhausted . Gas chromatographic analysis of products of incubating 2-butyloctyl sulfate with DP1 in gels revealed the formation of 2-butyloctanol, showing the enzyme to be a true sulfatase . In contrast, Pseudomonas sp . strain C12B, well known for its ability to degrade linear SDS, was unable to grow on 2-butyloctyl sulfate, and its alkylsulfatases responsible for initiating the degradation of SDS by releasing the parent alcohol exhibited no hydrolytic activity on 2-butyloctyl sulfate . DP1 and the analogous AP2 are thus new alkylsulfatase enzymes with novel specificity toward 2-butyloctyl sulfate.

J Am Coll Nutr, 2001 Dec, 20(6), 628 - 36
Alpha tocopheryl succinate, retinoic acid and polar carotenoids enhanced the growth-inhibitory effect of a cholesterol-lowering drug on immortalized and transformed nerve cells in culture; Kumar B et al.; OBJECTIVE: Statins (cholesterol lowering drugs) with a closed-ring structure (lovastatin, simvastatin and mevastatin) and an open-ring structure (pravastatin and fluvastatin) are currently used in the management of cardiac disease . Lovastatin and simvastatin inhibit the growth of tumor cells; however, the studies on the effect of a statin in combination with micronutrients such as alpha-tocopheryl succinate (alpha-TS), 13-cis retinoic acid (RA) and polar carotenoids (PC) have never been performed . The primary objective of this study was to investigate the effect of mevastatin alone and in combination with the above micronutrients on the growth of mouse neuroblastoma (NB) cells and rat immortalized dopamine (DA) neurons in culture . In addition, a comparative efficacy of mevastatin and pravastatin on the growth of NB cells was studied . METHODS: Cells were treated with mevastatin in combination with individual antioxidants, alpha-TS, RA and polar carotenoids, 24 hours after plating . Fresh growth medium and agents were changed at two days after treatment, and the viability in control and experimental groups was determined at three days after treatment by MTT assay . Each experiment was repeated three times with triplicate samples per treatment . Growth in experimental groups was expressed as % of untreated cells . RESULTS: Mevastatin inhibited the growth of neuroblastoma (NB) cells and immortalized, non-tumorigenic dopamine (DA) neurons in culture in a dose-dependent manner . Immortalized DA neurons were more sensitive to mevastatin than NB cells . Pravastatin at similar concentrations was ineffective in inhibiting the growth of NB cells . Mevastatin in combination with alpha-TS, RA or PC was more effective in reducing the growth of NB and DA neurons than the individual agents . CONCLUSIONS: Statins with a closed-ring structure can inhibit the growth of established cancer cells as well as immortalized cells (equivalent to pre-malignant lesion), whereas statins with an open-ring structure may be ineffective . A combination of a statin having a closed-ring structure with alpha-TS, RA and PC may be one of the potentially useful anti-cancer agents for prevention and treatment strategies.

Biol Chem, 2001 Nov, 382(11), 1563 - 73
Elimination of hydrocortisone from the medium enables tissue plasminogen activator gene expression by normal and immortalized nonmalignant human epithelial cells; Myohanen H et al.; Human cervical epithelial cells transfected and immortalized with human papillomavirus type 16 DNA (HCE16/3) can be, like many other epithelial cells, normally grown in medium supplemented with epidermal growth factor, cholera toxin, hydrocortisone, insulin, transferrin, thyroid hormone and serum . We found that hydrocortisone diminished tissue plasminogen activator (tPA) production to an undetectable level . The removal of hydrocortisone increased urokinase plasminogen activator (uPA) activity within 24-48 h and tPA activity within 48-72 h, and converted the cells to a more elongated and fibroblastic phenotype . Upregulation of uPA mRNA was seen as early as at 3 h and of tPA mRNA within 48-72 h . Higher molecular weight forms (97-110 kDa) of plasminogen activators were seen in zymograms, apparently complexed with PAI-1, starting at 6 h both in the presence and absence of hydrocortisone . Immunoprecipitation with a PAI-1 monoclonal antibody confirmed that both uPA and tPA were complexed . We also studied normal diploid human bronchial epithelial cells (NHBE) and NHBE cells transformed with an adeno-12/SV40 hybrid virus (BEAS-2B) . In both types of nonmalignant epithelial cells, the removal of hydrocortisone increased uPA activity . The omission of hydrocortisone increased tPA levels significantly in BEAS-2B cell cultures, and in NHBE cell cultures tPA became detectable at 72 h . No PA complexes were seen in these two cell types . We conclude that normal and immortalized nonmalignant epithelial cells produce tPA, but only if hydrocortisone is omitted in the growth medium.

Res Microbiol, 2001 Dec, 152(10), 849 - 59
A transcriptional luxAB reporter fusion responding to fluorene in Sphingomonas sp . LB126 and its initial characterisation for whole-cell bioreporter purposes; Bastiaens L et al.; The promoter probe mini-Tn5-luxAB-tet was used to create a luxAB transcriptional fusion responding to fluorene in the fluorene utilising bacterium Sphingomonas sp . LB126 . The mutant strain, named L-132, was impaired in fluorene utilisation and strongly emitted light upon addition of fluorene to the growth medium . L-132 was initially characterised and examined for its potential use as a whole-cell biosensor in the perspective of quantifying fluorene in environmental samples . Activity of the reporter gene as a response to fluorene was detectable after 30 min and was optimal after 4 h . A linear response to fluorene concentrations within the water solubility range was achieved, with a detection limit of 200 microg per litre . Besides fluorene, L-132 weakly responded to the polycyclic aromatic hydrocarbons phenanthrene and dibenzothiophene, whereas strong responses were obtained with 9-fluorenone, 9-hydroxyfluorene, phthalic acid and protocatechuic acid . The latter four compounds are metabolites formed in course of fluorene degradation, which suggested that a fluorene metabolite rather than fluorene itself was the true inducer of the luxAB fusion in L-132.

Folia Histochem Cytobiol, 2001, 39(4), 295 - 300
Diversity of myosin heavy chain expression in satellite cells from mouse soleus and EDL muscles; Bryla K et al.; Postnatal myoblasts, the satellite cells, originating from slow and fast skeletal muscle fibres differentiate and fuse into myotubes expressing different phenotype of myosin heavy chain (MyHC) isoforms . Little is known, however, of factors which establish and maintain this phenotypic diversity . We used immunofluorescent labelling and Western blotting to examine the expression of slow and fast MyHC isoforms in myotubes formed in vitro from satellite cells isolated from mouse fast twitch extensor digitorum longus (EDL) and slow twitch soleus muscles . Satellite cells were cultured in serum-rich growth medium promoting myoblast proliferation until cross-striated and self-contracting myotubes were formed . We report that in both cultures myotubes expressed slow as well as fast MyHC isoforms, but the level of slow MyHC was higher in soleus culture than in EDL culture . Hence, the pattern of expression of slow and fast MyHC was characteristic of the muscle fibre type from which these cells derive . These results support the concept of phenotypic diversity among satellite cells in mature skeletal muscles and suggest that this diversity is generated in vitro irrespectively of serum mitogens.

Can J Microbiol, 2001 Nov, 47(11), 1053 - 7
Salicylate inhibits the translation and transcription of ompF in Escherichia coli; Ramani N et al.; OmpF is a major outer membrane protein in Escherichia coli whose expression is regulated by a large number of factors, including the osmolarity of the growth medium and the concentration of salicylate . We have previously shown that at low osmolarity, OmpF is post-transcriptionally regulated by micF mRNA, and that at high osmolarity, regulation occurs primarily by the inhibition of transcription by OmpR (Ramani et al . 1994) . In contrast, salicylate was reported to alter OmpF expression solely by blocking translation primarily through micF mRNA (Rosner et al . 1991) . We examined the effect of salicylate by analyzing the levels of OmpF in wild-type and micF strains grown with salicylate . At low concentrations of salicylate (0-4 mM), OmpF levels were inhibited strongly in wild-type cells, whereas no inhibition of OmpF was observed in the micF strain . At high concentrations of salicylate (10-20 mM), both the wild type and the micF strain showed strong inhibition of OmpF . To study the effect of salicylate on transcription, ompF mRNA and micF mRNA were analyzed in wild-type cells . micF mRNA levels increased during growth with 1, 2, and 4 mM salicylate . In contrast, ompF mRNA levels were not affected by low concentrations of salicylate, but decreased strongly at 10 and 20 mM salicylate . Taken together, these results suggest that similar to osmoregulation, salicylate inhibits both the translation and transcription of ompF.

Res Microbiol, 2001 Nov, 152(9), 793 - 8
Effect of O2, H2 and redox potential on the activity and synthesis of hydrogenase 2 in Escherichia coli; Laurinavichene TV et al.; The aim of this work was to study the influence of O2 with special emphasis on low oxygen tension, the effect of H2 under various conditions of oxygen tension and the influence of the redox potential in the growth medium on hydrogenase 2 of Escherichia coli . The hydrogenase activity and the content of the large (HybC) and small (HybO) subunits of hydrogenase 2 were compared during turbidostat cultivation in a wild strain and mutant HDK103 lacking hydrogenases 1 and 3 . No hydrogenase 2 activity in the mutant HDK103 was observed under aerobic conditions, but it was maximal under anaerobic conditions and half-maximal at an oxygen tension of approximately 4 mbar as is common for enzymes of anaerobic respiration . The content of hydrogenase 2 in both the strains was maximal under anaerobic conditions . In the wild strain, H2 addition enhanced hydrogenase activity and the HybO content under microaerobic conditions only . Under anaerobic conditions endogenous H2 production hindered this effect . Under aerobic conditions, the 02-related negative effect seemed to dominate over the H2-related positive effect . By contrast, in the mutant HDK103, hydrogen influenced neither hydrogenase 2 activity nor its content . A possible role of hydrogenase I in the response of hydrogenase 2 to hydrogen is discussed . Under conditions of different O2 tension, hydrogenase activity in both strains correlated inversely with the value of the redox potential of the medium . The presence of H2 changed this dependence . Thus, the value of the redox potential itself is not a controlling factor for hydrogenase 2.

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 2001 Sep, 15(5), 265 - 8
{Culture of human muscle satellite cell}; Huang HW et al.; OBJECTIVE: To investigate the biological characteristics of human muscle satellite cell cultured in vitro . METHODS: Human muscle satellite cells were obtained from skeletal muscle biopsies of six patients during corrective orthopedic surgery, cultivated in growth medium for ten days, then in differentiation medium for additional five days . Human satellite cells were identified with monoclonal antibody against desmin . Cells were observed under phase contrast microscopy . RESULTS: Human muscle satellite cells proliferated in growth medium, and fused to form myotubes in differentiation medium . After 24 hours in differentiation medium, the confluent satellite cells began to fuse actively and achieved the top level at 72 hours . CONCLUSION: Human muscle satellite cell can proliferate and differentiate in appropriate culture condition . Immunocytochemical detection of desmin is the effective early method to determine satellite cell.

Antonie Van Leeuwenhoek, 2001 Oct, 80(1), 93 - 9
Bioprospecting for novel oxylipins in fungi: the presence of 3-hydroxy oxylipins in Pilobolus; Kock JL et al.; As previously found in various members of the Mucorales, 3-hydroxy oxylipins in Mucor genevensis are associated with the sporangia, i.e . mainly the columella structure and between aggregating sporangiospores . To determine if this phenomenon is also true in distantly related members, the mucoralean fungus Pilobolus was examined . This fungus is characterized by relatively large sub sporangial-columella structures which actively eject sporangia in a sticky liquid for attachment onto herbage surrounding its growth medium--in this case horse dung . Strikingly, this fungus produced a novel oxylipin i.e . a 3-hydroxy monounsaturated fatty acid, possibly a nonenoic acid, which is mainly associated with the sub sporangial-columella structure and aggregating sporangiospores . The specificity of the antibody against 3-hydroxy oxylipins used in immunofluorescence mapping of the mucoralean fungi, was further confirmed in the yeast, Saccharomycopsis malanga which produces 3-hydroxy palmitate in crystal form . These crystals occur between aggregating yeast cells . On the basis of the available data, we hypothesize that 3-hydroxy oxylipins probably function as adhesives, attaching fungal cells to each other or to other surfaces through entropic based hydrophobic forces and/or hydrogen bonds.

