J Chem Ecol, 2001 Aug, 27(8), 1691 - 700 Allelochemicals in wheat (Triticum aestivum L.): production and exudation of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one; Wu H et al.; An analytical technique employing gas chromatography and tandem mass spectrometry (GC/MS/MS) was employed to systematically screen fifty-eight wheat accessions for their differential production of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) from three consecutive sources, i.e., the shoots, roots, and in the associated agar growth medium (collected as root exudates) of 17-day-old wheat seedlings . DIMBOA content differed significantly in the shoots, roots, or in the agar growth medium between accessions . DIMBOA accumulated differentially within the plant, with roots containing more DIMBOA than the shoots . Only 19% of accessions were able to exude DIMBOA from living roots into their growth medium, indicating the exudation of DIMBOA is accession-specific . DIMBOA level in root tissues is expected to be high when a high level of DIMBOA content is detected in the shoots . Wheat seedlings did not release detectable amounts of DIMBOA when the DIMBOA level was low in the root tissues . The valuable genetic material with high levels of DIMBOA in the shoots or roots identified in the present research could be used to breed for wheat cultivars with elevated allelopathic activity. Syst Appl Microbiol, 2001 Jul, 24(2), 166 - 76 Taxonomic characterization of new alkaliphilic and alkalitolerant methanotrophs from soda lakes of the Southeastern Transbaikal region and description of Methylomicrobium buryatense sp.nov; Kaluzhnaya M et al.; Five strains of obligate methanotrophic bacteria (4G, 5G, 6G, 7G and 5B) isolated from bottom sediments of Southeastern Transbaikal soda lakes (pH 9.5-10.5) are taxonomically described . These bacteria are aerobic, Gram-negative monotrichous rods having tightly packed cup-shaped structures on the outer cell wall surface (S-layers) and Type I intracytoplasmic membranes . All the isolates possess particulate methane monooxygenase (pMMO) and one strain (5G) also contains soluble methane monooxygenase (sMMO) . They assimilate methane and methanol via the ribulose monophosphate pathway (RuMP) . The isolates are alkalitolerant or facultatively alkaliphilic, able to grow at pH 10.5-11.0 and optimally at pH 8.5-9.5 . These organisms are obligately dependent on the presence of sodium ions in the growth medium and tolerate up to 0.9-1.4 M NaCl or 1 M NaHCO3 . Although being mesophilic, all the isolates are resistant to heating (80 degrees C, 20 min), freezing and drying . Their cellular fatty acids profiles primarily consist of C(16:1) . The major phospholipids are phosphatidylethanolamine and phosphatidylglycerol . The main quinone is Q-8 . The DNA G+C content ranges from 49.2-51.5 mol % . Comparative 16S rDNA sequencing showed that the newly isolated methanotrophs are related to membres of the Methylomicrobium genus . However, they differ from the known members of this genus by DNA-DNA relatedness . Based on pheno- and genotypic characteristics, we propose a new species of the genus Methylomicrobium Methylomicrobium buryatense sp . nov. Leukemia, 2001 Sep, 15(9), 1433 - 41 Regulation of the acute myeloid leukemia cell line OCI/AML-2 by endothelial nitric oxide synthase under the control of a vascular endothelial growth factor signaling system; Koistinen P et al.; It is generally accepted that the vascular endothelial growth factor (VEGF) signal system has no role in the maintenance of normal blood cell formation, although it obviously regulates the development of primitive hematopoiesis during an early stage of embryogenesis . The VEGF signaling pathway, however, might have some role in malignant hematopoiesis, since malignant hematopoietic cells, including acute myeloid leukemia (AML) cells, have been shown to express VEGF and its receptors . In endothelial cells, the VEGF/Flk-1/KDR signal system is a very important generator of nitric oxide (NO) through the activation of its downstream effectors phosphatidylinositol-3-OH-kinase (PI3-K), Akt kinase and endothelial NO synthase (eNOS) . It is known that NO regulates hematopoiesis and modulates AML cell growth . The role of the VEGF signaling pathway in the control of AML cell growth through eNOS, however, has not been studied . By using the OCI/AML-2 cell line, which expresses VEGF receptor-2, ie Flk-1/KDR, eNOS and VEGF, as analyzed by flow cytometry, and produces VEGF into growth medium, as analyzed by ELISA, we showed that the Akt kinase and NOS activities in these cells were decreased by the inhibitors of VEGF, Flk-1/KDR and PI3-K, and NOS activity also by the direct inhibitor of NOS . The decreased NOS activity led to inhibition of clonogenic cell growth and, to some extent, induction of apoptosis . We also found that blast cells of bone marrow samples randomly taken from 14 AML patients uniformly expressed Flk-1/KDR and to varying degrees eNOS and VEGF, as analyzed by immunohistochemistry . We conclude that autocrine VEGF through Flk-1/KDR, by activating eNOS to produce NO through PI3-K/Akt kinase, maintains clonogenic cell growth in the OCI/AML-2 cell line . Since the patient samples did not express VEGF in all cases, it is possible that in vivo the regulatory connection between these two signal systems is also mediated via endocrine VEGF in addition to autocrine or paracrine VEGF. Mol Biol Cell, 2001 Aug, 12(8), 2534 - 45 Actin polymerization is essential for pollen tube growth; Vidali L et al.; Actin microfilaments, which are prominent in pollen tubes, have been implicated in the growth process; however, their mechanism of action is not well understood . In the present work we have used profilin and DNAse I injections, as well as latrunculin B and cytochalasin D treatments, under quantitatively controlled conditions, to perturb actin microfilament structure and assembly in an attempt to answer this question . We found that a approximately 50% increase in the total profilin pool was necessary to half-maximally inhibit pollen tube growth, whereas a approximately 100% increase was necessary for half-maximal inhibition of cytoplasmic streaming . DNAse I showed a similar inhibitory activity but with a threefold more pronounced effect on growth than streaming . Latrunculin B, at only 1--4 nM in the growth medium, has a similar proportion of inhibition of growth over streaming to that of profilin . The fact that tip growth is more sensitive than streaming to the inhibitory substances and that there is no correlation between streaming and growth rates suggests that tip growth requires actin assembly in a process independent of cytoplasmic streaming. Neurosci Lett, 2001 Aug 31, 309(3), 193 - 6 Phospholipid fatty acids and neurotoxicity in human neuroblastoma SH-SY5Y cells; Reynolds LM et al.; The fatty acid composition of phospholipids from differentiated human neuroblastoma SH-SY5Y cells, human and rat brain tissue and rat brain synaptosomes was determined using high pressure liquid chromatography . Comparison of the fatty acid composition of the cells with that derived from brain tissue identified differences in the cells including a profound deficit of docosahexaenoic acid and an elevation of arachidonic acid . The phospholipid fatty acid content could be modified by addition of free fatty acids to the growth medium, and this was shown to influence the susceptibility of the SH-SY5Y cells to the cell death induced by a mitochondrial toxin, 3-nitropropionic acid. J Agric Food Chem, 2001 Aug, 49(8), 3742 - 5 Allelochemicals in wheat (Triticum aestivum L.): cultivar difference in the exudation of phenolic acids; Wu H et al.; Analysis by GC-MS/MS showed that a worldwide collection of 58 wheat accessions differed significantly in the amounts of 7 known phenolic acids exuded by the living roots of 17-day-old wheat seedlings . The quantities of exuded allelochemicals varied with the specific compound and ranged from 2.3 to 18.6, from 0.6 to 17.5, from 0.1 to 4.9, from 0.0 to 52.7, from 0.33 to 12.7, from 1.5 to 20.5, and from 1.6 to 23.4 microg/L of water/agar for p-hydroxybenzoic, vanillic, cis-p-coumaric, syringic, cis-ferulic, trans-p-coumaric, and trans-ferulic acids, respectively . The concentrations of p-hydroxybenzoic and vanillic acids exuded by wheat seedlings were normally distributed in the 58 accessions . The level of each phenolic acid in root exudates did not correlate well to that previously observed in wheat . In comparison with weakly allelopathic accessions, strongly allelopathic accessions exuded larger quantities of allelochemicals into the growth medium . The chemical basis for wheat seedling allelopathy is an area for further investigation. Biochemistry, 2001 Aug 28, 40(34), 10350 - 9 Mutations of arginine 64 within the putative Ca(2+)-binding lumenal interhelical a-b loop of the photosystem II D1 protein disrupt binding of the manganese stabilizing protein and cytochrome c(550) in Synechocystis sp . PCC6803; Li ZL et al.; Mutations D1-R64E, D1-R64Q, and D1-R64V in the putative calcium-binding lumenal interhelical a-b loop of the photosystem II (PSII) D1 protein were characterized in terms of impact on growth, extrinsic protein binding, photoactivation, and properties of the H(2)O-oxidation complex . The D1-R64E charge reversal mutation greatly weakened the binding of the extrinsic manganese-stabilizing protein (MSP) and, to a considerably lesser extent, weakened the binding of cytochrome c(550) (c550) . Both D1-R64Q and D1-R64E exhibited an increased requirement for Ca(2+) in the cell growth medium . Bare platinum electrode measurements of O(2)-evolving membranes showed a retarded appearance of O(2) following single turn-over flashes, especially in the case of the D1-R64E mutant . The D1-R64E mutant also had a pronounced tendency to lose O(2) evolution activity in the dark and exhibited an increased relative quantum yield of photoactivation, which are characteristics shared by mutants that lack extrinsic proteins . S(2) and S(3) decay measurements in the isolated membranes indicate that D1-R64E and D1-R64Q have faster decays of these higher S-states as compared to the wild-type . However, fluorescence decay in the presence of DCMU, which monitors primarily Q(A)(-) charge recombination with PSII donors, showed somewhat slower decays . Taken together, the fluorescence and S-state decay indicate that the midpoint of either Q(B)(-) has been modified to be more negative in the mutants or that a recombination path presumably involving either Q(B)(-) or Y(D) has become kinetically more accessible. Arch Microbiol, 2001 Sep, 176(3), 211 - 6 Analysis of phenotypic diversity among host-independent mutants of Bdellovibrio bacteriovorus 109J; Barel G et al.; Host-independent (H-I) mutants of the obligate bacterial parasite Bdellovibrio bacteriovorus were isolated from wild-type strain 109J . Seven H-I mutants differed in morphological features such as cell length (2-30 microm) and shape (short or long spirals or rod-like), plaque size, and pigmentation (from almost colorless to bright orange) . The mutants exhibited widely different growth capabilities in rich medium, with biomass doubling times and final biomass varying by a factor of two or more . Growth was always enhanced by the addition of host cell extract or divalent cations to the growth medium, but the effect varied widely between the mutants . Analysis of the hit region, mutations in which were previously proposed to be associated with the H-I phenotype, revealed that changes in the nucleotide sequence in this region occurred only in three of the seven mutants. Arch Microbiol, 2001 Sep, 176(3), 197 - 203 The C-terminal part of the surface-associated protein MopE of the methanotroph Methylococcus capsulatus (Bath) is secreted into the growth medium; Fjellbirkeland A et al.; A protein with an apparent molecular mass of 46 kDa was detected as the major polypeptide in the