Appl Microbiol Biotechnol, 2001 Oct, 57(3), 329 - 33
Highly efficient Aerococcus viridans L-alpha-glycerophosphate oxidase production in the presence of H2O2-decomposing agent: purification and kinetic characterization; Streitenberger SA et al.; Glycerophosphate oxidase was purified from Aerococcus viridans cells by phase partitioning in Triton X-114, ammonium sulfate fractionation, FPLC ion-exchange chromatography and FPLC hydrophobic-interaction chromatography . The purification achieved from a crude extract of A . viridans was 38-fold with a 32% recovery of activity . Under the growth conditions used, A . viridans strain CECT 978 proved to be an excellent glycerophosphate-oxidase producer, with enzyme production 2,800-fold greater than that described in the literature for the same microorganism . The culture medium used in the present work is that commonly used for cultivation of this microorganism, except that an H2O2-decomposing enzyme was added . The addition of catalase to the growth medium had a clear effect on the growth rate . Furthermore, methylglyoxal, a metabolite that is formed enzymatically from triose phosphates, was found to be an inactivator of glycerophosphate oxidase activity.

J Biol Chem, 2002 Mar 8, 277(10), 8187 - 93 Epub 2001 Dec 18.
Site-directed mutagenesis reveals the essentiality of the conserved residues in the putative diiron active site of the trypanosome alternative oxidase; Ajayi WU et al.; Trypanosoma brucei possesses a non-cytochrome, salicylhydroxamic acid (SHAM)-sensitive ubiquinol:oxygen oxidoreductase known as trypanosome alternative oxidase (TAO) . TAO and similar SHAM-sensitive alternative oxidases (AOXs) contain 2-3 conserved diiron-binding motifs (EXXH) . Site-directed mutagenesis of residues H165A, E214A, E266A, and H269L within the conserved EXXH motif abolished the ability of TAO to complement the heme-deficient Escherichia coli strain GE1387 . These mutations also reduced the growth of this E . coli auxotroph to about 85% of the control cells containing wild type TAO . In contrast, mutation of residues outside the EXXH motifs, e.g . V205A, L243A, C261A, and V271A, had little effect on complementation, and the reduction in the cell growth was about 5-10% . Mutations of the putative iron-binding residues within the EXXH motifs of TAO abolished the ability to confer SHAM-sensitive respiration to E . coli heme mutant, whereas mutations of the non-conserved/non-iron binding residues resulted in 20-30% reduction of SHAM-sensitive respiration of the E . coli auxotroph . Immunoblot analysis of the total cellular protein of transformed E . coli revealed that the expression level of mutated and wild type TAO (35 kDa) remained unaltered . Mutation at C261A produced a truncated but functional protein of 28 kDa . The addition of ortho-phenanthroline to the growth medium produces a non-functional TAO . The effect of ortho-phenanthroline on the activity of TAO was completely alleviated by the addition of iron in the medium, which suggests that iron is needed for the activity of TAO . This work demonstrates that His-165, Glu-214, Glu-266, and His-269 and the presence of iron are essential for the activity of TAO.

Biotechnol Bioeng, 2001 Dec 5, 75(5), 504 - 9
Acetate-inducible protein overexpression from the glnAp2 promoter of Escherichia coli; Farmer WR et al.; The Ntr regulon in Escherichia coli has previously been engineered to control the expression of a heterologous metabolic pathway . In this study, we reengineered the same system for protein production . In the absence of NRII (glnL gene product), we showed that glnAp2 can be an effective promoter for protein production that is inducible by exogenous acetate, but both the induction ratio and the range of modulation are low . To deal with this issue, we inactivated phosphotransacetylase (pta gene product), which disrupts the acetate pathway and denies the cell the ability to synthesize acetate . With this additional modification, gene expression from glnAp2 can be controlled by directly adding acetate into the growth medium . Using a lacZ reporter fusion, we found that glnAp2 induction was modulatable over a range of potassium acetate concentrations, and the induction/noninduction ratio increased to 77 in the absence of pta . The extracellular acetate required for maximal induction is lower than the concentration that causes toxicity, and thus growth inhibition by acetate addition was not a matter of concern . Furthermore, compared to the P(tac) promoter, overexpression of a model protein using the modified glnAp2 promoter system did not cause significant growth inhibition, although a higher level of protein expression was achieved .

Bull Exp Biol Med, 2001 Sep, 132(3), 856 - 60
Structure of cell clusters formed in cultures of dissociated human embryonic brain; Revishchin AV et al.; Cell clusters in a culture of dissociated brain from human fetuses at 8-12 weeks gestation in a serum-free growth medium were studied by immunohistochemical methods and electron microscopy . Heterogeneity of cell population in culture was demonstrated . Despite the influence of proliferation-stimulating factors, cell clusters contained not only nestin-immunopositive stem cells, but also beta-tubulin-, vimentin-, and GFAP-positive cells differentiating by the neural pathway . Stem cells were localized on the surface of clusters . The percentage of stem cells in large clusters was lower than in small clusters.

Eur J Biochem, 2001 Dec, 268(24), 6358 - 68
Antiproliferative effect of nitric oxide on rat glomerular mesangial cells via inhibition of mitogen-activated protein kinase; Chin TY et al.; The effect of nitric oxide (NO) donors and lipopolysaccharide (LPS) on the proliferation of rat glomerular mesangial cells was characterized . Exogenous application of a NO donor inhibited serum-induced proliferation in a time- and dose-dependent manner . S-Nitrosoglutathione (GSNO) also increased cGMP generation and arachidonic acid release, but it did not cause any measurable increase in the cytosolic Ca2+ concentration . Chelation of cytosolic Ca2+ or inhibition of mitogen-activated protein kinase (MAPK) kinase had an inhibitory effect on proliferation, but neither enhanced the antiproliferative effect of GSNO . In contrast, inhibition of guanylate cyclase or phospholipase A2 had no effect on proliferation, but partially reversed GSNO-induced antiproliferation by approximately 98 and 65%, respectively . GSNO did not cause cell death . Incubation of cells with LPS induced endogenous NO generation and had an antiproliferative effect . LPS-induced antiproliferation was reversed completely by inhibition of nitric oxide synthase and partially by inhibition of guanylate cyclase or phospholipase A2 . GSNO or LPS inhibited serum-induced MAPK activation, and both effects were partially reversed by inhibition of guanylate cyclase or phospholipase A2 . Inclusion of 8-bromo-cGMP or arachidonic acid in the growth medium resulted in a similar antiproliferative effect . In conclusion, in rat glomerular mesangial cells, MAPK inhibition and an antiproliferative effect could be induced by either an increase in the cellular concentration of NO or exposure of the cells to LPS . Part of the effect of NO was attributable to the increased cellular cGMP generation and arachidonic acid release.

Br J Haematol, 2001 Dec, 115(3), 595 - 604
Matrix metalloproteinase and tissue inhibitors of metalloproteinase secretion by haematopoietic and stromal precursors and their production in normal and leukaemic long-term marrow cultures; Marquez-Curtis LA et al.; Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate the turnover of the extracellular matrix and may modulate the biology of haematopoietic cells . We investigated whether MMPs and TIMPs are produced in long-term marrow cultures (LTMCs) established from normal donors and acute myelogenous leukaemia (AML) patients, and by fibroblast- (F), granulocyte macrophage- (GM) and megakaryocyte- (Meg) colony-forming unit (CFU) and erythroid burst-forming unit (BFU-E)-derived precursor cells . ProMMP-9 levels were highest (> 400 ng/ml) at week 1 of normal LTMC, whereas proMMP-2, TIMP-1, TIMP-2 and TIMP-3 levels peaked (up to 1000 ng/ml) after the establishment of the adherent layer . In LTMC from AML patients, these patterns of secretion were reversed . Moreover, we found that after a 24 h incubation in serum-free media, normal CFU-GM-, BFU-E- and CFU-Meg-derived cells secreted proMMP-9 and CFU-F-derived cells proMMP-2, in contrast to cells from LTMC adherent layer which secreted both active and latent forms of MMP-2 and MMP-9 under serum-free conditions . However, when these adherent cells were incubated in 12.5% fetal calf or horse serum or complete LTMC growth media, active forms of MMP-2 and MMP-9 were no longer detectable, and TIMP levels increased . Hence, we concluded that (i) MMPs/TIMPs are secreted by normal human bone marrow haematopoietic and stromal cells and may play an important role in intercellular cross-talk in haematopoiesis; and (ii) only latent forms of MMPs are present under LTMC conditions, indicating that the specific media used for weekly re-feeding of LTMC can block activation of MMP-2 and MMP-9, maintaining the integrity of the stromal layer and supporting haematopoiesis in vitro.

J Microbiol Methods, 2002 Jan, 48(1), 87 - 93
Development of the sexual reproductive cycle of Xanthophyllomyces dendrorhous; Retamales P et al.; Conditions inducing the development of holobasidia with terminal basidiospores in wild-type and astaxanthin mutant strains of Xanthophyllomyces dendrorhous were reexamined . Important factors for the development of holobasidia were the incubation temperature and the medium composition . A temperature of 9 degrees C was demonstrated to enhance holobasidia formation . Minimal growth medium with glucose as sole carbon source at concentrations between 80 and 120 mM, and ammonium nitrate with concentrations of 28 mM gave optimal results . A period of 20 or more days was needed for the formation of holobasidia with basidiospores . Additionally, mutant strains of X . dendrorhous were observed to have different abilities to produce holobasidia and strains obtained after protoplast fusion, which have been called fusant in this study, to have an increased capacity to form holobasidia.

Traffic, 2001 Nov, 2(11), 820 - 30
Sgf1p, a new component of the Sec34p/Sec35p complex; Kim DW et al.; Here we report the identification of SGF1 as a high-copy suppressor of the sec35-1 mutant . SGF1 encodes an essential hydrophilic protein of approximately 100 kDa . Using the yeast two-hybrid system and coprecipitation studies, we demonstrate that Sgf1p is a new subunit of the multiprotein Sec34p/Sec35p complex . Reduced levels of Sgf1p lead to the accumulation of a variety of membranes as well as a kinetic block in endoplasmic reticulum to Golgi traffic . Immunofluorescence studies demonstrate that Sec34p is found throughout the Golgi, with a high concentration on early Golgi . Although an earlier study suggested that Sec34p (Grd20p) is not required for protein secretion, we show here that the sec34-2 and sec35-1 mutations lead to a pleiotropic block in the secretion of all proteins into the growth medium.

Sci Prog, 2001, 84(Pt 3), 205 - 33
Extracellular sensing and signalling pheromones switch-on thermotolerance and other stress responses in Escherichia coli; Rowbury RJ et al.; The findings reviewed here overturn a major tenet of bacterial physiology, namely that stimuli which switch-on inducible responses are always detected by intracellular sensors, with all other components and stages in induction also being intracellular . Such an induction mechanism even applies to quorum-sensed responses, and some others which involve functioning of extracellular components, and had previously been believed to occur in all cases . In contrast, for the stress responses reviewed here, triggering is by a quite distinct process, pairs of extracellular components being involved, with the stress sensing component (the extracellular sensing component, ESC) and the signalling component, which derives from it and induces the stress (the extracellular induction component, EIC), being extracellular and the stimulus detection occurring in the growth medium . The ESCs and EICs can also be referred to as extracellular sensing and signalling pheromones, since they are not only needed for induction in the stressed culture, but can act as pheromones in the same region activating other organisms which fail to produce the extracellular component (EC) pair . They can also diffuse to other regions and there act as pheromones influencing unstressed organisms or those which fail to produce such ECs . The cross-talk occurring due to such interactions, can then switch-on stress responses in such unstressed organisms and in those which cannot form the ESC/EIC pair . Accordingly, the ESC/EIC pairs can bring about a form of intercellular communication between organisms . If the unstressed organisms, which are induced to stress tolerance by such extracellular components, are facing impending stress challenge, then the pheromonal activities of the ECs provide an early warning system against stress . The specific ESC/EIC pairs switch-on numerous responses; often these pairs are proteins, but non-protein ECs also occur and for a few systems, full induction needs two ESC/EIC pairs . Most of the above ECs needed for response induction are highly resistant to irreversible inactivation by lethal agents and conditions and, accordingly, many killed cultures still contain ESCs or EICs . If these killed cultures come into contact with unstressed living organisms, the ECs again act pheromonally, altering the tolerance to stress of the living organisms . It has been claimed that bacteria sense increased temperature using ribosomes or the DnaK gene product . The work reviewed here shows that, for thermal triggering of thermotolerance and acid tolerance in E . coli, it is ESCs which act as thermometers.

Plant Mol Biol, 2001 Nov, 47(5), 633 - 40
Organ specificity of a vacuolar Ca2+-binding protein RVCaB in radish and its expression under Ca2+-deficient conditions; Yuasa K et al.; Radish vacuoles contain a new type of Ca2+-binding protein (RVCaB) with high capacity and low affinity for Ca2+ . The protein is able to stimulate Ca2+ uptake into vacuoles, which is driven by Ca2+-ATPase and Ca2+/H+ antiporter . In the present study, we found that the level of RVCaB mRNA is high in seedling hypocotyls and mature taproots but low in young roots and mature leaves . The RVCaB protein was abundant in hypocotyls and taproots but absent in leaves . The levels of the transcript and protein of RVCaB in taproots were gradually increased during maturation . The level of RVCaB mRNA in seedling hypocotyls doubled within a few hours when the growth medium was changed from 10 mM CaCl2 to water, although the level was strongly suppressed in 100 mM CaCl2 . This response of the RVCaB gene was specific to Ca2+ and did not occur with other ions including K+ and Mg2+ . RVCaB functioning as a Ca2+-sequestering protein in taproot vacuoles to provide for the Ca2+ deficiency is discussed.

J Vet Diagn Invest, 2001 Nov, 13(6), 508 - 13
Application of a PCR assay to enhance the detection and identification of Tritrichomonas foetus in cultured preputial samples; Parker S et al.; The traditional diagnostic test for Tritrichomonas foetus involves collection of preputial or vaginal samples followed by culture in a growth media and microscopic examination . Recently, polymerase chain reaction (PCR) techniques have been described for use as a diagnostic assay . The objective of this study was to evaluate a previously described PCR assay for detecting T . foetus in cultured preputial material . The detection limits of the assay for T . foetus organisms in a growth medium, in samples prepared from washing microscope slides, and in preputial material cultured in a growth medium were determined . Preputial samples were collected from 13 bulls uninfected with T . foetus . The PCR assay was able to detect 5 T . foetus organisms in the growth medium and the cultured preputial material . Amplification products were obtained from samples prepared from washes of microscope slides containing as few as 3 visualized organisms . The PCR assay was able to detect organisms in culture at a lower concentration than was possible by direct microscopic examination . This low detection limit may allow the PCR assay to be used to enhance the sensitivity of the current diagnostic test . In addition, the assay could be used to confirm the identification of T . foetus organisms observed by direct microscopic examination when other confirmation techniques, such as staining and phase microscopy, are not practical.

Mol Biotechnol, 2001 Nov, 19(3), 239 - 44
A quantitative ELISA for monitoring the secretion of ZZ-fusion proteins using SpA domain as immunodetection reporter system; Mergulhao FJ et al.; A sandwich-type enzyme-linked immunosorbent assay (ELISA) was established for monitoring the secretion of ZZ-fusion proteins . Two antibodies, a monoclonal mouse anti-human proinsulin and a rabbit anti-bovine IgG (strongly binding to the ZZ-domain), were used to quantify the secretion of recombinant human ZZ-proinsulin to the growth medium of Escherichia coli cultures . The method here reported conjugates the advantages of sandwich-type ELISA assays, namely, high sensitivity, specificity, and throughput, with the possibility of quantifying small protein molecules (e.g., peptides) . A further advantage of gene fusion techniques integrating both downstream processing and product detection and quantitation is highlighted . The method is capable of detecting levels of 0.05 ng of ZZ-proinsulin.

J Chem Neuroanat, 2001 Nov, 22(4), 263 - 73
Mouse retina explants after long-term culture in serum free medium; Caffe AR et al.; The neonatal mouse retina remains viable as an explant in serum-supplemented growth media for more than 4 weeks . Interpretation of drug effects on this tissue is compromised by the enigmatic composition of the serum . We sought to remove this ambiguity by culturing neonatal as well as late postnatal mouse retina in serum-free nutrient medium . In this study three important observations were made, (1) there is histotypic development of neonatal as well as preservation of late postnatal mouse retinal structure during long-term culture in serum-free medium, although the late postnatal tissue tends to show some loss of cells in the outer nuclear layer . (2) Protein expression in explant photoreceptor cells was similar to that in the litter-matched ones, except for green cone opsin and interphotoreceptor retinoid-binding protein, although mRNA of the latter is present at similar amounts as in age-matched in vivo controls . (3) Cells of the inner retina stained by antibodies to calcium-binding proteins display some novel sprouting of processes . The results show that the mouse retina can be cultured as an explant for more than 4 weeks in a serum-free medium . This represents an important step forward because, (1) the possibility of interference of drug effects by unknown serum factors has been eliminated; and (2) the spent culture medium can be analyzed to investigate biomolecules released by the retina in vitro.

J Microbiol Methods, 2001 Dec, 47(3), 307 - 13
Cryopreservation of basidiomycete strains using perlite; Homolka L et al.; A new alternative method using perlite as a particulate solid carrier in the growth medium with a cryoprotectant was successfully tested for cryopreservation of several basidiomycete species from different genera (Armillaria, Pleurotus, Pluteus, Polyporus) which failed to survive or retain their properties in cryopreservation procedures routinely used in our laboratory . Frozen basidiomycete strains were kept in cryovials submerged in liquid nitrogen and were either immediately after the freezing process or after a 6-month storage thawed and checked for viability, purity and changes in growth, morphology and biochemical characteristics . All cultures survived the cryopreservation procedure and no negative effects of cryopreservation by this method have been observed after 6 months of storage in liquid nitrogen.

In Vitro Cell Dev Biol Anim, 2001 Oct, 37(9), 618 - 23
Lipid peroxidation and growth inhibition of human microvascular endothelial cells; Hostmark AT et al.; Peroxidation products of polyunsaturated fatty acids may cause growth inhibition of cells in culture . This study was carried out to elucidate to what extent peroxidation products may be found in growth media, with and without cells and albumin, using thiobarbituric acid-reactive substances (TBARS) and protein carbonyl groups as measures of peroxidation . The growth of human microvascular endothelial cells was studied as influenced by docosahexaenoic (C22:6, n - 3), arachidonic acid (C20:4 . n - 6), and serum albumin . Cell growth was strongly inhibited by the fatty acids, and the inhibition was related to the concentration of TBARS in the medium . Defatted albumin (0.5 g/100 ml) nullified the increase of TBARS in the medium and released the growth inhibition by the fatty acids . With polyunsaturated fatty acids (PUFA) there was a time- and concentration-dependent increase in media TBARS, observed both with and without cells, but the TBARS increase was somewhat greater in the presence of cells . Surprisingly, TBARS in cell-free media also increased somewhat upon increasing the albumin concentration from 0.5 to 5 g/100 ml, and the TBARS increase differed among various preparations of albumin . Unexpectedly, the albumin that had not been defatted gave the lowest TBARS values . The amount of protein carbonyl groups did not differ among various albumin preparations . It is concluded that PUFA may autooxidize in media used for cell cultures, and thereby cause an unspecific growth inhibition, which can be prevented by a low albumin concentration . However, even defatted albumin preparations may contain lipid peroxidation products, the causes and implications of which remain to be elucidated.

J Exp Bot, 2001 Dec, 52(365), 2291 - 300
Cellular compartmentation of nickel in the hyperaccumulators Alyssum lesbiacum, Alyssum bertolonii and Thlaspi goesingense; Kupper H et al.; Nickel uptake and cellular compartmentation were investigated in three Ni hyperaccumulators: Alyssum bertolonii (Desv), Alyssum lesbiacum (Candargy) and Thlaspi goesingense (Halacsy) . The three species showed similar hyperaccumulation of Ni, but T . goesingense was less tolerant to Ni than the two Alyssum species . An addition of 500 mg Ni kg(-1) to a nutrient-rich growth medium significantly increased shoot biomass of all three species, suggesting that the Ni hyperaccumulators have a higher requirement for Ni than normal plants . Energy-dispersive X-ray microanalysis (EDXA) was performed on frozen-hydrated tissues of leaves (all species) and stems (Alyssum only) . In all species analysed, Ni was distributed preferentially in the epidermal cells, most likely in the vacuoles, of the leaves and stems . In stems, there was a second peak of Ni in the boundary cells between the cortical parenchyma and the vascular cylinder . The non-glandular trichomes on the leaf surfaces of the two Alyssum species were highly enriched with Ca, but contained little Ni except in the base . In the leaves of T . goesingense, the large elongated epidermal cells contained more Ni than the cells of the stomatal complexes . The role of cellular compartmentation in Ni hyperaccumulation is discussed.

Sheng Wu Gong Cheng Xue Bao, 2001 Jul, 17(4), 463 - 6
{Optional growth medium and conditions for mass production of Pandora delphacis mycelia in submerged culture}; Liu ZQ et al.; The entomophthoraceous fungi are important microbial control agents for insect control but their mass production is usually difficult and expensive . To produce in large quantity the mycelia of the entomophthoraceous fungus, Pandora delphacis (isolate F95129), this study was aimed at replacing expensive components of liquid medium that is usually used in laboratory only with cheap materials easily available . Based on comparative experiments with primary components of several media designed and optional conditions for submerged culture, an appropriate medium was recognized that included(per liter) 10 g of homemade yeast extract, 10 g industrial fish meal, 10 g wheat bran, 15 g corn meal, 1.0 g KH2PO4, 3.0 g NH4NO3, and 0.25 g MgSO(4).7H2O . The optional conditions for submerged culture was 25-30 degrees C, initial pH 6.5, and 40% (V/V) of flask filled with the medium and 10% of initial inoculum (liquid culture containing mycelia) for inoculation . Using the selected medium and the conditions considered, 48 h culture resulted in a considerably high yield of dry mycelia(> 25 mg/mL) with desirable capacity of spore production.

Am J Pathol, 2001 Nov, 159(5), 1877 - 87
Histological organization in hepatocyte organoid cultures; Michalopoulos GK et al.; Hepatocytes and other cellular elements isolated by collagenase perfusion of the liver and maintained in defined culture conditions undergo a series of complex changes, including apoptosis and cell proliferation, to reconstruct tissue with specific architecture . Cultures in collagen-coated pleated surface roller bottles, with hepatocyte growth medium medium and in the presence of hepatocyte growth factor (HGF) and epidermal growth factor (EGF), form characteristic and reproducible tissue architecture composed of a superficial layer of biliary epithelial cells, an intermediate layer of connective tissue and hepatocytes, and a basal layer of endothelial cells . Dexamethasone, EGF, and HGF are required for the complete histological organization . Analysis of the structures formed demonstrates that the receptor tyrosine kinase ligands HGF and EGF are required for the presence, growth, and phenotypic maturation of the biliary epithelium on the surface of the cultures and for the formation of connective tissue in the cultures . Dexamethasone, in the presence of HGF and EGF, was required for the phenotypic maturation of hepatocytes . The results demonstrate the role of these molecules for the formation and phenotypic maturation of specific histological elements of the liver and suggest roles for these signaling molecules in the formation and structure of the in vivo hepatic architecture.

Nat Biotechnol, 2001 Nov, 19(11), 1060 - 5
Selection analyses of insertional mutants using subgenic-resolution arrays; Badarinarayana V et al.; We describe a method of genome-wide analysis of quantitative growth phenotypes using insertional mutagenesis and DNA microarrays . We applied the method to assess the fitness contributions of Escherichia coli gene domains under specific growth conditions . A transposon library was subjected to competitive growth selection in Luria-Bertani (LB) and in glucose minimal media . Transposon-containing genomic DNA fragments from the selected libraries were compared with the initial unselected transposon insertion library on DNA microarrays to identify insertions that affect fitness . Genes involved in the biosynthesis of nutrients not provided in the growth medium were found to be significantly enriched in the set of genes containing negatively selected insertions . The data also identify fitness contributions of several uncharacterized genes, including putative transcriptional regulators and enzymes . The applicability of this high-resolution array selection in other species is discussed.

J Basic Microbiol, 2001, 41(5), 231 - 40
Subcellular level variation in protein accumulation of the halophylic yeast Debaryomyces hansenii grown under conditions of high osmolarity; Calderon-Torres M et al.; We have analyzed electrophoretic profiles of polypeptides extracted from various cell compartments of the yeast Debaryomyces hansenii, cultured under high osmolarity and under control conditions . We tested the effect of high concentrations of solutes with an osmotic component (sorbitol), and with osmotic and ionic components combined (NaCl or KCl) . Densitometric analyses of the extracted polypeptides indicated that the stressing solutes had a differential effect on the relative concentration of total proteins as well as in proteins extracted from three subcellular compartments . Sorbitol caused a significant decrease in the concentration of various polypeptides associated with the mitochondria and the cytoplasm . By contrast, sodium ions elicited marked increases in concentration in four cytoplasmic polypeptides . KCl did not have a major effect in any of the subcellular compartments . Polypeptides were grouped as having a general osmotic response, or as having a response apparently modulated by the particular ionic environment of the growth medium . In all treatments, the number of polypeptides with an increase in their relative concentration was roughly similar to the number of polypeptides with a decrease in concentration, both relative to controls . Our results agree with previous observations on the complexity of the osmoregulatory response involving proteins whose concentration depends on the solute causing the stress . The results also indicate that subcellular compartments respond differently to stressors.