culture medium of the biotechnologically important methanotrophic bacterium Methylococcus capsulatus (Bath) . The protein cross-reacted with polyclonal antibodies raised against the outer-membrane-associated protein MopE . The antiserum was used to identify a positive clone from a lambda gt11 library . The nucleotide sequence determined for the clone demonstrated that MopE and the secreted protein are encoded by the same gene, and that the secreted protein represents an N-terminally truncated form of MopE . By using monospecific antibodies against MopE in immunogold electron microscopy, the protein was localized at the cell surface and cell periphery . The mopE gene was expressed in Escherichia coli . The MopE protein synthesized was found in the periplasmic space of E . coli . No protein with sequence similarity over the entire length of MopE was detected in the databases, but some sequence similarity to the copper-repressible CorA protein of the methanotroph Methylomicrobium albus (Berson and Lidstrom 1997) was observed for the C-terminal region of MopE. Br J Nutr, 2001 May, 85 Suppl 2, S175 - 9 Highly nutrient-dense spreads: a new approach to delivering multiple micronutrients to high-risk groups; Briend A; Using a highly fortified food is the most attractive option to bringing missing nutrients to vulnerable groups . The recent development of a highly nutrient-dense spread (HNDS) for the treatment of malnourished children may have some relevance for other high-risk groups . Traditionally, severely malnourished children are fed for 3-4 weeks during their recovery with adapted milk feeds prepared by mixing dried skimmed milk, oil and sugar with a vitamin and mineral complex . This approach, however, is difficult to implement, since these feeds are excellent growth media for bacteria, and they must be prepared and fed under close supervision . This constraint led to the development of a HNDS, which is obtained by replacing part of the dried skimmed milk with a mixture of groundnut butter and powdered lactoserum . This spread can be eaten without dilution with water and preliminary trials showed that children preferred this HNDS to traditional liquid diets . In HNDS all powdered ingredients are embedded in fat which protects vitamins against oxidation and increases the shelf life of this product . Spreads also have a very low humidity and bacteria do not grow in it . Attempts to use spreads to supplement other vulnerable groups such as moderately malnourished children and pregnant women are discussed. Biometals, 2001 Jun, 14(2), 143 - 51 Pattern of cadmium accumulation and essential cations during growth of cadmium-tolerant fungi; Gharieb MM; The present study evaluates the growth response of two strains of filamentous fungi; a Fusarium sp . and Alternaria tenuis, grown on both solid and liquid Czapek Dox medium amended with different concentrations of CdCl2 . Colony extension and the mycelial dry weight of both fungi were significantly inhibited by high concentrations of cadmium . Extended lag phases and low growth rates resulted from cadmium administration . Cadmium drastically affected fungal morphogenesis by the production of stunted sterile thick mycelial filaments of the Fusarium sp . and chains of uncharacterized swellings instead of conidia in A . tenuis . Experiments showed that cadmium accumulation by the Fusarium sp . grown in liquid medium was a concentration dependent, and over the incubation time it displayed a plateau pattern . The cells grown on medium containing 0.25 mmol l(-1) CdCl2 accumulated up to 89 +/- 12 ,umol Cd (gm dw)(-l) after two days, falling to approximately 29 +/- 10 mol Cd (gm dw)(-1) after five days . At 0.5 mmol l(-1) CdCl2 treatment the maximum cellular cadmium content was approximately 132 +/- 14 micromol (gm dw)(-1), attained after 3 days, and decreased to degrees 98 +/- 9 micromol (gm dw)(-1) at the end of the incubation time . There was a simultaneous marked drop in cadmium content and pH of the growth medium during the first few days . The presence of cadmium markedly altered the cellular essential cations; K+ and Mg2+ being decreased while Na+ increased during the growth period . Such findings resulted a reverse pattern of cellular Na+/K+ ratio for cells grown on cadmium-containing medium in respect to the control treatment . The results are discussed in relation to a further dimension of cadmium effects that might reflect its toxicity, as well as the implication of cadmium extrusion for tolerance during fungal growth. Drug Resist Updat, 1999 Dec, 2(6), 363 - 369 Active transport of siderophore-mimicking antibacterials across the outer membrane; Braun V; The outer membrane of gram-negative bacteria forms a permeability barrier that usually reduces antibiotic access to intracellular targets and renders gram-negative bacteria less susceptible to antibiotics than gram-positive bacteria, which lack an outer membrane . However, gram-negative bacteria become highly susceptible to antibiotics that are actively transported across the outer membrane . Some antibiotics use active transport systems of substrates with which they share structural features . Examples are naturally occurring sideromycins and synthetic derivatives of Fe(3+)-siderophores, which are taken up across the outer membrane by transport systems for Fe(3+)-siderophores . A well-studied example is albomycin, which has structural similarities to the natural substrate ferrichrome; albomycin and ferrichrome are both transported by the FhuA protein . A semisynthetic rifamycin derivative, CGP 4832, is also taken up by the FhuA transport protein, although its structure is completely different from that of ferrichrome . The crystal structures of FhuA with bound ferrichrome, albomycin, or rifamycin CGP 4832 reveal that the three compounds occupy the same site on FhuA; this site is accessible from the growth medium by a surface cavity that accommodates the antibiotic moieties . There is a rather strict stereochemical requirement for the portion that fits into the active site of FhuA, but a rather large tolerance regarding the portion that is located in the cavity . These data provide precise structural information for the design of highly active antibiotics composed of an antibiotically active moiety connected by a linker to a transported carrier . A number of Fe(3+)-siderophore carriers of the hydroxamate and catechol type linked to antibiotics have been isolated from microbes and synthesized; their superior efficacy has been demonstrated in vitro and in mice . Although none have been therapeutically employed, it is proposed that this alternative method of synthesizing useful antibiotics should be tested in light of the increasing problem of resistant pathogens . Mol Plant Microbe Interact, 2001 Aug, 14(8), 969 - 79 Phenazine-1-carboxamide production in the biocontrol strain Pseudomonas chlororaphis PCL1391 is regulated by multiple factors secreted into the growth medium; Chin-A-Woeng TF et al.; Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f . sp . radicis-lycopersici . The production of phenazine-1-carboxamide (PCN) is crucial for this biocontrol activity . In vitro production of PCN is observed only at high-population densities, suggesting that production is under the regulation of quorum sensing . The main autoinducer molecule produced by PCL1391 was identified structurally as N-hexanoyl-L-homoserine lactone (C6-HSL) . The two other autoinducers that were produced comigrate with N-butanoyl-L-homoserine lactone (C4-HSL) and N-octanoyl-L-homoserine lactone (C8-HSL) . Two PCL1391 mutants lacking production of PCN were defective in the genes phzI and phzR, respectively, the nucleotide sequences of which were determined completely . Production of PCN by the phzI mutant could be complemented by the addition of exogenous synthetic C6-HSL, but not by C4-HSL, C8-HSL, or any other HSL tested . Expression analyses of Tn5luxAB reporter strains of phzI, phzR, and the phz biosynthetic operon clearly showed that phzI expression and PCN production is regulated by C6-HSL in a population density-dependent manner . The introduction of multiple copies of the regulatory genes phzI and phzR on various plasmids resulted in an increase of the production of HSLs, expression of the PCN biosynthetic operon, and consequently, PCN production, up to a sixfold increase in a copy-dependent manner . Surprisingly, our expression studies show that an additional, yet unidentified factor(s), which are neither PCN nor C4-HSL or C8-HSL, secreted into the growth medium of the overnight cultures, is involved in the positive regulation of phzI, and is able to induce PCN biosynthesis at low cell densities in a growing culture, resulting in an increase of PCN production. New Microbiol, 2001 Jul, 24(3), 243 - 7 Establishment of a swine monocyte cell line; Kadoi K et al.; A swine monocyte cell line was established from peripheral blood sample collected from a healthy adult male pig . The cloned cells grow actively in forming monolayers in both glass and plastic cell culture flasks with the growth medium reported previously (Kadoi, 2000) at 36.5 degrees C incubation . The plating efficiency is more than 95% . Densely grown cells in flasks show an epithelioid morphology . The fundamental properties of the cells were examined for cytological definition as monocytes . A positive property detected was guinea pig complement receptor, porcine IgG receptor, non-specific esterase, and acid phosphatase . A significant phagocytic activity proved by the inoculation of Saccharomyces cerevisiae is also one of the characteristics observed in the LPS-activated cells. J Ind Microbiol Biotechnol, 2001 May, 26(5), 283 - 9 Biodegradation of polycyclic aromatic hydrocarbons by Sphingomonas strains isolated from the terrestrial subsurface; Shi T et al.; Several strains of Sphingomonas isolated from deep Atlantic coastal plain aquifers at the US Department of Energy Savannah River Site (SRS) near Aiken, SC were shown to degrade a variety of aromatic hydrocarbons in a liquid culture medium . Sphingomonas aromaticivorans strain B0695 was the most versatile of the five strains examined . This strain was able to degrade acenaphthene, anthracene, phenanthrene, 2,3-benzofluorene, 2-methylnaphthalene, 2,3-dimethylnaphthalene, and fluoranthene in the presence of 400 mg l(-1) Tween 80 . Studies involving microcosms composed of aquifer sediments showed that S . aromaticivorans B0695 could degrade phenanthrene effectively in sterile sediment and could enhance the rate at which this compound was degraded in nonsterile sediment . These findings indicate that it may be feasible to carry out (or, at least, to enhance) in situ bioremediation of phenanthrene-contaminated soils and subsurface environments with S . aromaticivorans B0695 . In contrast, strain B0695 was unable to degrade fluoranthene in microcosms containing aquifer sediments, even though it readily degraded this polynuclear aromatic hydrocarbon (PAH) in a defined liquid growth medium. Biotechnol Prog, 2001 Jul-Aug, 17(4), 618 - 23 Factors affecting the production of a single-chain antibody fragment by Aspergillus awamori in a stirred tank reactor; Sotiriadis A et al.; A recombinant strain of Aspergillus awamori expressing anti-lysozyme single chain antibody fragments (scFv), under the control of a xylanase promoter, was studied in order to investigate the impact of medium, induction regime and protease production on the expression of the product . Experiments with the time of induction showed that the optimum results are achieved when induction is started in the late exponential phase (21 h after inoculation) improving the titer of the product from 14.5 mg L(-1), obtained in the early exponential phase (7 h after inoculation), to 16.2 mg L(-1) . A 100% increase of the carbon (fructose) and nitrogen (ammonium sulfate) sources in the growth medium resulted in an increase in product concentration from 16.2 to 108.9 mg L(-1) and an increase in maximum dry cell weight from 7.5 to 11.5 g L(-1) . A 50% reduction in the concentration of the inducer resulted in an increase in the product yield from 10 mg g(-1) dry cell weight to 12 mg g(-1) . Proteolytic enzymes were produced during the fermentation up to concentrations equivalent to 1.4 g L(-1) trypsin, but they had no detrimental effect on the concentration of the antibody fragment. J Biomed Mater Res, 2001 Nov, 57(2), 232 - 7 Cell response to herpes simplex virus type 1 infection mediated by biphasic calcium-phosphate ceramics: in vitro approach; Varadinova TL et al.; Based on well-documented data showing that bioactive ions (such as Ca2+, PO4-, etc.) released by BCPC induce various cell responses and on the significance of herpes outbreaks in human pathology, we investigated whether BCPC can modify cell response to HSV-1 infection . The roles of some physical and chemical properties of ceramics were evaluated using three BCPC samples--French commercial macro-microporous (FR) and two Bulgarian microporous laboratory samples--one of which was modified with magnesium (BG and BG + Mg) . Samples only washed in 0.9% NaCl were designated as nonconditioned while those resuspended in cell growth medium for 10 days after washing were designated as conditioned . Experiments were done on cells from the continuous MDBK line precultured on BCPC surfaces for different time intervals and thereafter HSV-1 infected . The yield of infectious virus progeny, measured as virus titers, was the parameter used to determine the cell response to HSV-1 infection mediated by BCPC as compared to that of the virus control, that is, virus yield in cells cultured without BCPC . The data obtained show that all three nonconditioned BCPC samples were able to modify cells to resist HSV-1 infection . The prolongation of the resistant state depended on the specific physical and chemical properties of the particular BCPC sample: as the data show that the conditioning procedure (1) increased the ability of BG + Mg to promote cell resistance, and (2) reduced the ability of FR samples to modify cells to resist HSV-1 infection . The data obtained show that apart from Ca2+ and PO4-, ions of biometals such as Mg2+ also are responsible for the induction and maintenance of cell resistance to HSV-1 infection . Neuroscience, 2001, 105(1), 131 - 7 Post-insult activity is a major cause of delayed neuronal death in organotypic hippocampal slices exposed to glutamate; Lahtinen H et al.; We investigated the pathophysiological mechanisms of glutamate-induced delayed neuronal damage in rat hippocampal slice cultures {Stoppini et al . (1991) J . Neurosci . Methods 37, 173-182}, with propidium iodide as a marker of cell death . Exposure of the cultures to growth medium containing 10 mM glutamate for 30 min resulted in a slowly developing degeneration of hippocampal principal cells, starting from the medial end of the CA1 region and reaching the dentate gyrus by 48 h . By 24 h, most pyramidal cells in CA1 were damaged . An acute phase of degeneration preceded the delayed damage at 2-6 h, affecting cells in a spatially diffuse manner . When tetrodotoxin (0.5 microM) was present during the glutamate insult, a marked protection (mean 57%, P<0.001) of the CA1 damage was observed . Rather strikingly, when tetrodotoxin was applied immediately following or even with a delay of 30 min after the insult, a similar amount of protection was achieved . In field recordings carried out after the insult, the glutamate-treated slices exhibited spontaneously occurring negative shifts with a duration of 1-10 s and an amplitude of up to 400 microV in the CA3 region, whereas the control slices were always quiescent . Taken together, the results suggest that post-insult neuronal network activity, rather than the direct action of exogenous glutamate, is a major cause of delayed CA1 pyramidal cell death in the organotypic slices . These observations may have implications in the design of neuroprotective strategies for the treatment of brain traumas which are accompanied by delayed and/or distal neuronal damage. J Antimicrob Chemother, 2001 Aug, 48(2), 163 - 77 Susceptibility testing of pathogenic fungi with itraconazole: a process analysis of test variables; Rambali B et al.; A 2(10-5) fractional factorial model was used to investigate the influence of 10 process variables in broth microdilution susceptibility tests with itraconazole against eight isolates of Candida species and six isolates of filamentous fungi in two growth media . An analysis of variance (ANOVA) indicated that glucose concentration and incubation time both significantly influenced control turbidity optical density (OD) values for most of the Candida spp . isolates, while incubation in >10% CO(2) versus ambient air, incubation temperature and inoculum size significantly influenced these OD values for about half of the yeast isolates . Control OD values for the mould isolates were most influenced by incubation time and temperature, and by occlusion of the wells with an adhesive sticker . Three statistical approaches, ANOVA, rank transformation and Mann-Whitney U-test, were used to assess the influence of the variable combinations on MIC, determined with a 50% growth reduction end-point . Incubation temperature and time, glucose concentration and inoculum size were the variables that most often affected susceptibility results to the level of statistical significance; however, the supplier of RPMI 1640 medium, the use of adhesive stickers and the atmosphere of incubation significantly influenced the MIC for some isolates . The medium used to prepare the test inoculum, the solvent used to prepare the stock solution and the shape of the microdilution plate wells significantly affected outcome, but only sporadically . A principal component analysis of the data matrix confirmed this order of relative influence of the test variables on the MIC . Since each fungal isolate responded differently to combinations of process variables in the test, we conclude that any unified method for antifungal susceptibility determination represents a compromise, rather than an idealized system. J Mol Neurosci, 2001 Apr-Jun, 16(2-3), 263 - 72; discussion 279-84 Plasmalogens, phospholipase A2, and docosahexaenoic acid turnover in brain tissue; Farooqu AA et al.; Plasmalogens are glycerophospholipids of neural membranes containing vinyl ether bonds . Their synthetic pathway is located in peroxisomes and endoplasmic reticulum . The rate-limiting enzymes are in the peroxisomes and are induced by docosahexaenoic acid (DHA) . Plasmalogens often contain arachidonic acid (AA) or DHA at the sn-2 position of the glycerol moiety . The receptor-mediated hydrolysis of plasmalogens by cytosolic plasmalogen-selective phospholipase A2 generates AA or DHA and lysoplasmalogens . AA is metabolized to eicosanoids . The mechanism of signaling with DHA is not known . The plasmalogen-selective phospholipase A2 differs from other intracellular phospholipases A2 in molecular mass, kinetic properties, substrate specificity, and response to glycosaminoglycans, gangliosides, and sialoglycoproteins . A major portion of {3H}DHA incorporated into neural membranes is found at the sn-2 position of ethanolamine glycerophospholipids . Studies with a mutant cell line defective in plasmalogen biosynthesis indicate that the incorporation of DHA is reduced in this RAW 264.7 cell line by 50% . In contrast, the incorporation of AA remains unaffected . This is reversed completely when the growth medium is supplemented with sn-1-hexadecylglycerol, suggesting that DHA can be selectively targeted for incorporation into plasmalogens . We suggest that deficiencies of DHA and plasmalogens in peroxisomal disorders, Alzheimer's disease (AD), depression, and attention deficit hyperactivity disorders (ADHD) may be responsible for abnormal signal transduction associated with learning disability, cognitive deficit, and visual dysfunction . These abnormalities in the signal-transduction process can be partially corrected by supplementation with a diet enriched with DHA. J Biol Chem, 2001 Oct 12, 276(41), 37815 - 20 Epub 2001 Jul 26. Expression of p21Waf1/Cip1 and cyclin D1 is increased in butyrate-resistant HeLa cells; Derjuga A et al.; Sodium butyrate induced cell cycle arrest in mammalian cells through an increase in p21Waf1/Cip1, although another study showed that this arrest is related to pRB signaling . We isolated variants of HeLa cells adapted to growth in 5 mm butyrate . One of these variants, clone 5.1, constitutively expressed elevated levels of p21Waf1/Cip1 when incubated in regular growth medium and in the presence of butyrate . Despite this elevated level of p21Waf1/Cip1, the cells continue to proliferate, albeit at a slower rate than parental HeLa cells . Western blot analyses showed that other cell cycle regulatory proteins were not up-regulated to compensate for the elevated expression of p21Waf1/Cip1 . However, cyclin D1 was down-regulated by butyrate in HeLa cells but not in clone 5.1 . We conclude that continued expression of cyclin D1 allowed clone 5.1 to grow in the presence of butyrate and elevated levels of p21Waf1/Cip1. Physiol Plant, 2001 Jul, 112(3), 397 - 402 Isolation of aluminum-tolerant cell lines of tobacco in a simple calcium medium and their responses to aluminum; Rama Devi S et al.; Aluminum (Al)-tolerant cell lines (ALT301 and ALT401) of tobacco were isolated in a simple calcium (Ca) solution from ethyl methane sulfonate (EMS)-treated suspension cultured tobacco cells (Nicotiana tabacum L . cv . Samsun, a cell line SL) at the logarithmic phase of growth . A highly tolerant cell line ALT301 exhibited the accumulation of Al and the deposition of callose to the same extent as the parental SL cells during the exposure to Al . However, the Al-treated ALT301 cells grew much better than the Al-treated SL cells after transfer to Al-free growth medium . Compared to SL cells, ALT301 cells were more tolerant to toxicity of copper and iron, but not to that of lanthanum . These results suggest that ALT301 cells have an internal tolerance mechanism, which makes cells grow normally in spite of Al accumulation and Al-induced lesion represented by the deposition of callose . This tolerance mechanism seems also to be effective against copper and iron toxicity . A slightly tolerant cell line ALT401 also accumulated Al to the same degree as SL cells, but deposited significantly less callose than did SL cells (43% of SL) . The growth of ALT401 cells after Al treatment was only slightly better than that of SL cells . Thus, it seems that ALT401 cells have a mechanism to protect cells only from the Al-induced deposition of callose, which is not enough to overcome the Al-induced inhibition of growth . The different phenotypes exhibited by these Al-tolerant cell lines suggest that the deposition of callose is not directly related to the inhibition of growth in Al-treated cells. Physiol Plant, 2001 Jul, 112(3), 334 - 342 Effect of embryonic axis removal and exogenous calcium on carboxypeptidase I of mung bean seedling cotyledons; Singh S et al.; Removal of the embryonic axis prevents the normal decline of carboxypeptidase (Cpase) I in mung bean seedling cotyledons . Cpase I activity and protein, the latter manifested on western blots, almost completely disappear about 24 h before the cotyledon abscises . Of the 3 proteolytic enzyme patterns, only that of Cpase I can be restored by an exogenous supply of 10 mM CaCl2 in the agar growth medium . The calcium effect is dependent on {CaCl2} and is not manifested in the presence of chelators and calcium channel blockers . For detached cotyledons to show the normal low level of Cpase I by the eighth day of growth, calcium had to be supplied during seed imbibition and throughout the entire time from removal of the axis . The difference between detached cotyledons in the absence and