Appl Environ Microbiol, 2001 Nov, 67(11), 5017 - 24
Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli; Waterfield N et al.; Previous attempts to express the toxin complex genes of Photorhabdus luminescens W14 in Escherichia coli have failed to reconstitute their oral toxicity to the model insect Manduca sexta . Here we show that the combination of three genes, tcdA, tcdB, and tccC, is essential for oral toxicity to M . sexta when expression in E . coli is used . Further, when transcription from native toxin complex gene promoters is used, maximal toxicity in E . coli cultures is associated with the addition of mitomycin C to the growth medium . In contrast, the expression of tcdAB (or the homologous tcaABC operon) with no recombinant tccC homolog in a different P . luminescens strain, K122, is sufficient to confer oral toxicity on this strain, which is otherwise not orally toxic . We therefore infer that P . luminescens K122 carries a functional tccC-like homolog within its own genome, a hypothesis supported by Southern analysis . Recombinant toxins from both P . luminescens K122 and E . coli were purified as high-molecular-weight particulate preparations . Transmission electron micrograph (TEM) images of these particulate preparations showed that the expression of tcdAB (either with or without tccC) in E . coli produces visible approximately 25-nm-long complexes with a head and tail-like substructure . These data are consistent with a model whereby TcdAB constitutes the majority of the complex visible under TEM and TccC either is a toxin itself or is an activator of the complex . The implications for the potential mode of action of the toxin complex genes are discussed.

Biosens Bioelectron, 2001 Dec, 16(9-12), 773 - 82
A quartz crystal microbalance cell biosensor: detection of microtubule alterations in living cells at nM nocodazole concentrations; Marx KA et al.; The quartz crystal microbalance (QCM) was used to create a piezoelectric biosensor utilizing living endothelial cells (ECs) as the biological signal transduction element . ECs adhere to the hydrophilically treated gold QCM surface under growth media containing serum . At 24 h following cell addition, calibration curves were constructed relating the steady state Deltaf and DeltaR shift values observed to the numbers of electronically counted cells requiring trypsinization to be removed from the surface . We then utilized this EC QCM biosensor for the detection of the effect of {nocodazole} on the steady state Deltaf and DeltaR shift values . Nocodazole, a known microtubule binding drug, alters the cytoskeletal properties of living cells . At the doses used in these studies (0.11-15 microM), nocodazole, in a dose dependent fashion, causes the depolymerization of microtubules in living cells . This leads a monolayer of well spread ECs to gradually occupy a smaller area, lose cell to cell contact, exhibit actin stress fibers at the cell periphery and acquire a rounded cell shape . We observed the negative Deltaf shift values and the positive DeltaR shift values to increase significantly in magnitude over a 4-h incubation period following nocodazole addition, in a dose dependent fashion, with a transition midpoint of 900 nM . Fluorescence microscopy of the ECs, fixed on the gold QCM surface and stained for actin, demonstrated that the shape and cytoskeleton of ECs were affected by as little as 330 nM nocodazole . These results indicate that the EC QCM biosensor can be used for the study of EC attachment and to detect EC cytoskeletal alterations . We suggest the potential of this cellular biosensor for the real time identification or screening of all classes of biologically active drugs or biological macromolecules that affect cellular attachment, regardless of their molecular mechanism of action.

Mutat Res, 2001 Nov 15, 498(1-2), 129 - 33
The role of adrenal corticosteroids in induction of micronuclei by morphine; Sawant SG et al.; It has previously been shown that morphine can increase the frequency of micronucleated splenocytes when administered to mice, but not when cells are exposed to the opiate in vitro . Morphine treatment is also known to increase circulating levels of glucocorticosteroids, which have been reported to produce genetic damage in vivo and in vitro . In order to determine whether adrenal hormones might mediate the genotoxic effects of morphine, adrenalectomized and sham-operated mice were treated with morphine sulfate . In sham-operated animals administration of morphine produced a dose-related increase in the frequency of micronucleated cells, whereas adrenalectomy abolished the effect . When plasma from morphine-treated mice was used to supplement growth medium of untreated splenocytes, the frequency of micronucleated cells increased, an effect partially blocked by the steroid antagonist RU 486 . The N-methylmorphine, which does not stimulate the release of corticosterone from adrenal glands, induced micronuclei formation in splenocytes, and administration of metyrapone, an inhibitor of corticosterone biosynthesis, blocked the morphine-induced increase in corticosterone secretion, but had no effect on the frequency of micronuclei formation . These results indicate that basal levels of glucocorticosteroids are required for induction of micronuclei by morphine in murine splenocytes, but activation of the hypothalamo-pituitary-adrenal (HPA) axis by morphine does not contribute to the observed response.

Inorg Chem, 1997 Mar 12, 36(6), 1143 - 1148
Synthesis and Structure of One-, Two-, and Three-Dimensional Alkaline Earth Metal Gallium Nitrides: Sr(3)Ga(2)N(4), Ca(3)Ga(2)N(4), and Sr(3)Ga(3)N(5); Clarke SJ et al.; We report the structures of three new alkaline earth metal gallium nitrides synthesized as crystals from the elements in sealed Nb tubes at 760 degrees C using Na/Sr and Na/Ca melts as growth media . The materials are all transparent insulators . Yellow Sr(3)Ga(2)N(4) is isostructural with the previously reported Ba(3)Ga(2)N(4) and Sr(3)Al(2)N(4) . It crystallizes in Pnna (No . 52) with a = 5.9552(6) A, b = 10.2753(8) A, c = 9.5595(9) A, and Z = 4 . It contains infinite chains, {GaN(4/2)(3)(-)}, of trans-edge-shared tetrahedra extending along the b axis . Colorless Ca(3)Ga(2)N(4) is isostructural with Ca(3)Al(2)As(4) and gamma-Ca(3)Al(2)N(4) . It crystallizes in C2/c (No . 15) with a = 10.6901(11) A, b = 8.3655(7) A, c = 5.5701(4) A, beta = 91.194(6) degrees, and Z = 4 . It contains infinite sheets, {GaN(4/2)(3)(-)}, of edge- and corner-sharing tetrahedra in the bc plane . Orange-yellow Sr(3)Ga(3)N(5) has a new structure; it crystallizes in P&onemacr; (No . 2) with a = 5.9358(6) A, b = 7.2383(8) A, c = 8.6853(12) A, alpha = 108.332(10) degrees, beta = 103.783(9) degrees, gamma = 95.326(8) degrees, and Z = 2 . It is one of the few materials prepared from Na melts which has an infinite three-dimensional framework structure . It contains a {Ga(3)N(5)(6)(-)} framework of edge- and corner-sharing GaN(4) tetrahedra.

Inorg Chem, 1996 Nov 20, 35(24), 7009 - 7012
Synthesis and Structure of Two New Strontium Germanium Nitrides: Sr(3)Ge(2)N(2) and Sr(2)GeN(2); Clarke SJ et al.; We report the structures of two new strontium germanium nitrides synthesized as crystals from the elements in sealed Nb tubes at 750 degrees C using liquid Na as a growth medium . Sr(3)Ge(2)N(2) is isostructural with the previously reported Ba analogue . It crystallizes in P2(1)/m (No . 11), with a = 9.032(2) A, b = 3.883(1) A, c = 9.648(2) A and beta = 112.42(3) degrees, and has two formula units per unit cell . It contains GeN(2)(4)(-) units and additionally |Ge(2)(-) zigzag chains . Sr(2)GeN(2) crystallizes in P4(2)/mbc (No . 135) with a= b = 11.773(2) A and c= 5.409(1) A and has Z = 8 . It also contains GeN(2)(4)(-) units which have 18 valence electrons and, consequently are bent, like the isoelectronic molecule SO(2).

Proc Natl Acad Sci U S A, 1994 Feb 1, 91(3), 952 - 6
The protein phosphatase inhibitor calyculin A mimics elicitor action in plant cells and induces rapid hyperphosphorylation of specific proteins as revealed by pulse labeling with {33P}phosphate; Felix G et al.; Suspension-cultured tomato cells react to microbial signals, so-called elicitors, with rapid alkalinization of the growth medium and increased biosynthesis of the stress hormone ethylene . These responses to elicitors can be blocked by staurosporine and K-252a, two specific inhibitors of protein kinases . Here we show that calyculin A, a potent inhibitor of protein phosphatases, mimics the action of elicitors and, at nanomolar concentrations, induces medium alkalinization as well as a strong increase in the activity of 1-aminocyclopropane-1-carboxylate synthase, the key enzyme of ethylene biosynthesis . Both responses were strongly inhibited by K-252a, and calyculin A induced both responses more rapidly than did a fungal elicitor, xylanase . For example, the lag phase for medium alkalinization was only 0.2-0.4 min for calyculin A, compared with 2 min for xylanase . To study changes in the dynamics of protein phosphorylation, cells were labeled with 30-sec pulses of {33P}orthophosphate . Calyculin A strongly increased phosphorylation of several polypeptide bands within 40 sec of treatment . The same phosphorylated bands also appeared in response to xylanase, but only after a lag phase of 2-3 min . These results show that the protein phosphatase inhibitor calyculin A leads to rapid hyperphosphorylation of specific proteins in cultured cells and indicate that elicitor action could be based on inhibition of a protein phosphatase as well as on activation of a protein kinase.

Hum Immunol, 2001 Oct, 62(10), 1092 - 8
A new method for in vitro expansion of cytotoxic human CD3-CD56+ natural killer cells; Carlens S et al.; Adoptive transfer of immunocompetent cells may induce anti-tumor effects in vivo . However, a significant obstacle to the development of successful cellular immunotherapy has been the availability of appropriate cytotoxic cells . Among the immunologic effector cells that are considered mediators of anti-tumor effects, those with the highest per-cell cytotoxic capacity express a natural killer (NK) cell phenotype, i.e., CD56(+)CD3(-) . However, such cells are normally present only in low numbers in peripheral blood mononuclear cells (PBMCs), lymphokine activated killer (LAK), and cytokine induced killer (CIK) cell preparations . To optimize the expansion of human NK cells, PBMCs were cultured in different serum free medium supplemented with monoclonal anti-CD3 antibodies and interleukin (IL)-2 at varying concentrations . By using Cellgro stem cell growth medium supplemented with 5% human serum and IL-2 (500 U/ml) cells expanded 193-fold (median, range 21-277) after 21 days, and contained 55% (median, range 7-92) CD3(-)CD56(+) cells . The remaining cells were CD3(+) T cells, 22% (median, range 2-68) of which co-expressed CD56 . The expanded cell population lysed 26 to 45% of K562 targets in a 1:1 effector to target ratio, signifying substantial cytotoxic efficacy . The described method is a simple and efficient way of expanding and enriching human NK cells . We have termed these high-yield CD3(-)CD56(+) cells cytokine-induced natural killer (CINK) cells.

Anticancer Drugs, 2001 Oct, 12(9), 769 - 79
Establishment and characterization of adriamycin-resistant human colorectal adenocarcinoma HCT-15 cell lines with multidrug resistance; Uchiyama-Kokubu N et al.; The multidrug resistance (MDR) phenotype, either intrinsic and/or acquired, is discussed in relation to several MDR-associated markers such as P-glycoprotein (P-gp) encoded by mdr1, multidrug-resistance-associated protein (MRP) encoded by MRP and lung-resistance-associated protein (LRP) encoded by LRP . Well-characterized in vitro models are required to elucidate the mechanisms of MDR . The aim of the present study is the establishment of a drug-resistant subline from human colorectal adenocarcinoma HCT-15 that intrinsically expresses moderate levels of P-gp, MRP and LRP . Three adriamycin-resistant sublines (HCT-15/ADM1, HCT-15/ADM2 and HCT-15/ADM2-2) were established by stepwise exposure in growth medium that was supplemented with 25-200 ng/ml adriamycin-resulting in a 2.2- to 7.8-fold increase in IC(50) values by using the XTT assay . They were cross-resistant to MDR-related drugs, epirubicin, mitoxantrone, vincristine, etoposide and taxol, but not the MDR-unrelated drug, mytomycin C . The resistance to adriamycin was confirmed in vivo by a lack of sensitivity in athymic nude mice . Gene expression data for mdr1/P-gp, MRP/MRP and LRP/LRP on both mRNA and protein levels demonstrated that the molecules contributing to MDR in resistant sublines are mainly P-gp and partially MRP . The newly established adriamycin-resistant sublines of HCT-15 will provide clinically relevant tools to investigate how to overcome drug resistance and elucidate possible mechanisms of acquired MDR in human colon cancer.