presence of calcium was greatest when the cotyledons were detached 4-6 days after seed imbibition . Loss of Cpase I activity and protein can be demonstrated in vitro, with the maximum level of Cpase I-degrading activity measured 4 days after seed imbibition under the same growth conditions used to study the calcium effect . It is sensitive to pepstatin and has a pH optimum of 3, suggesting that this Cpase I-degrading activity is due to an aspartic protease. Appl Environ Microbiol, 2001 Aug, 67(8), 3750 - 2 Evidence for iron-dependent nitrate respiration in the dissimilatory iron-reducing bacterium Geobacter metallireducens; Senko JM et al.; The dissimilatory iron-reducing bacterium Geobacter metallireducens was found to require iron at a concentration in excess of 50 microM for continuous cultivation on nitrate . Growth yield (approximately 3-fold), cytochrome c content (approximately 7-fold), and nitrate (approximately 4.5-fold) and nitrite (approximately 70-fold) reductase activities were all increased significantly when the growth medium was amended with 500 microM iron. Appl Environ Microbiol, 2001 Aug, 67(8), 3455 - 62 Isolation and characterization of a gene specific to lager brewing yeast that encodes a branched-chain amino acid permease; Kodama Y et al.; We found two types of branched-chain amino acid permease gene (BAP2) in the lager brewing yeast Saccharomyces pastorianus BH-225 and cloned one type of BAP2 gene (Lg-BAP2), which is identical to that of Saccharomyces bayanus (by-BAP2-1) . The other BAP2 gene of the lager brewing yeast (cer-BAP2) is very similar to the Saccharomyces cerevisiae BAP2 gene . This result substantiates the notion that lager brewing yeast is a hybrid of S . cerevisiae and S . bayanus . The amino acid sequence homology between S . cerevisiae Bap2p and Lg-Bap2p was 88% . The transcription of Lg-BAP2 was not induced by the addition of leucine to the growth medium, while that of cer-BAP2 was induced . The transcription of Lg-BAP2 was repressed by the presence of ethanol and weak organic acid, while that of cer-BAP2 was not affected by these compounds . Furthermore, Northern analysis during beer fermentation revealed that the transcription of Lg-BAP2 was repressed at the beginning of the fermentation, while cer-BAP2 was highly expressed throughout the fermentation . These results suggest that the transcription of Lg-BAP2 is regulated differently from that of cer-BAP2 in lager brewing yeasts. J Biol Chem, 2001 Oct 26, 276(43), 39512 - 21 Epub 2001 Jul 24. Recruitment of a foreign quinone into the A1 site of photosystem I . In vivo replacement of plastoquinone-9 by media-supplemented naphthoquinones in phylloquinone biosynthetic pathway mutants of Synechocystis sp . PCC 6803; Johnson TW et al.; Interruption of the phylloquinone (PhQ) biosynthetic pathway by interposon mutagenesis of the menA and menB genes in Synechocystis sp . PCC 6803 results in plastoquinone-9 (PQ-9) occupying the A(1) site and functioning in electron transfer from A(0) to the FeS clusters in photosystem (PS) I (Johnson, T . W., Shen, G., Zybailov, B., Kolling, D., Reategui, R., Beauparlant, S., Vassiliev, I . R., Bryant, D . A., Jones, A . D., Golbeck, J . H., and Chitnis, P . R . (2000) J . Biol . Chem . 275, 8523-8530 . We report here the isolation of menB26, a strain of the menB mutant that grows in high light by virtue of a higher PS I to PS II ratio . PhQ can be reincorporated into the A(1) site of the menB26 mutant strain by supplementing the growth medium with authentic PhQ . The reincorporation of PhQ also occurs in cells that have been treated with protein synthesis inhibitors, consistent with a displacement of PQ-9 from the A(1) site by mass action . The doubling time of the menB26 mutant cells, but not the menA mutant cells, approaches the wild type when the growth medium is supplemented with naphthoquinone (NQ) derivatives such as 2-CO(2)H-1,4-NQ and 2-CH(3)-1,4-NQ . Since PhQ replaces PQ-9 in the supplemented menB26 mutant cells, but not in the menA mutant cells, the phytyl tail accompanies the incorporation of these quinones into the A(1) site . Studies with menB26 mutant cells and perdeuterated 2-CH(3)-1,4-NQ shows that phytylation occurs at position 3 of the NQ ring because the deuterated 2-methyl group remains intact . Therefore, the specificity of the phytyltransferase enzyme is selective with respect to the group present at ring positions 2 and 3 . Supplementing the growth medium of menB26 mutant cells with 1,4-NQ also leads to its incorporation into the A(1) site, but typically without either the phytyl tail or the methyl group . These findings open the possibility of biologically incorporating novel quinones into the A(1) site by supplementing the growth medium of menB26 mutant cells. FEMS Microbiol Lett, 2001 Jul 24, 201(2), 265 - 9 Isolation and characterization of Mn(III) tartrate from Phanerochaete chrysosporium culture broth; Podgornik H et al.; High initial Mn(II) concentration results in accumulation of a Mn(III) tartrate complex in the growth medium of Phanerochaete chrysosporium . Since Mn(III) is the major oxidant in ligninolysis by manganese peroxidase, the role of accumulated complex should not be neglected when degradation experiments by a crude culture filtrate are performed . To study the Mn(III) complex oxidative potential it was isolated by absorption to polyamide followed by desorption with an alkaline methanol solution . High performance liquid chromatography analysis and atomic absorption spectroscopy confirmed that the isolate was Mn(III) tartrate . Oxidation of 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonate) was used for testing the temperature and pH stability of the isolate that also intensively oxidized 2,6-dimethoxyphenol . In comparison with the non-isolated complex in the culture filtrate, the isolate showed increased temperature and pH stability . The oxidative potential of the isolated Mn(III) tartrate was additionally tested by decolorization of the synthetic dye Indigo carmine. FEMS Microbiol Lett, 2001 Jul 24, 201(2), 157 - 62 Choline deficiency induced by Mycoplasma fermentans enhances apoptosis of rat astrocytes; Ben-Menachem G et al.; A choline uptake system accumulating free choline in an energy-dependent process is described in Mycoplasma fermentans . The uptake system has a K(m) of 2.2x10(-5) M and a V(max) of 0.15 nmol 10 min(-1) mg(-1) cell protein and the choline incorporated could be recovered in the soluble fraction as free choline, phosphorylcholine and CDP-choline . Choline accumulation by M . fermentans resulted in a marked choline depletion of the growth medium . The choline depletion of an astrocyte cell culture induced by M . fermentans was associated with the apoptotic death of the cells . Apoptosis was not obtained with heat-inactivated mycoplasmas and could be reversed by the addition of free choline to the growth medium. J Surg Res, 2001 Aug, 99(2), 381 - 4 Nicotine induces endothelial TNF-alpha expression, which mediates growth retardation in vitro; Albaugh G et al.; PURPOSE: Atherosclerosis is understood as the common pathologic manifestation of arterial injury caused by a variety of etiologies . One well-established etiologic agent is nicotine . We hypothesized that cytokines of endothelial origin are involved with the pathologic changes found in atherosclerosis associated with smoking . We chose to assay for TNF-alpha due to its many biologic actions that are similar to those found in peripheral vascular disease . METHODS: Human umbilical vein endothelial cells (HUVEC) were plated in endothelial growth medium (EGM-2) on plastic coverslips until 75% confluent . Free base nicotine (FBN) was diluted in EGM-2 to a concentration of 10(-8) M and added to experimental cells . At 1, 3, and 24 h, coverslips were removed and fixed . Immunohistochemical staining was performed using anti-TNF-alpha . Digital image analysis (DIA) was performed to quantify expression of TNF-alpha . An intensity stain index measuring area and intensity of stain/total cellular area was determined for each time point (n = 5) . Additional HUVEC were plated in 12-well plates in EGM-2 at 2 x 10(3) cells/cm(2) on T(-2) day . FBN was diluted in medium to 10(-9) M and added to wells with and without 0.9 microg/ml anti-TNF-alpha on T(0) day . Cell counts were performed in triplicate on days T(2-5) utilizing hemocytometry . Data was analyzed using Student's t test and ANOVA, with a 95% confidence interval . RESULTS: Dose response determinations showed that the minimal concentration required to show statistically significant cell retardation is 10 (-9) M . Accordingly, this concentration was used for subsequent proliferation studies . DIA showed a threefold increase in TNF-alpha activity at 1 h and a twofold increase at 3 h . Activity returned to baseline by 24 h . Cell growth was significantly decreased in cells exposed to nicotine when compared to controls on days T(2)-T(5) (P < 0.05) . In cells exposed to anti-TNF-alpha and nicotine there was inhibition of the growth retardation seen in the cells containing nicotine alone . Differences between the control group and the anti-TNF-alpha group were not statistically significant . CONCLUSION: These data demonstrate the ability of endothelial cells to secrete TNF-alpha in response to nicotine at levels found in serum after smoking and also shows that endothelial cell growth retardation as a consequence of nicotine exposure may be TNF-alpha mediated . Planta, 2001 Jun, 213(2), 318 - 22 Oxytropism: a new twist in pollen tube orientation; Blasiak J et al.; Chemical gradients and structural features within the pistil have been previously proposed as factors determining the directionality of pollen tube growth . In this study, we examine the behavior of pollen of eight species germinated in a dynamic oxygen gradient . While the germination rates of some species decreased directly with decreasing oxygen tension, other species showed no decrease in germination at oxygen tensions as low as 2 kPa . In one species, germination was consistently greater at decreased oxygen tensions than at ambient atmospheric levels . In three of the eight species tested, the developing pollen tube showed clear directional growth away from the more-oxygenated regions of the growth medium, while in one species growth was towards the more-oxygenated region . The remaining four species showed random tube growth . The pattern of oxytropic responses among the taxa suggests that this tropic behavior is both widespread and phylogenetically unpredictable. J Bacteriol, 2001 Aug, 183(16), 4848 - 59 Structure-function analysis of BfpB, a secretin-like protein encoded by the bundle-forming-pilus operon of enteropathogenic Escherichia coli; Schmidt SA et al.; Production of type IV bundle-forming pili by enteropathogenic Escherichia coli (EPEC) requires BfpB, an outer-membrane lipoprotein and member of the secretin protein superfamily . BfpB was found to compose a ring-shaped, high-molecular-weight outer-membrane complex that is stable in 4% sodium dodecyl sulfate at temperatures of < or = 65 degrees C . Chemical cross-linking and immunoprecipitation experiments disclosed that the BfpB multimeric complex interacts with BfpG, and mutational studies showed that BfpG is required for the formation and/or stability of the multimer but not for the outer-membrane localization of BfpB . Formation of the BfpB multimer also does not require BfpA, the repeating subunit of the pilus filament . Functional studies of the BfpB-BfpG complex revealed that its presence confers vancomycin sensitivity, indicating that it may form an incompletely gated channel through the outer membrane . BfpB expression is also associated with accumulation of EPEC proteins in growth medium, suggesting that it may support both pilus biogenesis and protein secretion. J Virol, 2001 Aug, 75(16), 7651 - 61 Duck hepatitis B virus replication in primary bile duct epithelial cells; Lee