Biotechnol Bioeng, 2001 Nov 5, 75(3), 322 - 33
Galactomannanases Man2 and Man5 from Thermotoga species: growth physiology on galactomannans, gene sequence analysis, and biochemical properties of recombinant enzymes; Parker KN et al.; The enzymatic hydrolysis of mannan-based hemicelluloses is technologically important for applications ranging from pulp and paper processing to food processing to gas and oil well stimulation . In many cases, thermostability and activity at elevated temperatures can be advantageous . To this end, the genes encoding beta-mannosidase (man2) and beta-mannanase (man5) from the hyperthermophilic bacteria Thermotoga neapolitana 5068 and Thermotoga maritima were isolated, cloned, and expressed in Escherichia coli . The amino acid sequences for the mannosidases from these organisms were 77% identical and corresponded to proteins with an M(r) of approximately 92 kDa . The translated nucleotide sequences for the beta-mannanase genes (man5) encoded polypeptides with an M(r) of 76 kDa that exhibited 84% amino acid sequence identity . The recombinant versions of Man2 and Man5 had similar respective biochemical and biophysical properties, which were also comparable to those determined for the native versions of these enzymes in T . neapolitana . The optimal temperature and pH for the recombinant Man2 and Man5 from both organisms were approximately 90 degrees C and 7.0, respectively . The presence of Man2 and Man5 in these two Thermotoga species indicates that galactomannan is a potential growth substrate . This was supported by the fact that beta-mannanase and beta-mannosidase activities were significantly stimulated when T . neapolitana was grown on guar or carob galactomannan . Maximum cell densities increased by at least tenfold when either guar or carob galactomannan was added to the growth medium . For T . neapolitana grown on guar at 83 degrees C, Man5 was secreted into the culture media, whereas Man2 was intracellular . These localizations were consistent with the presence and lack of signal peptides for Man5 and Man2, respectively . The identification of the galactomannan-degrading enzymes in these Thermotoga species adds to the list of biotechnologically important hemicellulases produced by members of this hyperthermophilic genera .

Biotechnol Bioeng, 2001 Nov 5, 75(3), 292 - 304
Quantification of random motility and chemotaxis bacterial transport coefficients using individual-cell and population-scale assays; Lewus P et al.; A number of individual-cell and population-scale assays have been introduced to quantify bacterial motility and chemotaxis . The transport coefficients reported in the literature, however, span several orders of magnitude, making it difficult to ascertain to what degree variations in bacterial species/strain, growth medium, growth and experimental conditions, and experiment type contribute to the reported differences in coefficient values . We quantified the random motility of Escherichia coli AW405 using the capillary assay, stopped-flow diffusion chamber (SFDC), and tracking microscope . We obtained good agreement for the random motility coefficient between these assays when using the same bacterial strain and consistent growth and experimental conditions . Chemotaxis of E . coli toward the attractant alpha-methylaspartate was quantified using the SFDC and capillary assay . Good agreement for the chemotactic sensitivity coefficient between the SFDC and the capillary assay was obtained across a limited attractant concentration range . Three different mathematical models were considered for analyzing capillary assay data to obtain a chemotactic sensitivity coefficient . These models differed by their treatment of the bacterial concentration in the chamber and the attractant concentration at the mouth . Results from our study indicate that the capillary assay, the most commonly used bacterial random motility and chemotaxis assay, can be used to accurately quantify bacterial transport coefficients over a limited range of attractant concentrations, provided experiments are performed carefully and appropriate mathematical models are used to interpret the experimental data .

Cell Biol Int, 2001, 25(10), 1021 - 4
Effect of phenol red and steroid hormones on cystic fibrosis transmembrane conductance regulator in mouse endometrial epithelial cells; Tsang LL et al.; Previous studies have demonstrated that cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-mediated Cl(-)channel found in most epithelia including reproductive tract, could be regulated by various culture conditions . The present study further investigated the effect of phenol red, a pH indicator widely used in growth medium, and steroid hormones, present in the supplement fetal bovine serum (FBS), on primary cultured endometrial epithelial cells by monitoring ion channel activities using the short-circuit current technique . When compared to the results obtained with normal medium supplemented with regular FBS, the forskolin-stimulated I(SC), presumably mediated by CFTR, obtained in phenol red-free medium was significantly reduced, from 16.95+/-1.53 microA/cm(2)(control) to 9.72+/-0.89 microA/cm(2)(medium without phenol red, P< 0.05) . The forskolin-activated I(SC)was further attenuated to 5.29+/-0.46 microA/cm(2)in the phenol red-free medium when supplemented with charcoal/ dextran-treated FBS where steroid hormones were removed . Our data suggest that phenol red and steroid hormones present in culture medium and FBS supplement, respectively, may somehow upregulate CFTR expression in vitro . Our study demonstrates the need for carefully choosing the culture media and supplements due to the effect of steroid hormones .

Biotechnol Prog, 2001 Sep-Oct, 17(5), 791 - 7
Minimal reaction sets for Escherichia coli metabolism under different growth requirements and uptake environments; Burgard AP et al.; A computational procedure for identifying the minimal set of metabolic reactions capable of supporting various growth rates on different substrates is introduced and applied to a flux balance model of the Escherichia coli metabolic network . This task is posed mathematically as a generalized network optimization problem . The minimal reaction sets capable of supporting specified growth rates are determined for two different uptake conditions: (i) limiting the uptake of organic material to a single organic component (e.g., glucose or acetate) and (ii) allowing the importation of any metabolite with available cellular transport reactions . We find that minimal reaction network sets are highly dependent on the uptake environment and the growth requirements imposed on the network . Specifically, we predict that the E . coli network, as described by the flux balance model, requires 224 metabolic reactions to support growth on a glucose-only medium and 229 for an acetate-only medium, while only 122 reactions enable growth on a specially engineered growth medium.

Biochem Pharmacol, 2001 Sep 1, 62(5), 569 - 80
Enhanced activity of antisense phosphorothioate oligos against leishmania amastigotes: augmented uptake of oligo, ribonuclease H activation, and efficient target intervention under altered growth conditions; Mishra M et al.; Leishmania, a parasitic protozoan, infects human macrophages, often causing severe morbidity and mortality . The pathogenic form of this parasite, the amastigote, lives inside the acidic phagolysosomes of infected macrophages . In our attempt to develop anti-miniexon phosphorothioate oligodeoxyribonucleotides (S-oligos) as an alternative chemotherapy against Leishmania, we found that intracellular as well as 'axenic' amastigotes were more susceptible to these S-oligos than were the cultured promastigotes . Lower pH (4.5) and elevated temperature (35 degrees) of the medium were among the direct enhancing factors for killing . Addition of the cationic polypeptide poly-l-lysine (PLL) to the growth medium further enhanced the killing effect of the S-oligo at pH 4.5 . The enhancement of specific ablation of mRNA expression was directly correlated to the increased leishmanicidal activity of the S-oligo . This was shown by the increased inhibition of luciferase activity expressed in transgenic Leishmania amazonensis promastigotes by anti-miniexon S-oligo or anti-luciferase S-oligo at acidic pHs and in the presence of PLL . The leishmanicidal effects of S-oligos at acidic pH and in the presence of PLL were related to increased uptake of the S-oligos under these conditions . The rate of S-oligo uptake was enhanced up to 15-fold at pH 4.5 . The addition of PLL to the assay medium at acidic pH further enhanced the uptake of S-oligo up to 80-fold . RNase H is known to accentuate the antisense action of S-oligos . We found that at an elevated temperature RNase H activity in Leishmania cell extracts increased about 5-fold . Thus, enhanced uptake of S-oligos at the acidic pH of macrophage phagolysosomes and activation of RNase H may explain the efficient killing of the parasite in macrophages, both in tissue culture and in the animal model, by antisense miniexon oligonucleotide/PLL, when targeted directly to the parasite-containing phagolysosomes.

Biochemistry, 2001 Oct 9, 40(40), 12123 - 31
Expression, purification, characterization, and NMR studies of highly deuterated recombinant cytochrome c peroxidase; Savenkova MI et al.; Two forms of extensively deuterated S . cerevisiae cytochrome c peroxidase (CcP; EC 1.11.1.5) have been overexpressed in E . coli by growth in highly deuterated medium . One of these ferriheme enzyme forms (recDCcP) was produced using >97% deuterated growth medium and was determined to be approximately 84% deuterated . The second form {recD(His)CcP} was grown in the same highly deuterated medium that had been supplemented with excess histidine (at natural hydrogen isotope abundance) and was also approximately 84% deuterated . This resulted in direct histidine incorporation without isotope scrambling . Both of these enzymes along with the corresponding recombinant native CcP (recNATCcP), which was expressed in a standard medium with normal hydrogen isotope abundance, consisted of 294 amino acid polypeptide chains having the identical sequence to the yeast-isolated enzyme, without any N-terminal modifications . Comparative characterizations of all three enzymes have been carried out for the resting-state, high-spin forms and in the cyanide-ligated, low-spin forms . The primary physical methods employed were electrophoresis, UV-visible spectroscopy, hydrogen peroxide reaction kinetics, mass spectrometry, and (1)H NMR spectroscopy . The results indicate that high-level deuteration does not significantly alter CcP's reactivity or spectroscopy . As an example of potential NMR uses, recDCcPCN and recD(His)CcPCN have been used to achieve complete, unambiguous, stereospecific (1)H resonance assignments for the heme hyperfine-shifted protons, which also allows the heme side chain conformations to be assessed . Assigning these important active-site protons has been an elusive goal since the first NMR spectra on this enzyme were reported 18 years ago, due to a combination of the enzyme's comparatively large size, paramagnetism, and limited thermal stability.

J Biol Chem, 2001 Nov 30, 276(48), 44751 - 6 Epub 2001 Sep 28.
A novel sugar-stimulated covalent switch in a sugar sensor; Chen Q et al.; The bgl sensory system is composed of a membrane-bound sugar sensor, BglF, and a transcriptional regulator, BglG . The sensor BglF has several enzymatic activities: in its nonstimulated state, it acts as BglG phosphorylase; in the presence of beta-glucoside in the growth medium, it acts as BglG dephosphorylase and as the beta-glucoside phosphotransferase . The same active site on BglF, Cys-24, is responsible for the phosphorylation of both the stimulating sugar and the BglG protein . BglF is composed of three domains, two hydrophilic and one hydrophobic . Our previous results suggested that catalysis of the sugar-stimulated functions depends on specific interactions between the B domain, which contains the active site cysteine, and the integral membrane C domain . We report here that the stimulating sugar triggers the formation of a disulfide bond between the active site cysteine and another cysteine in the membrane-embedded domain of BglF . Inability of a mutant BglF protein to form the disulfide bridge between the B and C domains correlates with its inability to catalyze the sugar-stimulated functions . The ability of the cysteine residue in BglF to bind covalently either to a phosphoryl group or to another cysteine residue, depending on the protein stimulation state, suggests a novel way to control signaling by alternative bond formation.

Microbiology, 2001 Oct, 147(Pt 10), 2795 - 803
The transcriptional activator NhaR is responsible for the osmotic induction of osmC(p1), a promoter of the stress-inducible gene osmC in Escherichia coli; Toesca I et al.; Two overlapping promoters, osmC(p1) and osmC(p2), direct the transcription of the osmC gene of Escherichia coli . The proximal promoter, osmC(p2), is induced upon entry into stationary phase under the control of Esigma(s), the RNA polymerase that uses the sigma(s) (RpoS) sigma factor . Transcription from the distal promoter, osmC(p1), is independent of sigma(s) . Previous analysis demonstrated that the osmolarity of the growth medium modulates expression of both promoters . The use of an E . coli genomic library showed that the cloned nhaR gene was able to stimulate transcription of an osmC-lac reporter fusion . NhaR is a positive regulator of the LysR family, previously identified as an activator of nhaA, a gene encoding a Na+/H+ antiporter involved in adaptation to Na+ and alkaline pH in E . coli and other enteric bacteria . NhaR was shown to activate only the expression of osmC(p1) and to be necessary for the induction of this promoter by LiCl, NaCl and sucrose . Therefore, activation by NhaR is responsible for the osmotic induction of osmC(p1) . In contrast to its action on nhaA, NhaR activation of osmC(p1) is independent of H-NS . Activation of osmC(p1) by NhaR requires a site located just upstream of the atypical -35 region of the promoter.