JY et al.; Primary cultures of intrahepatic bile duct epithelial (IBDE) cells isolated from duckling livers were successfully grown for studies of duck hepatitis B virus (DHBV) . The primary IBDE cells were characterized by immunohistochemistry using CAM 5.2, a cytokeratin marker which was shown to react specifically to IBDE cells in duck liver tissue sections and in primary cultures of total duck liver cells . Immunofluorescence assay using anti-duck albumin, a marker for hepatocytes, revealed that these IBDE cultures did not appear to contain hepatocytes . A striking feature of these cultures was the duct-like structures present within each cell colony of multilayered IBDE cells . Normal duck serum in the growth medium was found to be essential for the development of these cells into duct-like structures . When the primary cultures of duck IBDE cells were acutely infected with DHBV, dual-labeled confocal microscopy using a combination of anti-DHBV core proteins and CAM 5.2 or a combination of anti-pre-S1 proteins and CAM 5.2 revealed that the IBDE cell colonies contained DHBV proteins . Immunoblot analysis of these cells showed that the DHBV pre-S1 and core proteins were similar to their counterparts in infected primary duck hepatocyte cultures . Southern blot analysis of infected IBDE preparations using a digoxigenin-labeled positive-sense DHBV riboprobe revealed the presence of hepadnavirus covalently closed circular (CCC) DNA, minus-sense single-stranded (SS) DNA, double-stranded linear DNA, and relaxed circular DNA . The presence of minus-sense SS DNA in the acutely infected IBDE cultures is indicative of DHBV reverse transcriptase activity, while the establishment of a pool of viral CCC DNA reveals the ability of these cells to maintain persistent infection . Taken collectively, the results from this study demonstrated that primary duck IBDE cells supported hepadnavirus replication as shown by the de novo synthesis of DHBV proteins and DNA replicative intermediates. Biotechnol Bioeng, 2001 Mar 20, 72(6), 611 - 9 Protein secretion biotechnology using Streptomyces lividans: large-scale production of functional trimeric tumor necrosis factor alpha; Pozidis C et al.; We evaluated the feasibility of large-scale production of biopharmaceuticals expressed as heterologous polypeptides from the Gram-positive bacterium Streptomyces lividans . As a model protein we used murine tumor necrosis factor alpha (mTNFalpha) . mTNFalpha fused C-terminally to the secretory signal peptide of the subtilisin-inhibitor protein from Streptomyces venezuelae . Under appropriate fermentation conditions, significant amounts of mature mTNFalpha (80-120 mg/L) can be recovered from spent growth media . Efficient downstream processing allowing rapid purification of mTNFalpha from culture supernatants was developed . Importantly, the protein is recovered from the spent growth medium in its native trimeric state as judged by biophysical analysis . Further, mTNFalpha secreted by S . lividans is significantly more active in an in vitro apoptosis tissue culture assay than a corresponding polypeptide produced in Escherichia coli . This pilot study provides the first validation of S . lividans protein secretion as an alternative bioprocess for large-scale production of oligomeric proteins of potential therapeutic value . Cell Tissue Res, 2001 Jun, 304(3), 383 - 9 Evidence that a copper-metallothionein complex is responsible for fluorescence in acid-secreting cells of the Drosophila stomach; McNulty M et al.; Copper cells were originally identified in Drosophila midgut epithelium by their striking orange fluorescence in copper-fed larvae . Here, we examined copper cell fluorescence in light of the previous observations that (1) a similar fluorescent signal in yeast is produced by a complex between copper and metallothionein, and (2) metallothionein is expressed constitutively in the copper cell region and inducibly in other regions of the Drosophila midgut . Pulse-feeding experiments with 1 mM CuCl2 revealed that fluorescence appeared rapidly in copper cells (<5 min) and slowly in other cells of the midgut (days), suggesting a constitutive cofactor in the former and an inducible cofactor in the latter . Fluorescence was also detected in Drosophila S2 tissue culture cells after induction of metallothionein synthesis by addition of CuCl2 to the growth medium . Thus, fluorescence coincided spatially and temporally with the expression of metallothionein . Fluorescence was also linked to the acid-secreting activity of copper cells . Fluorescence was not observed when acid secretion was inhibited by a mutation in the alpha spectrin gene and acidification was blocked in copper-fed wild-type larvae . However, acidification was restored after a 1-day chase period in which the fluorescent signal became sequestered within a vesicular compartment . We therefore conclude that copper cell fluorescence is most probably attributable to a cytoplasmic copper-metallothionein complex, suggesting an unanticipated role for metallothionein in acid-secreting cells. Appl Biochem Biotechnol, 2001 May, 94(2), 135 - 45 Fiber fractions from processing of barley in production and conservation of a biologic control agent; Tuomi T et al.; Carriers are frequently used to overcome problems associated with microbial survival in soil after inoculation . Moreover, the use of carriers can prolong the shelf lives and lessen dusting of both biofungicides and biologic fertilizers . This study investigated the suitability of barley-based fiber fractions as growth media and immobilization matrices in the cultivation of a Streptomyces griseoviridis biologic control agent, as well as for the conservation of obtained biomass in dehydrated hydrogel capsules . The second main ingredient in all the examined carrier matrices was alginate . The aim was to find a hydrogel formulation suited for a production process in which all individual steps, including cultivation of the organism; downstream processing; and formulation, storage, and application of the product (i.e., biologic control agent), are carried out in the hydrogel matrix . Of the tested fractions, brewer's spent grain was the best choice, when considering the price vs the nutrient contents as well as the storage time and ease of processing of the crude and the finished products . It seems that cereal fibers can be replenished with cereal fractions less rich in fiber but having a higher content of utilizable nutrients and, hence, better suited for the production of biomass . A high content of water-insoluble fiber favorably influenced the appearance as well as the applicability of the products. Semin Perinatol, 2001 Jun, 25(3), 133 - 8 The use of stable isotopes in drug metabolism studies; Abramson FP; Although there is a long history of stable isotopes use in drug metabolism research, it is appropriate to evaluate them in pregnancy drug studies in which safety takes highest priority . It is well established through a number of human and animal experiments that stable isotopes themselves rarely generate additional toxicities beyond the molecules to which they are attached . For the analysis of stable isotopes involved in metabolism studies, mass spectrometry plays the predominant role . Several mass spectrometry-based techniques now exist that enable the selective quantitative detection of stable isotopes with better sensitivity and better retention of chromatographic resolution than do in-line radioactivity monitors for 14C . Even mass balance studies can be performed by using stable isotopes, a type of experiment that still predominantly uses radioisotopes . Some of the newest developments in the use of stable isotopes involve biopolymers, in which fully isotope-labeled species can be generated from cells grown in isotopically labeled growth media . Having shown safety, sensitivity, specificity, and versatility, stable isotopes should play an important role in drug metabolism studies in pregnancy. Biofizika, 2001 May-Jun, 46(3), 452 - 9 {Identification of minor components of medusomycete exometabolites . 1H-NMR spectra in 13C and deuterium substitutions}; Kutyshenko VP et al.; High-resolution 1H-NMR experiments were performed on 2H/13C isotopically labelled metabolites of growing medusomycete culture . Some minor metabolites were identified and the degree of their deuterization was established . Some of these metabolites were shown to be present in isomeric forms . Our results demonstrated that glycerol was produced in the growth medium on the early stage of the system adaptation to the growth in D2O. Gynecol Endocrinol, 2001 Jun, 15(3), 225 - 33 Estrogenic and antiestrogenic effects of raloxifene on collagen metabolism in breast cancer MCF-7 cells; Wolczynski S et al.; We compared the effects of different concentrations of raloxifene (1, 4 and 10 microM) on collagen biosynthesis, gelatinolytic and prolidase activities and matrix metalloproteinase (MMP) expression (MMP-2 and MMP-9) in estradiol-stimulated (2 nM) breast cancer MCF-7 cells . Raloxifene inhibited in a dose-dependent manner the proliferation of MCF-7 cells, independently of the presence or absence of estradiol in the growth medium . Raloxifene at concentrations of 1 microM and 4 microM inhibited collagen biosynthesis by about 10-fold and prolidase activity by about 50%, while at a concentration of 10 microM it inhibited these processes by only about 25% . This phenomenon was accompanied by differences in gelatinolytic activity and MMP (MMP-2 and MMP-9) expression as demonstrated by zymography and Western immunoblot analysis, respectively . In estrogen-stimulated MCF-7 cells, cultured in the presence of 1 microM raloxifene, a dramatic increase in the activity of both collagenases was found . In contrast, addition of raloxifene at a concentration of 10 microM to the medium of the cells resulted in restoration of gelatinolytic activity to that found in control cells . Similarly, but at both doses (1 and 10 microM), raloxifene was able to reduce MMP-2 expression in the cells . However, when used alone (without estradiol) a concentration of 1 microM raloxifene strongly stimulated MMP-2 expression, while at a concentration of 10 microM the effect was not observed . In the case of MMP-9, only trace amounts of this gelatinase were detected, although in contrast to MMP-2, an increase in its expression was noticed at a concentration of 10 microM raloxifene . The data raise the possibility that in estrogen-stimulated MCF-7 cells, raloxifene at low concentrations (1 and 4 microM) evokes antiestrogenic effect on collagen biosynthesis and prolidase activity on the one hand, and an estrogenic effect on gelatinolytic activity on the other, while at higher concentrations (about 10 microM) it evokes an estrogenic effect on collagen biosynthesis and prolidase activity, and an antiestrogenic effect on gelatinolytic activity . Our data suggest that the effects of raloxifene on collagen synthesis, prolidase and metalloproteinase activities in breast cancer may explain its role in the prevention of breast cancer development. Infect Immun, 2001 Aug, 69(8), 4891 - 7 Nickel-responsive induction of urease expression in Helicobacter pylori is mediated at the transcriptional level; van Vliet AH et al.; The nickel-containing enzyme urease is an essential colonization factor of the gastric pathogen Helicobacter pylori, as it allows the bacterium to survive the acidic conditions in the gastric mucosa . Although urease can represents up to 10% of the total protein content of H . pylori, expression of urease genes is thought to be constitutive . Here it is demonstrated that H . pylori regulates the expression and activity of its urease enzyme as a function of the availability of the cofactor nickel . Supplementation of brucella growth medium with 1 or 100 microM NiCl(2) resulted in up to 3.5-fold-increased expression of the urease subunit proteins UreA and UreB and up to 12-fold-increased urease enzyme activity . The induction was specific for nickel, since the addition of cadmium, cobalt, copper, iron, manganese, or zinc did not affect the expression of urease . Both Northern hybridization studies and a transcriptional ureA::lacZ fusion demonstrated that the observed nickel-responsive regulation of urease is mediated at the transcriptional level . Mutation of the HP1027 gene, encoding the ferric uptake regulator (Fur), did not affect the expression of urease in unsupplemented medium but reduced the nickel induction of urease expression to only twofold . This indicates that Fur is involved in the modulation of urease expression in response to nickel . These data demonstrate nickel-responsive regulation of H . pylori urease, a phenomenon likely to be of importance during the colonization and persistence of H . pylori in the gastric mucosa. J Med Microbiol, 2001 Jul, 50(7), 602 - 12 Cytopathic effects of outer-membrane preparations of enteropathogenic Escherichia coli and co-expression of maltoporin with secretory virulence factor, EspB; Kumar SS et al.; Enteropathogenic Escherichia coli (EPEC) is an important aetiological agent of persistent infantile diarrhoea . EPEC pathogenicity is not mediated through known toxins and the role played by outer-membrane proteins (OMPs) in the initial adherence of the bacterium, intimate attachment to epithelial cells and ultimately in the effacement of the intestinal epithelium is being pursued vigorously . In this study of the different cellular fractions of the bacterium investigated, only the outer-membrane fraction was able to disrupt HEp-2 cells . The outer-membrane fraction was also found to be cytotoxic and caused actin accumulation around the periphery of the host cells . To understand the role of OMPs in pathogenesis, protein profiles of outer-membrane preparations of wild-type and attenuated mutants lacking either the EPEC adherence factor (EAF) mega-plasmid or EPEC attaching and effacing gene A (eaeA) coding for a 94-kDa OMP, intimin or EPEC secretory protein gene B (espB) coding for a 34-kDa translocated signal transducing protein were compared and correlated with their cytopathic effects . A 43-kDa protein seen along with intimin in the outer membrane of EPEC was identified as maltoporin, an E . coli outer-membrane porin normally expressed only in response to maltose in the growth medium . In the case of EPEC, not only was this regulation lost, but also the expression of maltoporin was found to be tightly coupled to the expression of the secretory virulence factor EspB . Maltoporin per se is not toxic, as evidenced by the treatment of HEp-2 cells with the outer-membrane preparation of E . coli DH5a grown in the presence of maltose and the significance of this pathogenic adaptation is not clear . However, when maltoporin and possibly other unidentified proteins were not present as a component of the outer-membrane preparation, as in the outer-membrane preparation of an espB-negative strain, cellular disruption as well as actin accumulation proceeded at a very slow rate even though the cytotoxic effects were comparable to those of the wild-type EPEC strains. Prikl Biokhim Mikrobiol, 2001 May-Jun, 37(3), 297 - 300 {Dependence of the activity of L-amino acid oxidase in the fungus Aspergillus niger R-3 on the source of nitrogen in growth media}; Papoian AR et al.; Various populations of peroxisomes in cells of Aspergillus niger R-3 were formed under the growth in media containing 0.5% glucose and various sources of nitrogen (1/4 of optimal concentrations of (NH4)2SO4, L-alanine, and L-methionine) . Different levels of of L-amino acid oxidase activity were found in these populations of peroxisomes. J Appl Microbiol, 2001 Jul, 91(1), 67 - 71 The role of non-Saccharomyces species in releasing glycosidic bound fraction of grape aroma components--a preliminary study; Mendes Ferreira A et al.; AIMS: The purpose of the study was to evaluate the effect of beta-glycosidase activity in wine yeasts in releasing terpene glycosides from grape juice . METHODS AND RESULTS: Glycosidase activity was screened in 160 yeasts by testing their ability to hydrolyse arbutine on agar plates . Only non-Saccharomyces species exhibited beta-glycosidase activity . Enzyme activity, based on hydrolytic activity on p-nitrophenyl-beta-glycoside, was mainly located in the whole cell fraction, with smaller amounts in permeabilized cells being released into the growth medium . The hydrolysis of glycosides was determined by HRGC-MS, confirming the role of yeast in the liberation of monoterpenols, especially linalool and geraniol . CONCLUSION: The results indicate the potential of microbial beta-glycosidases for releasing flavour compounds from glycosidically-bound, non-volatile precursors, with significant implications for wines made from less aromatic grapes . SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms the role of non-Saccharomyces species in enhancing wine aroma and flavour, suggesting that the future lies with controlled use of mixed cultures in winemaking. Genetika, 2001 May, 37(5), 624 - 30 {Effect of rye chromosomes on features of androgenesis in wheat-rye substituted lines of Triticum aestivum L . sort Saratovskaya 29/Secale cerale L . sort Onokhoiskaia and Triticale}; Dobrovolskaia OB et al.; The characteristic features of androgenesis in six wheat-rye substitution lines Triticum aestivum L . (cv . Saratovskaya 29)/Secale cereale L . (cv . Onokhoiskaya) and triticale (2n = 56) using anther culture at different concentrations of 2,4-D in the growth medium were studied . Under variable cultivation conditions, the significant effect of genotypic diversity on the variability of such androgenesis parameters as the frequency of productive anthers, the frequency of embryoid formation, and the frequency of total regenerated plantlets, was shown . It was demonstrated that chromosomes 1R, 3R, and 7R stimulated the formation of androgenous embryoids, while chromosome 5R produced an opposite effect . In triticale and substitution lines, the regeneration ability of androgenous embryoids induced by elevated 2,4-D concentrations was inhibited . Chromosome 1R of the Onokhoiskaya cultivar was suggested to contain genes suppressing regeneration of green plantlets, while chromosome 3R, conversely, stimulated their formation . Chromosomes 1R, 2R, 3R, and 7R of the Onokhoiskaya cultivar did not inhibit the spontaneous formation of androgenous hexaploids in the substitution lines. J Exp Bot, 2001 May, 52(358), 933 - 42 Changes in fatty acid composition during development of tissues of coconut (Cocos nucifera L.) embryos in the intact nut and in vitro; Lopez-Villalobos A et al.; Intact coconuts were germinated in situ and compared with excised zygotic embryos germinated in vitro . The growth of the embryonic tissue and their fatty acid compositions were measured . Haustoria, plumules and radicles of coconuts germinated in situ grew continuously and proportionately throughout the 120 d experiment with haustauria increasing to 45 g x nut(-1) and weighing 4-5-fold more than the other two tissues . The plumules and radicles of the seedlings cultured in vitro also grew continuously but the haustoria grew sporadically between 15 d and 75 d in culture and, at 250 mg x nut(-1) after 75 d, were smaller than the other two tissues . All the tissues of the nuts grown in situ contained significant amounts of lauric acid, the acid characteristic of coconut oil, as well as longer chain saturated and unsaturated fatty acids . The content of medium and long chain fatty acids increased in all growing tissues as the experiment proceeded, especially the haustorium which contained 24-35% of its fatty acid as lauric acid; the fat content of solid endosperm reduced during this period . Seedlings grown in vitro, on the other hand, failed to accumulate lauric acid in any of their tissues (haustorium contained 6-11% of its fatty acid as lauric acid) . The results may have implications for the design of growth media for growing zygotic and somatic cultures of coconut and may provide a marker for successful germination. Microbiology, 2001 Jul, 147(Pt 7), 1755 - 63 Differential regulation of laccase gene expression in Pleurotus sajor-caju; Soden DM et al.; Four laccase isozyme genes, Psc lac1, 2, 3 and 4 have been cloned from the edible mushroom, Pleurotus sajor-caju . The genes display a high degree of homology with other basidiomycete laccases (55-99%) at the amino acid level . Of the laccase genes isolated, Psc lac1 and 4 displayed the highest degree of similarity (85% at the amino acid level), while Psc lac3 showed the highest degree of divergence, exhibiting only 52-57% amino acid similarity to the other PL: sajor-caju laccase gene sequences . Laccase activity in PL: sajor-caju is affected by nutrient nitrogen and carbon, and by the addition of copper and manganese to the growth medium . In addition, 2,5-xylidine, ferulic acid, veratric acid and 1-hydroxybenzotriazole induced laccase activity in the fungus . Induction of individual laccase isozyme genes by carbon, nitrogen, copper, manganese and the two aromatic compounds, 2,5-xylidine and ferulic acid, occurred at the level of gene transcription . While Psc lac3 transcript levels appeared to be constitutively expressed, transcript levels for the other laccase isozyme genes, lac1, 2 and 4, were differentially regulated under the conditions tested. J Appl Toxicol, 2000 Dec, 20 Suppl 1, S87 - 91 Intervention of sulfur mustard toxicity by downregulation of cell proliferation and metabolic rates; Ray R et al.; Metabolically active and proliferating basal cells in the skin are most sensitive to the potent skin blistering chemical warfare compound HD (bis-(2-chloroethyl) sulfide) . We previously described a Ca2+-dependent mechanism of HD (0.3-1 mM) toxicity that was inhibited by the cell-permeant Ca2+ chelator BAPTA AM (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester) . We describe some cellular effects of BAPTA AM that suggest a mechanism for its protective action . Monolayer log-phase normal human epidermal keratinocytes were incubated (37 degrees C) first in keratinocyte growth medium (KGM) containing BAPTA AM (10-40 microM) for 30 min and then in KGM alone overnight prior to evaluation . The BAPTA AM inhibited cell growth in a concentration-dependent manner with some cellular degeneration above 30 microM (light microscopy) . At 20-30 microM, BAPTA AM also inhibited cellular metabolic processes, as evidenced by a lower incorporation of {3H}-thymidine (DNA synthesis, 54 +/- 5%), {3H}-uridine (RNA synthesis, 29 +/- 6%) and {14C}-valine (protein synthesis, 12 +/- 2%) as well as a lower protein content per culture (30 +/- 3%) compared with corresponding untreated controls . However, 20-30 microM BAPTA AM did not cause any demonstrable cytopathology based on morphological (electron microscopy) as well as biochemical (lactate dehydrogenase release, an indicator of cell viability loss) criteria, indicating a lack of acute toxicity . These results suggest that a mechanism of protection by BAPTA AM against HD may be via decreasing some metabolic, and therefore proliferative, rates. J Clin Microbiol, 2001 Jul, 39(7), 2732 - 5 Effects of two different growth media on the postantifungal effect induced by polyenes on Candida species; Shu M et al.; There are no data on the effects of different growth media on polyene-induced postantifungal effect (PAFE) in Candida species . Hence, the nystatin- and amphotericin B-induced PAFEs in six Candida species (26 isolates) grown in Sabouraud's dextrose broth (SAB) and RPMI broth were evaluated, following limited exposure to the MICs of the two polyenes, using an automated turbidometric method . For nystatin, PAFE varied between 1.88 and 4.87 h in SAB and 0.66 and 6.89 h in RPMI, and for amphotericin B, the equivalent values were 3.13 to 10.98 h in SAB and 0.97 to 7.01 h in RPMI . These highly