Plant J, 2001 Sep, 27(6), 561 - 9
Identification of a novel genetically controlled step in mycorrhizal colonization: plant resistance to infection by fungal spores but not extra-radical hyphae; David-Schwartz R et al.; Vesicular arbuscular mycorrhizal fungi infect plants by means of both spores and vegetative hyphae at early stages of symbiosis . Using 2500 M2 fast-neutron-mutagenized seeds of the miniature tomato (Lycopersicon esculentum) cultivar, Micro-Tom, we isolated a mutant, M161, that is able to resist colonization in the presence of Glomus intraradices spores . The myc(-) phenotype of the mutant was stable for nine generations, and found to segregate as a single Mendelian recessive locus . The mutant exhibited morphological and growth-pattern characteristics similar to those of wild-type plants . Alterations of light intensity and day/night temperatures did not eliminate the myc(-) characteristic . Resistance to mycorrhizal fungal infection and colonization was also evident following inoculation with the fungi Glomus mosseae and Gigaspora margarita . Normal colonization of M161 was evident when mutant plants were grown together with arbuscular mycorrhizal-inoculated wild-type plants in the same growth medium . During evaluation of the pre-infection stages in the mutant rhizosphere, spore germination and appressoria formation of G . intraradices were lower by 45 and 70%, respectively, than the rates obtained with wild-type plants . These results reveal a novel, genetically controlled step in the arbuscular mycorrhizal colonization process, governed by at least one gene, which significantly reduces key steps in pre-mycorrhizal infection stages.

Fungal Genet Biol, 2001 Oct, 34(1), 1 - 10
A fungal perspective on human inborn errors of metabolism: alkaptonuria and beyond; Penalva MA; Crucial for the establishment and development of biochemical genetics as a self-standing discipline was Beadle and Tatum's choice of Neurospora crassa as experimental organism some 60 years ago . Although Garrod's insights on biochemical genetics and his astonishingly modern concepts of biochemical individuality and susceptibility to disease had been ignored by their contemporaries, Beadle acknowledged on several occasions how close Garrod had come to the "one-gene-one-enzyme" hypothesis . In an unexpected turn of events, several genes involved in human inborn errors of metabolism, including the gene for Garrod's favorite disease, alkaptonuria, have been characterized by exploitation of the experimental advantages of another mold, Aspergillus nidulans, which shares with N . crassa the experimental advantages that prompted pioneers of biochemical genetics to use them: rapid growth, facile genetic manipulation, and an environment (the composition of the growth medium) that can be manipulated a la carte .

J Microbiol Methods, 2001 Oct, 47(1), 41 - 50
Factors influencing the cultivability of lake water bacteria; Bussmann I et al.; Counting bacteria in natural water samples by cultivation yields only low recovery efficiencies (ca . 1%), compared to total counts obtained after 4,6-diamidino-2-phenylindol (DAPI) staining . In order to optimize the cultivation of heterotrophic planktonic bacteria from Lake Constance (Germany), selected parameters of the medium composition were modified . The most important factor was the concentration of organic substrate (nutrient broth plus yeast extract), which significantly influenced the "most probable number" obtained in liquid growth medium . Reduced oxygen concentrations (3-12%) lowered the "most probable number" . Addition of N-acyl homoserine lactones to the medium increased the cultivability slightly . Low substrate concentrations {0.03-0.06% (w/v)}, an incubation atmosphere of 21% oxygen at 16 degrees C for 4 weeks were optimal and increased the cultivability ("most probable number" related to total bacterial counts) to an average cultivability of 18+/-11%, (n=8) . The results indicate that cultivabilities of heterotrophic bacteria from lakewater samples can be significantly increased by modifying the cultivation methods.

J Protein Chem, 2001 Apr, 20(3), 233 - 7
An E . coli expression system for the extracellular secretion of barley alpha-amylase; Lee CC et al.; Libraries of modified genes are often screened during the process of genetically engineering enzymes with specifically tailored activities . It is important, therefore, to create expression systems which allow for the rapid screening of many clones . We developed an Escherichia coli expression system which will secrete enzymes into the growth medium . We describe the first reported expression of barley alpha-amylase in E . coli . The enzyme is secreted onto solid media containing starch to produce easily visualized halos . In addition, the enzyme is secreted into liquid media in an intact, active form.

Environ Health Perspect, 2001 Aug, 109(8), 801 - 8
The immortalized UROtsa cell line as a potential cell culture model of human urothelium; Rossi MR et al.; The UROtsa cell line was isolated from a primary culture of normal human urothelium through immortalization with a construct containing the SV40 large T antigen . It proliferates in serum-containing growth medium as a cell monolayer with little evidence of uroepithelial differentiation . The working hypothesis in the present study was that this cell line could be induced to differentiate and express known features of in situ urothelium if the original serum-containing growth medium was changed to a serum-free formulation . We demonstrated that the UROtsa cells could be successfully placed into a serum-free growth medium consisting of a 1:1 mixture of Dulbeco's modified Eagle's medium and Ham's F-12 supplemented with selenium (5 ng/mL), insulin (5 microg/mL), transferrin (5 microg/mL), hydrocortisone (36 ng/mL), triiodothyronine (4 pg/mL), and epidermal growth factor (10 ng/mL) . Under serum-free growth conditions, confluent UROtsa cells were shown by light microscopy to produce raised, three-dimensional structures . Routine ultrastructural examination disclosed these three-dimensional areas to consist of a stratified layer of cells that strongly resembled in situ urothelium . The cells displayed numerous desmosomal connections, complex interactions of the lateral membranes, and abundant intermediate filaments within the cytoplasm . Freeze fracture analysis demonstrated that the cells possessed tight-junction sealing strands and gap junctions . The overall morphology was most consistent with that found in the intermediate layers of in situ urothelium . The basal expression patterns of the metallothionein (MT) and heat shock proteins 27, 60, and 70 were determined in these cells, and expression was in agreement with that known to occur for in situ urothelium . The cells were also successfully tested for their ability to be stably transfected using expression vectors containing the MT-3 or MT-2A genes . The findings suggest that the UROtsa cells grown with a serum-free medium could be a valuable adjunct for studying environmental insult to the human urothelium in general and for the stress response in particular.

Neuroscience, 2001, 106(1), 217 - 25
Primary culture of rat taste bud cells that retain molecular markers for taste buds and permit functional expression of foreign genes; Kishi M et al.; Taste buds are constituted of several kinds of cells which have distinct characteristics and play different roles . In this study, we have established an in vitro culture system by optimizing the method for isolating the cells and by selecting culture media and reagents effective for cell viability and adhesion . As a result, the taste bud cells were adhesive and viable for over 3 days when cultured onto Matrigel-coated dishes in medium based on keratinocyte growth medium . The cells retained molecular markers for both the cytoskeleton and intracellular signaling such as cytokeratin 8 and phospholipase Cbeta2 . In addition, three intracellular signaling molecules, gustducin, phospholipase Cbeta2, and inositol 1,4,5-trisphosphate receptor type 3, are expressed in the same correlation as those in vivo, although the ratio of signaling molecule-positive cells vs . total cells was somewhat lower in the culture than in vivo . Next, we tried several methods to introduce foreign genes into the cells, and obtained a greater than 90% efficiency of introduction using an adenovirus vector . Finally, we show that an exogenously expressed myc-tagged alpha1A-adrenoceptor sorts into the plasma membrane, and transduces a ligand-dependent signal resulting in intracellular {Ca(2+)} increase in about half of the infected cells.These results suggest that taste bud cells after 3 days of culture retain characteristic molecular markers, and may prove useful for describing the molecular and physiological features of taste bud cells, and that these cells can be further manipulated by adenovirus-mediated gene introduction.

J Invest Dermatol, 2001 Sep, 117(3), 647 - 53
Sodium dodecyl sulfate induces plasminogen activator inhibitor type 2 expression in epidermal keratinocytes in vivo and in vitro; Chung NM et al.; The detergent sodium dodecyl sulfate is a well-known inducer of irritant contact dermatitis . In this study we show that sodium dodecyl sulfate induces the serine proteinase inhibitor, plasminogen activator inhibitor type 2, in epidermal keratinocytes . The enhancement in plasminogen activator inhibitor type 2 mRNA and antigen is observed both when sodium dodecyl sulfate is applied topically to normal human skin as well as when it is added to the growth medium of cultured human keratinocytes . In vitro, plasminogen activator inhibitor type 2 mRNA is increased within 4-8 h after addition of the detergent, and the increase in plasminogen activator inhibitor type 2 antigen occurs slightly later . The enhancing effect of sodium dodecyl sulfate on plasminogen activator inhibitor type 2 is not related to nonspecific cell lysis nor is it secondary to induction of tumor necrosis factor alpha . Similarities between our in vitro and in vivo findings lead us to hypothesize that sodium dodecyl sulfate may exert its effect on epidermal plasminogen activator inhibitor type 2 via interaction with the keratinocyte.

Mikrobiologiia, 2001 Jul-Aug, 70(4), 543 - 51
{Electron microscopic study of the colonies of an alkylsulfonate-utilizing bacterial consortium}; Mogil'naia OA et al.; The light- and electron-microscopic analysis of the colonies of a bacterial consortium capable of utilizing alkylsulfonates, which are the main ingredients of sewage from the synthetic rubber industry, revealed the presence of eight types of cells . All types of cells were gram-negative and differed in shape, size, and the presence of capsule and cytoplasmic inclusions . Three types of cells were present in all the colonies that were studied . The presence of the other types of cells depended on the inoculum used and on the composition of the growth medium.

Mikrobiologiia, 2001 Jul-Aug, 70(4), 452 - 8
{Aerobic methylobacteria are capable of synthesizing auxins}; Ivanova EG et al.; Obligately and facultatively methylotrophic bacteria with different pathways of C1 metabolism were found to be able to produce auxins, particularly indole-3-acetic acid (IAA), in amounts of 3-100 micrograms/ml . Indole-3-pyruvic acid and indole-3-acetamide were detected only in methylobacteria with the serine pathway of C1 metabolism, Methylobacterium mesophilicum and Aminobacter aminovorans . The production of auxins by methylobacteria was stimulated by the addition of tryptophan to the growth medium and was inhibited by ammonium ions . The methylobacteria under study lacked tryptophan decarboxylase and tryptophan side-chain oxidase . At the same time, they were found to contain several aminotransferases . IAA is presumably synthesized by methylobacteria through indole-3-pyruvic acid.

J Biol Chem, 2002 Feb 15, 277(7), 5299 - 307 Epub 2001 Sep 10.
Alzheimer's disease-related overexpression of the cation-dependent mannose 6-phosphate receptor increases Abeta secretion: role for altered lysosomal hydrolase distribution in beta-amyloidogenesis; Mathews PM et al.; Prominent endosomal and lysosomal changes are an invariant feature of neurons in sporadic Alzheimer's disease (AD) . These changes include increased levels of lysosomal hydrolases in early endosomes and increased expression of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is partially localized to early endosomes . To determine whether AD-associated redistribution of lysosomal hydrolases resulting from changes in CD-MPR expression affects amyloid precursor protein (APP) processing, we stably transfected APP-overexpressing murine L cells with human CD-MPR . As controls for these cells, we also expressed CD-MPR trafficking mutants that either localize to the plasma membrane (CD-MPRpm) or to early endosomes (CD-MPRendo) . Expression of CD-MPR resulted in a partial redistribution of a representative lysosomal hydrolase, cathepsin D, to early endosomal compartments . Turnover of APP and secretion of sAPPalpha and sAPPbeta were not altered by overexpression of any of the CD-MPR constructs . However, secretion of both human Abeta40 and Abeta42 into the growth media nearly tripled in CD-MPR- and CD-MPRendo-expressing cells when compared with parental or CD-MPRpm-expressing cells . Comparable increases were confirmed for endogenous mouse Abeta40 in L cells expressing these CD-MPR constructs but not overexpressing human APP . These data suggest that redistribution of lysosomal hydrolases to early endocytic compartments mediated by increased expression of the CD-MPR may represent a potentially pathogenic mechanism for accelerating Abeta generation in sporadic AD, where the mechanism of amyloidogenesis is unknown.

Can J Anaesth, 2001 Sep, 48(8), 748 - 54
Anesthesia breathing circuits protected by the DAR Barrierbac S breathing filter have a low bacterial contamination rate; Vezina DP et al.; PURPOSE: In order to reuse the same anesthesia breathing circuit for more than one patient, it has been proposed to add a breathing filter between the Y-piece and the artificial airway . The purpose of this study was to evaluate the in vivo bacterial filtration efficacy of an anesthesia filter in a usual clinical anesthesia setting . METHODS: A sterile DAR Barrierbac S breathing filter was inserted at the Y-piece of a sterile single-use anesthesia breathing circuit before induction of general anesthesia . At the end of anesthesia, the breathing circuit connector of the filter and of the endotracheal tube connector were cultured separately on growth media (chocolate and blood agar) . These were incubated for 48 hr and bacterial identification was conducted using standard methods . RESULTS: Bacterial cultures were negative on both sides of the filter membrane of 1842 of the 2001 filters studied . Cultures were positive on the patient side of 104 filters . In two of those, the same bacteria were found on both the circuit side and the patient side of the filter . Therefore these data indicate a clinical effectiveness of 99.9% (confidence interval, CI 95%, 99.6-99.998%), and an in vivo filtration efficacy of 98.08% (CI 95%, 92.54-99.67%) . CONCLUSION: Using the upper limit of the CI, it can be assumed that the practice of using a sterile DAR Barrierbac S breathing filter for every patient while reusing the anesthesia breathing circuit would result in a cross contamination rate of the breathing circuit lower than once every 250 cases.