significant (P < 0.001) variations in the PAFE with both drugs, noted with most Candida strains grown in different media, call for standardization of intralaboratory methodology in measuring this parameter in order to obtain universally comparable data. Enzyme Microb Technol, 2001 Jul 5, 29(1), 13 - 19 Effects of growth medium composition, iron sources and atmospheric oxygen concentrations on production of luciferase-bacterial magnetic particle complex by a recombinant Magnetospirillum magneticum AMB-1; Yang C et al.; Growth conditions for mass production of luciferase-bacterial magnetic particles (BMPs) by a recombinant Magnetospirillum magneticum AMB-1 were investigated in a pH-regulated fed-batch culture system . Enrichment of growth medium with L-cysteine, yeast extract and polypeptone enhanced both bacterial growth and BMP production . The presence of L-cysteine in the medium was useful for induction of cell growth . Strict anaerobic conditions led to a prolonged lag phase and limited the final cell density . Trace oxygen enhanced cell growth with increasing BMP production . As iron sources, ferrous sulfate and ferric gallate dramatically enhanced BMP yield as compared with ferric quinate, an iron chelate conventionally used . The optimized conditions increased cell density to 0.59 +/- 0.03 g cell dry weight/liter and BMP production to 14.8 +/- 0.5 mg dry weight/liter in fed-batch culture for four days. Reproduction, 2001 May, 121(5), 745 - 51 Delayed effect of heat stress on steroid production in medium-sized and preovulatory bovine follicles; Roth Z et al.; During the autumn, the conception rate of dairy cattle in warm countries is low although ambient temperatures have decreased and cows are no longer exposed to summer thermal stress, indicating that there may be a delayed effect of heat stress on cattle fertility . Two experiments were conducted to examine possible delayed effects of heat stress on follicular characteristics and steroid production at two distinct stages of follicular growth: medium-sized and preovulatory follicles, 20 and 26 days after heat exposure, respectively . Lactating cows were subjected to heat stress for 12 h a day in an environmental chamber, during days 2-6 of a synchronized oestrous cycle . In Expt 1, ovaries were collected on day 3 of the subsequent cycle, before selection of the dominant follicle, and medium-sized follicles were classified as atretic or healthy . In Expt 2, on day 7 of the subsequent cycle, PGF(2a) was administered and preovulatory follicles were collected 40 h later . In both experiments, follicular fluid was aspirated, granulosa and thecal cells were incubated, and steroid production was determined . In healthy medium-sized follicles (Expt 1), oestradiol production by granulosa cells and androstenedione production by thecal cells were lower (P < 0.05) and the concentration of progesterone in the follicular fluid was higher in cows that had been previously heat-stressed than in control cows (P < 0.05) . In preovulatory follicles (Expt 2), the viability of granulosa cells was lower (P < 0.05) and the concentration of androstenedione in the follicular fluid and its production by thecal cells were lower (P < 0.05) in cows that had been previously heat-stressed than in control cows . In both experiments, the oestradiol concentrations in the follicular fluids were not altered by heat stress . These results demonstrate a delayed effect of heat stress on steroid production and follicular characteristics in both medium-sized and preovulatory follicles; this effect could be related to the low fertility of cattle in the autumn. J Biomed Mater Res, 2001 Apr, 55(1), 104 - 13 Fibronectin facilitates adhesion of K562 leukemic cells normally growing in suspension to cationic surfaces; Rainaldi G et al.; The role of protein adsorption in the forced adhesive growth of K562 leukemic cells onto a cationic surface composed of polylysine was investigated . Numerous studies have demonstrated that adhesion in anchorage-dependent cells is mediated in vitro by adsorption of serum proteins {particularly proteins of the extracellular matrix (ECM) such as fibronectin and vitronectin} present in the growth medium . Specifically, adhesion has been shown to occur when ECM proteins attach to the substratum and act as ligands for specific receptors located on the surface of cells . K562 cells are human erythroleukemic cells that normally grow in suspension . These cells are not involved in the same cell adhesion processes as anchorage-dependent cells and do not need to be attached to ECM proteins in order to survive and grow . Thus, with these systems, it is possible to better determine the role of protein adsorption in the adhesion of cells, growing in suspension such as blood cells, onto charged surfaces . The results presented show that adhesion of K562 cells onto the positively charged polylysine surface in the presence of serum is mediated through specific interactions between fibronectin receptors present on K562 cells and fibronectin adsorbed onto that cationic surface . Specifically, determination of cell adhesion under different experimental conditions indicates that nonspecific charge interactions do not take place directly between the cells and polylysine, but rather take place between polylysine and fibronectin, which adsorbs onto the cationic polymer . In addition, flow cytometric analyses reveal that only fibronectin receptors are present on these cells and, consequently, only fibronectin can be responsible for the actual adhesion of these cells onto the cationic surface . In view of the data presented, the possibility should be considered that ECM components adsorbed onto surfaces with specific charges and/or belonging to certain functional groups are involved in structural and functional modifications in cells . These cells grow in suspension and are normally not involved in adhesion phenomena, though these components should be considered . These considerations should be made especially when designing biomaterials that can modulate the response of cells growing in suspension, such as blood cells, and also in tissue engineering of blood substitutes. Appl Environ Microbiol, 2001 Jul, 67(7), 3041 - 5 Characterization of a heme-dependent catalase from Methanobrevibacter arboriphilus; Shima S et al.; Recently it was reported that methanogens of the genus Methanobrevibacter exhibit catalase activity . This was surprising, since Methanobrevibacter species belong to the order Methanobacteriales, which are known not to contain cytochromes and to lack the ability to synthesize heme . We report here that Methanobrevibacter arboriphilus strains AZ and DH1 contained catalase activity only when the growth medium was supplemented with hemin . The heme catalase was purified and characterized, and the encoding gene was cloned . The amino acid sequence of the catalase from the methanogens is most similar to that of Methanosarcina barkeri. J Ind Microbiol Biotechnol, 1999 Oct, 23(4-5), 332 - 335 Degradation of the phenoxy acid herbicide diclofop-methyl by Sphingomonas paucimobilis isolated from a Canadian prairie soil; Adkins A; Sphingomonas paucimobilis, isolated from a soil in Manitoba, Canada, was able to utilize diclofop-methyl, (R,S)-methyl-2-{4-(2,4-dichlorophenoxy)phenoxy}propionate, as the sole source of carbon and energy . An actively growing aerobic culture completely degraded 1.5 &mgr;g diclofop-methyl ml(-1) to diclofop acid within 54 h, at 25 degrees C . A biphasic growth pattern indicated that this organism was capable of degrading diclofop acid to 4-(2,4-dichlorophenoxy)phenol and 2,4-dichlorophenol and/or phenol . The accumulation of 2,4-dichlorophenol in the growth medium, however, suggested that Sphingomonas paucimobilis was unable to utilize this compound as a source of carbon and energy. J Biol Chem, 2001 Aug 17, 276(33), 30987 - 94 Epub 2001 Jun 21. Heterogeneous fatty acylation of Src family kinases with polyunsaturated fatty acids regulates raft localization and signal transduction; Liang X et al.; Fatty acylation of Src family kinases is essential for localization of the modified proteins to the plasma membrane and to plasma membrane rafts . It has been suggested that the presence of saturated fatty acyl chains on proteins is conducive for their insertion into liquid ordered lipid domains present in rafts . The ability of unsaturated dietary fatty acids to be attached to Src family kinases has not been investigated . Here we demonstrate that heterogeneous fatty acylation of Src family kinases occurs and that the nature of the attached fatty acid influences raft-mediated signal transduction . By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we show that in addition to 14:0 (myristate), 14:1 and 14:2 fatty acids can be attached to the N-terminal glycine of the Src family kinase Fyn when the growth media are supplemented with these dietary fatty acids . Moreover, we synthesized novel iodinated analogs of oleate and stearate, and we showed that heterogeneous S-acylation can occur on cysteine residues within Fyn as well as Galpha, GAP43, and Ras . Modification of Fyn with unsaturated or polyunsaturated fatty acids reduced its raft localization and resulted in decreased T cell signal transduction . These studies establish that heterogeneous fatty acylation is a widespread occurrence that serves to regulate signal transduction by membrane-bound proteins. J Antimicrob Chemother, 2001 Jul, 48 Suppl 1, 81 - 5 Instrumentation in antimicrobial susceptibility testing; Felmingham D et al.; Studies in the 1960s demonstrated the problems of variability in susceptibility testing methods, especially those affecting the performance of disc diffusion procedures . These studies made apparent the need for standardization and resulted in more clearly defined performance limits for growth medium, incubation conditions, inoculum concentration, disc content for diffusion methods, the setting of interpretative MIC breakpoints and the establishment of quality control parameters . More recently, there has been a growing interest in the use of instrumentation for reading disc diffusion tests and the endpoints of agar or broth dilution MIC determinations . Instrumentation ranges in complexity from the simple optical reading of zones of inhibition or growth endpoints, requiring operator interpretation, to more sophisticated devices for reading, recording and 'expert system' analysis of results with interfacing of instruments to laboratory information management systems . Some of the more developed systems are fully automated and can also identify the organisms tested . The pressure to reduce labour costs and provide results earlier favours the use of more automated systems whilst the requirement for resistance surveillance provides impetus for the use of systems that provide quantitative results and electronic data handling. Curr Genet, 2001 May, 39(3), 137 - 49 Diauxic shift-induced stress resistance against hydroperoxides in Saccharomyces cerevisiae is not an adaptive stress response and does not depend on functional mitochondria; Maris AF et al.; Respiring Saccharomyces cerevisiae cells grown on a non-fermentable carbon source are intrinsically more resistant to several stresses, including oxidative stress . The mechanisms leading to increased stress resistance are not yet well understood . Active mitochondria are the major source of intracellular reactive oxygen species (ROS), which could cause the up-regulation of the antioxidant defense systems . We investigated the role of mitochondria in the intrinsic stress resistance against the hydroperoxides H2O2 and tert-butylhydroperoxide 4 h after a shift in carbon source . We found that, independently of functional mitochondria, the yeast acquired the intrinsic resistance of respiring cells against hydroperoxides solely as a response to a change of carbon source in the growth medium . Furthermore, utilizing reporter gene