Clin Experiment Ophthalmol, 2001 Aug, 29(4), 248 - 52
Effects of varying 5-fluorouracil exposure duration on tenon's capsule fibroblasts; Merriman MB et al.; PURPOSE: 5-Fluorouracil (5-FU) is used intraoperatively to improve the success of trabeculectomy Common practice is a 5-min application of 5-FU delivered via a Weck Cel sponge.The aim of this study was to determine if shorter exposure times were as effective in inhibiting Tenon's capsule fibroblast proliferation . METHODS: Samples of Tenon's capsules, obtained from young patients undergoing strabismus surgery, were immersed In vitro in 5-FU (50 mg/mL) for periods ranging from 1 to 10 min . Control samples were exposed either to growth medium (M 199) or 5-FU vehicle alone . Explants were cultured for up to 7 days and analysed for cell density, cell viability using fluoroprobe detection, and cell proliferation determined by proliferating cell nuclear antigen (PCNA)-staining . RESULTS: Exposure to 5-FU for 1 and 5 min resulted in a similar and significant inhibition of the culture-induced increase in stromal cell density (P < 0.01), a significant reduction in the percentage of viable cells (P < 0.02), and reduced PCNA indices, compared with controls . Exposure times of 3 and 10 min produced similar results . CONCLUSIONS: In vitro, under conditions of organ culture, a 1-min exposure to 5-FU had a significant antiproliferative effect on fibroblasts of Tenon's capsule, and was similar to 5 min of exposure . Optimal intraoperative 5-FU exposure time may be as little as 1 minute.

J Bacteriol, 2001 Oct, 183(19), 5675 - 83
External-pH-dependent expression of the maltose regulon and ompF gene in Escherichia coli is affected by the level of glycerol kinase, encoded by glpK; Chagneau C et al.; The expression of the maltose system in Escherichia coli is regulated at both transcriptional and translational levels by the pH of the growth medium (pHo) . With glycerol as the carbon source, transcription of malT, encoding the transcriptional activator of the maltose regulon, is weaker in acidic medium than in alkaline medium . malT transcription became high, regardless of the pHo, when glycerol-3-phosphate or succinate was used as the carbon source . Conversely, malT expression was low, regardless of the pHo, when maltose was used as the carbon source . The increase in malT transcription, associated with the pHo, requires the presence of glycerol in the growth medium and the expression of the glycerol kinase (GlpK) . Changes in the level of glpK transcription had a great effect on malT transcription . Indeed, a glpFKX promoter-down mutation has been isolated, and in the presence of this mutation, malT expression was increased . When glpK was expressed from a high-copy-number plasmid, the glpK-dependent reduced expression of the maltose system became effective regardless of the pHo . Analysis of this repression showed that a malTp1 malTp10 promoter, which is independent of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex, was no longer repressed by glpFKX amplification . Thus, GlpK-dependent repression of the maltose system requires the cAMP-CRP complex . We propose that the pHo may affect a complex interplay between GlpK, the phosphotransferase-mediated uptake of glucose, and the adenylate cyclase.

New Phytol, 1999 Apr, 142(1), 59 - 66
Nuclear magnetic resonance imaging in studies of gravitropism in soil mixtures; Antonsen F et al.; Gravitropic responses of oat coleoptiles were measured in different growth media; humid air, natural soil and artificial soil (glass beads) . The oat coleoptiles in soil and glass beads were monitored by NMR imaging, while those in humid air were imaged in darkness with an infrared-sensitive charge-coupled device (CCD) camera . The present study shows for the first time that gravitropic experiments can be performed in artificial soil using NMR imaging as a convenient and suitable recording method . Not only was it possible to follow the gravitropic curvatures in natural soil, but the artificial soil allowed plant images of sufficient spatial and temporal resolution to be recorded . The advantages of using artificial soil in magnetic resonance imaging studies are that the iron content of glass beads is very low compared with natural soil, and that the artificial soil matrix can easily, be standardized with regard to particle size distribution and nutrient content . Two types of glass beads were used, the diameter of the small and the large beads being 300-400 and 420-840 micrometers, respectively . The growth rate of the coleoptiles in soil and in big beads was roughly the same and only slightly lower than in humid air, whereas small beads reduced the growth rate by approx . 16% . The bending rate of the coleoptiles during the gravitropic response was reduced by c . 65% in soil and 75% in bead mixtures relative to bending in air . It should be noted, however, that the maximum curvature of the coleoptile tip was of the same order in all cases, about 35 degrees . This value may represent the largest possible curvature of the organ . The potential of NMR imaging to study, how plant organs penetrate the soil under the influence of gravitropism, mechanical impedance and thigmotropism is also discussed.

J Plant Res, 1999 Dec, 112(1108), 391 - 6
Effects of natural and synthetic auxins on the gravitropic growth habit of roots in two auxin-resistant mutants of Arabidopsis, axr1 and axr4: evidence for defects in the auxin influx mechanism of axr4; Yamamoto M et al.; The partially agravitropic growth habit of roots of an auxin-resistant mutant of Arabidopsis thaliana, axr4, was restored by the addition of 30-300 nM 1-naphthaleneacetic acid (NAA) to the growth medium . Neither indole 3-acetic acid (IAA) nor 2,4-dichlorophenoxyacetic acid (2,4-D) showed such an effect . Growth of axr4 roots was resistant to IAA and 2,4-D, but not at all to NAA . The differential effects of the three auxins suggest that the defects of axr4 result from a lower auxin influx into its cells . The partially agravitropic growth habit of axr1 roots, which was less severe than that of axr4 roots, was only slightly affected by the three auxins in the growth medium at concentrations up to 300 nM; growth of axr1 roots was resistant to all three of the auxins . These results suggest that the lesion of axrl mutants is different from that of axr4.

Adv Space Res, 1998, 22(10), 1413 - 8
Particulated growth media for optimal liquid and gaseous fluxes to plant roots in microgravity; Jones SB et al.; An important and yet relatively under researched area of plant growth in microgravity, deals with the rooting environment of plants . A comprehensive approach for selecting the physical characteristics of root growth media which optimizes the dynamic availability of water and dissolved nutrients, and gases to plant roots was developed and tested . Physically-based and parametric models describing the relationship between content and fluxes of liquids and gases were used to cast a multi-objective optimization problem . This methodology reveals that a medium's ability to supply liquid and gas fluxes optimally is dependent upon physiological target values, system operation limits and root module design which dictate the medium's range of soil water characteristic and particle size distribution . Optimized media parameters designate a particle size distribution from which a particulated growth media was constructed and matched to the optimized media parameters . This methodology should improve the selection of optimal media properties for plant growth in microgravity as well as other porous media applications.

Adv Space Res, 1998, 22(10), 1407 - 12
A capillary-driven root module for plant growth in microgravity; Jones SB et al.; A new capillary-driven root module design for growing plants in microgravity was developed which requires minimal external control . Unlike existing systems, the water supply to the capillary-driven system is passive and relies on root uptake and media properties to develop driving gradients which operate a suction-induced flow control valve . A collapsible reservoir supplies water to the porous membrane which functions to maintain hydraulic continuity . Sheet and tubular membranes consisting of nylon, polyester and sintered porous stainless steel were tested . While finer pore sized membranes allow greater range of operation, they also reduce liquid flux thereby constraining system efficiency . Membrane selection should consider both the maximum anticipated liquid uptake rate and maximum operating matric head (suction) of the system . Matching growth media water retention characteristics to the porous membrane characteristics is essential for supplying adequate liquid flux and gas exchange . A minimum of 10% air-filled porosity (AFP) was necessary for adequate aeration . The capillary-driven module maintained hydraulic continuity and proper gas exchange rates for more than 80 days in a plant growth experiment.

Adv Space Res, 1997, 20(10), 1905 - 8
CO2 enrichment influences yields of 'Florunner,' 'Georgia Red' and 'New Mexico' peanut cultivars; Mortley DG et al.; Three peanut cultivars, 'Florunner,' 'Georgia Red,' and 'New Mexico,' were grown in reach-in chambers to determine response to CO2 enrichment . CO2 treatments were ambient (400 micromol mol-1) and 700 micromol mol-1 . Growth chamber conditions included 700 micromol m-2 s-1 photosynthetic photon flux (PPF), 28/22C, 7O% RH, and 12/12 h photoperiod . Growth media consisted of a 1:1 mixture (v/v) of vermiculite and sterilized sand . Six 10 L pots of each cultivar were fertilized three times per week with 250 mL of nutrient solution containing additional Ca (10 mM) and NO3 (25 mM) and watered well . Beginning 21 days after planting (DAP) and every three weeks thereafter up to 84 days, the second leaf from the growing axis (main stem) was detached to determine CO2 effect on leaf area, specific leaf area (SLA) and dry weight . Plants were harvested 97 DAP, at which time total leaf area, leaf number, plant and root weights and pod production data were taken . Numbers of pods per plant, pod fresh and dry weights, fibrous root and plant dry weights were higher for all cultivars grown at 700 micromol mol-1 than at ambient CO2 . Also, leaf area for all cultivars was larger with CO2 enrichment than at ambient . SLA tended to decline with time regardless of CO2 treatment . Percentage of total sound mature kernels (%TSMK) was similar for both treatments . Plants grown at 700 micromol mol-1 CO2 had slightly more immature pods and seeds at final harvest.

Arch Microbiol, 1985, 142, 317 - 25
Spirochaeta bajacaliforniensis sp . n . from a microbial mat community at Laguna Figueroa, Baja California Norte, Mexico; Fracek SP Jr et al.; A new anaerobic spirochete was isolated from anaerobic muds beneath the laminated sediment in the evaporite flat at Laguna Figueroa, Baja California Norte, Mexico . The organism is a member of the stratified microbial community involved in the deposition of the laminated sediments in the lagoon . The size of the spirochete is 0.3 by 30 micrometers, with a wave amplitude of 0.5 micrometer and a wavelength of 1.25 micrometers . The periplasmic flagella have a 1-2-1 arrangement . The outer membrane of the modified Gram-negative cell wall (the sheath) is irregularly crenulated and has a sillon . The growth medium contained yeast extract, trypticase, cellobiose, sodium thioglycolate and at least 20% natural seawater . Chemically defined artificial seawater media did not support growth . Optimal growth occurred with a seawater concentration of 80% at 36 degrees C and a pH of 7.5 . Glucose was fermented to acetate, ethanol, carbon dioxide and hydrogen . The guanine + cytosine content of the DNA was 50 mol % . The spirochete body reacts positively to antibodies raised against eukaryotic brain tubulin protein . On the basis of its free-living anaerobic habitat, its unique morphological and physiological characteristics and G+C ratio, it is proposed that this isolated be considered a new species and names Spirochaeta bajacaliforniensis.

Adv Space Res, 1998, 21(8-9), 1225 - 8
Calcium balance in pea root statocytes under both clinorotation and Ca2+ channel blockers' influence; Belyavskaya NA et al.; We have previously demonstrated that space flight and clinorotation conditions increase cytoplasmic Ca2+ level in pea root statocytes . A rise in {Ca2+}i may be a serious problem for plants in microgravity environment . It is hypothesized that involvement of Ca2+ channel blockers in the growth medium may rescue a plant from abundance of Ca2+ ions . Indeed, combination of clinorotation (2 rpm, 5 days) and any Ca2+ channel blocker (1 micromole D600 or nicardipine, 12 hr) causes decreasing the Ca2+ concentration in pea root statocytes in comparison with clinorotation alone . Redistribution of Ca(2+)-ATPase activities observed under clinorotation comes to normal after D600 application whereas following by nicardipine action the pattern of the cytochemical staining is intermediate between those in stationary control and under clinorotation . Our data support the hypothesis that Ca2+ channel blockers may act as protectors for plants against rise in {Ca2+}i . The role for Ca2+ channels in graviperception and in microgravity effects as well as ways for stabilization of Ca2+ balance in plant cells in space flights are discussed.

Am J Bot, 1992 Nov, 79(11), 1247 - 58
Gravity effects on cellulose assembly; Brown RM Jr et al.; The effect of microgravity on cellulose synthesis using the model system of Acetobacter xylinum was the subject of recent investigations using The National Aeronautics and Space Administration's Reduced Gravity Laboratory, a modified KC-135 aircraft designed to produce 20 sec of microgravity during the top of a parabolic dive . Approximately 40 parabolas were executed per mission, and a period of 2 x g was integral to the pullout phase of each parabola . Cellulose biosynthesis was initiated on agar surfaces, liquid growth medium, and buffered glucose during parabolic flight and terminated with 2.0% sodium azide or 50.0% ethanol . While careful ground and in-flight controls indicated normal, compact ribbons of microbial cellulose, data from five different flights consistently showed that during progression into the parabola regime, the cellulose ribbons became splayed . This observation suggests that some element of the parabola (the 20 sec microgravity phase, the 20 sec 2 x g phase, or a combination of both) was responsible for this effect . Presumably the cellulose I alpha crystalline polymorph normally is produced under strain, and the microgravity/hypergravity combination may relieve this stress to produce splayed ribbons . An in-flight video microscopy analysis of bacterial motions during a parabolic series demonstrated that the bacteria continue to synthesize cellulose during all phases of the parabolic series . Thus, the splaying may be a reflection of a more subtle alteration such as reduction of intermicrofibrillar hydrogen bonding . Long-term microgravity exposures during spaceflight will be necessary to fully understand the cellulose alterations from the short-term microgravity experiments.

Eur J Protistol, 1994 Feb 18, 30(1), 18 - 24
Effects of heavy metals on motility and gravitactic orientation of the flagellate, Euglena gracilis; Stallwitz E et al.; The effects of copper, mercury, cadmium and lead on the gravitactic orientation of the photosynthetic flagellate Euglena gracilis were investigated . The first two heavy metals reverse the direction of downward swimming (positive gravitaxis) in young cultures (up to 8 days) to an upward swimming (negative gravitaxis); cadmium produced a less pronounced effect . Higher concentrations of heavy metals decrease the precision of orientation as compared to the control due to frequent deviations of the cells from straight paths . Higher concentrations also decrease the swimming velocity of the populations . When the cells were growing in the presence of the heavy metal, copper was effective at > or = 50 microM, cadmium at > or = 3 microM and mercury at > or = 1 microM . Since lead formed insoluble precipitations with the acetate in the growth medium it was tested after the cells were transferred into Tris buffer . Under these conditions lead did not affect the direction of movement or the precision of orientation up to a concentration of 300 microM in the time up to 24 h after the addition of the heavy metal . However, high concentrations of lead strongly decreased the swimming speed of the cells, which was partially reversed with time.

HortScience, 1993 Oct, 28(10), 981 - 4
Stabilization of pH in solid-matrix hydroponic systems; Frick J et al.; 2-{N-morpholino}ethanesulfonic acid (MES) buffer or Amberlite DP-1 (cation-exchange resin beads) were used to stabilize substrate pH of passive-wicking, solid-matrix hydroponic systems in which small canopies of Brassica napus L . (CrGC 5-2, genome : ACaacc) were grown to maturity . Two concentrations of MES (5 or 10 mM) were included in Hoagland 1 nutrient solution . Alternatively, resin beads were incorporated into the 2 vermiculite : 1 perlite (v/v) growth medium at 6% or 12% of total substrate volume . Both strategies stabilized pH without toxic side effects on plants . Average seed yield rates for all four pH stabilization treatments (13.3 to 16.9 g m-2 day-1) were about double that of the control (8.2 g m-2 day-1), for which there was no attempt to buffer substrate pH . Both the highest canopy seed yield rate (16.9 g m-2 day-1) and the highest shoot harvest index (19.5%) occurred with the 6% resin bead treatment, even though the 10 mM MES and 12% bead treatments maintained pH within the narrowest limits . The pH stabilization methods tested did not significantly affect seed oil and protein contents.

Plant Physiol, 1990, 94, 1512 - 21
Changes in membrane lipid composition during saline growth of the fresh water cyanobacterium Synechococcus 6311; Huflejt ME et al.; Growth of Synechococcus 6311 in the presence of 0.5 molar NaCl is accompanied by significant changes in membrane lipid composition . Upon transfer of the cells from a low salt' (0.015 molar NaCl) to high salt' (0.5 molar NaCl) growth medium at different stages of growth, a rapid decrease in palmitoleic acid (C16:1 delta 9) content was accompanied by a concomitant increase in the amount of the two C18:1 acids (C18:1 delta 9, C18:1 delta 11), with the higher increase in oleic acid C18:1 delta 9 content . These changes began to occur within the first hour after the sudden elevation of NaCl and progressed for about 72 hours . The percentage of palmitic acid (C16:0) and stearic acid (C18:0) remained almost unchanged in the same conditions . High salt-dependent changes within ratios of polar lipid classes also occurred within the first 72 hours of growth . The amount of monogalactosyl diacylglycerol (bilayer-destabilizing lipid) decreased and that of the digalactosyl diacylglycerol (bilayer-stabilizing lipid) increased . Consequently, in the three day old cells, the ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol in the membranes of high salt-grown cells was about half of that in the membranes of low salt-grown cells . The total content of anionic lipids (phosphatidylglycerol and sulfoquinovosyl diacylglycerol) was always higher in the isolated membranes and the whole cells from high salt-grown cultures compared to that in the cells and membranes from low salt-grown cultures . All the observed rearrangements in the lipid environment occurred in both thylakoid and cytoplasmic membranes . Similar lipid composition changes, however, to a much lesser extent, were also observed in the aging, low salt-grown cultures . The observed changes in membrane fatty acids and lipids composition correlate with the alterations in electron and ion transport activities, and it is concluded that the rearrangement of the membrane lipid environment is an essential part of the process by which cells control membrane function and stability.

Adv Space Res, 1989, 9(8), 161 - 8
Design for a bioreactor with sunlight supply and operations systems for use in the space environment; Mori K et al.; An experiment was carried out to determine the characteristics of an operations system that can support fast cultivation of algae at high densities in the weightlessness of space . The experiment was conducted in glass bioreactor tanks, in which light was supplied by radiator rods connected to optical fiber cables . The illumination areas of the tanks were 2600 cm2, 6000 cm2, and 9200 cm2 per liter of solution . The characteristics of O2-CO2 gas exchange, concentration and separation of chlorella in the growth medium, dialysis of ionic salts in the growth medium, etc . were examined . Chloralla ellipsoidea was used in the experiment, yielding the following results: (1) By increasing the ratio of illumination area to volume, growth rates of up to approximately 0.6 g/L h could be obtained in a highly concentrated solution (one that contains 20 g/L or more of algae) . (2) The most suitable proportions of carbon dioxide and oxygen gases for growing algae quickly at high concentrations were found to be 10% CO2 and 10% O2 (by volume) . (3) There was a high optimum concentration for fast cultivation, and the data obtained resembled the theoretical curve postulated by P . Behrens et al . (4) It was possible to exchange carbon dioxide and oxygen using gas-permeable membrane modules . (5) It was possible to separate the chlorella from the growth medium and recycle the medium.

J Nucl Med, 2001 Sep, 42(9), 1412 - 7
Dual time point 18F-FDG PET imaging for differentiating malignant from inflammatory processes; Zhuang H et al.; The aim of this study was to investigate the difference in the rates of FDG uptake between malignant and inflammatory cells and processes . METHODS: In vitro studies: (18)F-FDG uptake by different tumor cell lines (human mesothelioma {REN}; rat mesothelioma {II45}; mice melanoma {B18F10}; mice mesothelioma {AB12}; human myeloma {GM1500}; and human ovarian cancer {SKOV3}) and peripheral blood mononuclear cells isolated from 8 healthy human volunteers was measured 20 and 60 min after FDG was added into growth medium . Animal studies: II45 cells were implanted into the left flank of rats (n = 5) and a focal inflammatory reaction (mechanical irritation) was generated in the right flank . PET images at 45 and 90 min after injection of FDG were obtained and standardized uptake values (SUVs) were determined . Patient studies: Seventy-six patients who had dual time FDG PET scans were retrospectively analyzed . All results were expressed as the percentage change in SUV of the later time image from that of the earlier time (mean +/- SD) . RESULTS: In vitro studies: Except for the SKOV3 cell line, which had only minimally increased FDG uptake (+10% +/- 26%; P > 0.3), all other tumor cell lines tested showed significantly increased FDG uptake over time (GM1500, +59% +/- 19%; B18F10, +81% +/- 15%; AB12, 93% +/- 21%; II45, +161% +/- 21%; REN, +198% +/- 48%; P < 0.01 for all) . By contrast, FDG uptake in mononuclear cells was decreased in 7 of 8 donors . Animal studies: SUVs of tumors from 90-min images were significantly higher than those from 45-min images (+18% +/- 8%; P < 0.01), whereas the SUVs of inflammatory lesions decreased over time (-17% +/- 13% of the early images; P < 0.05) . Clinical studies: The SUVs of delayed images from the known malignant lesions compared with those of earlier scans increased over time (+19.18% +/- 9.58%; n = 31; P < 0.001; 95% confidence interval, 15.8%-22.6%) . By contrast, the SUVs of benign lung nodules decreased slightly over time (-6.3% +/- 8.1%; n = 12; P < 0.05; 95% confidence interval, -10.9% to -1.7%) . The SUV of inflammatory lesions caused by radiation therapy (+1.16% +/- 7.23%; n = 8; P > 0.05; 95% confidence interval, -3.9%-6.2%) and the lesions of painful lower limb prostheses (+4.03% +/- 11.32%; n = 25; P > 0.05; 95% confidence interval, -0.4%-8.5%) remained stable over time . CONCLUSION: These preliminary data show that dual time imaging appears to be useful in distinguishing malignant from benign lesions . Further research is necessary to confirm these results.

Prikl Biokhim Mikrobiol, 2001 Jul-Aug, 37(4), 418 - 23
{Effects of exogenous factors on the activity of enzymes involved in carbon metabolism in thermoacidophilic bacteria of the genus Sulfobacillus}; Krasil'nikova EN et al.; Aerobic thermoacidophilic chemolithotrophic bacteria Sulfobacillus thermosulfidooxidans 1269T and Sulfobacillus thermosulfidooxidans subsp . asporogenes 41 were shown to be resistant to stress factors, including high concentrations of Zn2+ (0.8 M) and H+ (pH 1.2) that exceeded the optimum values . The growth and biomass gain rates decreased, but bacteria retained their functions . The activity of nearly all enzymes involved in carbon metabolism decreased . Glucose was primarily metabolized via the Entner--Doudoroff pathway . The activity tricarboxylic acid cycle enzymes decreased compared to that in cells grown under normal conditions . After saturation of the growth medium with 5 vol % CO2, sulfobacteria utilized glucose by the Embden-Meyerhof and pentose phosphate pathways under mixotrophic conditions.

Exp Parasitol, 2001 Jul, 98(3), 145 - 51
Entamoeba invadens: enhancement of excystation and metacystic development by cytochalasin D; Makioka A et al.; Effects of three actin-modifying drugs, cytochalasin D, latrunculin A, and jasplakinolide, on the excystation and metacystic development in vitro of Entamoeba invadens were examined by transfer of the cysts to growth medium with the drugs . Cytochalasin D unexpectedly increased the number of metacystic amoebae of E . invadens strain IP-1 during incubation . Metacystic development, which was determined by the number of nuclei of metacystic amoebae, was faster in the culture with cytochalasin D than in the culture without the drug . These results suggest that cytochalasin D enhances the excystation and metacystic development . In contrast, latrunculin A and jasplakinolide inhibited these process . No excystation occurred in encystation medium even in the presence of cytochalasin D, suggesting that growth medium is essential for excystation . Excystation was further enhanced when the cysts were incubated with cytochalasin D before culture in growth medium with the drug . The enhancing effect of cytochalasin D on the excystation and metacystic development was abrogated by jasplakinolide . Thus, the results indicate that cytochalasin D, unlike latrunculin A and jasplakinolide, caused enhancement of the excystation and metacystic development of this parasite .

Curr Genet, 2001 Jul, 39(5-6), 335 - 9
Cloning and expression analysis of a new hydrophobin cDNA from the ectomycorrhizal basidiomycete Pisolithus; Duplessis S et al.; Hydrophobins are fungal cell wall proteins which play a crucial role in cell adhesion and aggregative processes . We have identified a new hydrophobin cDNA (hydPt-3) in the symbiotic mycelium of Pisolithus tinctorius (putative P . albus) during the formation of ectomycorrhizae around eucalypt roots . This sequence is highly divergent from two other previously identified Pisolithus symbiosis-regulated hydrophobins, hydPt-1 and hydPt-2 . Also, expression analyses demonstrated that hydPt-3 is up-regulated during the formation of ectomycorrhizae . In contrast to phytopathogenic fungi, changes in glucose or ammonium concentrations in the growth medium did not influence the accumulation of any Pisolithus hydrophobin mRNAs . This suggests that other factors act as regulators of hydrophobin gene expression in ectomycorrhizae.

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