fusion constructs, we monitored the expression of the gamma-glutamylcysteinyl synthetase (encoded by GSH1) and the two superoxide dismutases (encoded by SOD1 and SOD2) during the metabolic transition from fermentation to respiration; and we detected an up-regulation of all three genes during the diauxic shift . Overall available data allowed us to propose that the antioxidant system of S . cerevisiae could be considered as a class of genes under glucose/carbon catabolite regulation . This control system is different from the well-known adaptive response to oxidative stress. Lett Appl Microbiol, 2001 Jun, 32(6), 407 - 11 Laccase production by Phanerochaete chrysosporium--an artefact caused by Mn(III)? Podgornik H, Stegu M, Zibert E, Perdih A. AIMS: The possibility of laccase production by Phanerochaete chrysosporium was studied . METHODS AND RESULTS: A relatively high initial Mn(II) concentration (1-4 mM) in the growth medium leads to the development of reddish-brown coloration and intensive oxidation of 2.2'-azino-bis(3-etilbenz-tiazolin-6-sulfonate) (ABTS) . The peak of ABTS oxidation was obtained approximately 1 day after the peak of MnP activity . CONCLUSION: ABTS oxidation was not caused by manganese peroxidase (MnP) nor by laccase but was the consequence of the action of Mn(III) which was stabilised in the growth medium . Decomposition of the complex took place after the biomass was removed from the growth medium and especially after the aeration of the culture was interrupted . Significance and Impact of the Study: Mn(III) seems to be the cause of false positive laccase reactions . More reliable data on MnP activity can be obtained if the complex is decomposed by the fungus before MnP activity is measured in the medium. J Eukaryot Microbiol, 2001 May-Jun, 48(3), 374 - 81 Polyamine synthesis and interconversion by the Microsporidian Encephalitozoon cuniculi; Bacchi CJ et al.; Polyamines are small cationic molecules necessary for growth and differentiation in all cells . Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e . antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms . Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans . Organisms were grown in RK-13 cells and harvested using Percoll gradients . Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils . Intact purified pre-emergent spores incubated with {1H} ornithine and methionine synthesized putrescine, spermidine, and spermine, while {14C}spermine was converted to spermidine and putrescine . Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA) . Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities . ODC activity was inhibited by DFMO, but not by DFMA . AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected . It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines . Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity. Curr Microbiol, 2001 May, 42(5), 330 - 8 Sensitivity of partially purified ice nucleation activity of Fusarium acuminatum SRSF 616; Humphreys TL et al.; Factors that affect bacterial ice nucleation, including growth medium, growth phase, nutrient deprivation, and cold-temperature exposure, were investigated in the ice nucleation active (INA) fungus Fusarium acuminatum SRSF 616 . Ice nucleation activity remained relatively constant throughout the growth cycle, and the cell-free culture supernatant consistently displayed higher ice nucleation activity than the hyphal pellet . Although nutrient starvation and low-temperature exposure enhance bacterial ice nucleation activity, reducing the concentration of C, N, or P in synthetischer nahrstoffarmer broth (SNB) did not increase fungal ice nucleation activity, nor did exposure to 4 degrees C or 15 degrees C . From the SNB supernatant, selected INA chromatography fractions were obtained that demonstrated increased sensitivity to proteinase K and heat compared with culture supernatant . We propose that partial purification of the fungal ice nuclei resulted in removal of low-molecular-weight stabilizing factors. J Biotechnol, 2001 Jun 15, 88(2), 173 - 6 The effect of agitation and nitrogen concentration on lignin peroxidase (LiP) isoform composition during fermentation of Phanerochaete chrysosporium; Podgornik H et al.; Convective Interaction Media (CIM) monolithic columns were applied for the HPLC monitoring of Phanerochaete chrysosporium lignin peroxidase (LiP) isoforms during cultivation . The influence of the agitation mode (circular, elliptic) and rate (130 and 200 rpm), as well as the initial nitrogen concentration (1.6-6 mM) in the growth medium was investigated . Identical rotation rate but different agitation modes resulted in different LiP activities and isoenzyme compositions . On the other hand, at different agitation types and rates, similar LiP activities were obtained at different isoenzyme compositions . Although LiP H2 and LiP H6/H7 were predominant isoenzymes obtained at various cultivation conditions, relative isoenzyme amounts differ considerably when initial nitrogen concentration was changed between 1.6 and 5 mM. Parasitol Res, 2001 May, 87(5), 371 - 5 Inhibition of encystation of Entamoeba invadens by wortmannin; Makioka A et al.; Using an axenic encystation system in vitro, we examined the effect of wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), which is a signaling molecule responsible for numerous cellular responses, on the encystation of Entamoeba invadens . Wortmannin inhibited both encystation and growth of E . invadens strain IP-1 in a dose-dependent manner, the former being more resistant to the drug than the latter . There was little decrease in the number of trophozoites after 3 days of culture in encystation medium containing wortmannin; and the cells remained motile, suggesting that the inhibitory effect of the drug on encystation was not due to its toxic effect on trophozoites . The addition of wortmannin after the induction of encystation was also inhibitory for encystation . Trophozoites incubated for 1 day in encystation medium with wortmannin did not encyst after removal of the drug, suggesting that the drug effect was not reversible in encystation medium . In contrast, trophozoites cultured in growth medium with wortmannin did encyst after their transfer to encystation medium without the drug . Encystation with wortmannin was more strongly inhibited among trophozoites grown in the presence of the drug than among those grown in the absence of the drug . The process of cyst maturation was slightly affected by wortmannin . These results suggest a possible role for PI 3-kinase in the signaling involved in the encystation of E . invadens. Cell Biol Int, 2001, 25(5), 451 - 65 Utilization of amino acids in growing kidney proximal tubule cell cultures; Ali AR et al.; The growth of rat kidney proximal tubule cells was monitored continuously by the cellular incorporation of {methyl-(14)C} thymidine using scintillating microplates . The radioisotope had no effect on cell proliferation over a 5 day period, neither was it extensively converted to thymine . Leibovitz L-15 medium supplemented with bicarbonate proved a good growth medium and its high levels of carbohydrates and amino acids facilitated the appearance of intermediates in the cells' metabolism of additional radioactive amino acids . Kidney proximal tubule cells had a greater potential to process amino acids than BHK-21 cells . The utilization of amino acids by proximal tubule cells differed from that of other organs . The amino acids could be classified into three classes . Members of the first type were only used for protein synthesis (arginine, lysine, histidine and tyrosine) . The second class of amino acids yielded only one or two metabolites (leucine and isoleucine), while the last type gave more than two metabolites (alanine, aspartate, glycine, methionine, proline and valine) . Can J Microbiol, 2001 May, 47(5), 417 - 23 Production of transferrin receptors by Histophilus ovis: three of five strains require two signals; Ekins A et al.; Five strains of Histophilus ovis (9L, 642A, 714, 5688T, and 3384Y) were investigated with respect to iron acquisition . All strains used ovine, bovine, and goat transferrins (Tfs), but not porcine or human Tfs, as iron sources for growth . In solid phase binding assays, total membranes from only two (9L and 642A) of the five strains, grown under iron-restricted conditions, were able to bind Tfs (ovine, bovine, and goat, but not porcine or human) . However, when the organisms were grown under iron-restricted conditions in the presence of bovine transferrin (Tf), total membranes from all strains exhibited Tf binding (as above); competition experiments demonstrated that all three Tfs (ovine, bovine, and goat) were bound by the same receptor(s) . Membranes from organisms grown under iron-replete conditions in the presence or absence of bovine Tf failed to bind any of the test Tfs . An affinity-isolation procedure allowed the isolation of two putative Tf-binding polypeptides (78 and 66 kDa) from total membranes of strains 9L and 642A grown under iron-restricted conditions, and from membranes of all strains if the growth medium also contained Tf . It is concluded that all strains tested acquire Tf-bound iron by means of siderophore-independent mechanisms involving surface receptors analogous to the Tf-binding proteins (TbpA and TbpB) found in comparable organisms; although iron restriction alone is sufficient to promote the expression of these proteins by strains 9L and 642A, their production by strains 714, 5688T, and 3384Y appears to require two signals, iron restriction and the presence of Tf. Appl Microbiol Biotechnol, 2001 May, 55(4), 486 - 91 Constitutive and inducible hydroxylase activities involved in the degradation of naphthalene by Cunninghamella elegans; Faber BW et al.; The non-ligninolytic fungus Cunninghamella elegans was investigated for its ability to produce naphthalene hydroxylase (NAH) and naphthol hydroxylase (NOH) activities under various conditions . When the organism was cultivated on a rich growth medium, the mycelia exhibited significant constitutive NAH activity in the late exponential growth phase, but not in the early-exponential-growth-phase . On incubating the early-exponential-growth-phase mycelia with naphthalene, NAH activity was increased five-fold; however, this increase did not occur in the presence of the protein synthesis inhibitor cycloheximide . Since incubation of the late-phase mycelia with naphthalene did not lead to a higher degradation rate of naphthalene, mycelia in this physiological state have apparently lost the ability to induce synthesis of the enzyme exhibiting NAH activity . This is not due to an overall inability to perform de novo protein synthesis, since NOH activity, non-constitutive at all growth phases, could be induced by incubating late-phase mycelia with naphthalene . Whether inducible and constitutive NAH activity originate from one and the same enzyme remains to be elucidated . It is suggested that naphthalene oxidizing enzyme(s) may also oxidize pyrene, but not anthracene or benzo{a}pyrene, although the latter are degradable by C . elegans. Phytochemistry, 2001 Jul, 57(5), 643 - 52 Endoplasmic oleoyl-PC desaturase references the second double bond; Schwartzbeck JL et al.; The regiospecificity for the gene product of fad2,(1) the microsomal oleoyl-PC desaturase from higher plants, differs from some previous suggestions . Rather than only referencing the carboxyl group (a Delta(12) desaturase) or the methyl terminus (an omega-6 desaturase), this desaturase locates the second double bond in its substrates by first referencing the existing double bond . This specificity was demonstrated for the oleoyl-PC desaturase cDNA from the developing seeds of peanut (Arachis hypogaea L) expressed in yeast (Saccharomyces cerevisae) . The expressed enzyme was capable of desaturating monounsaturated fatty acyl groups in membrane lipids . Endogenous palmitoleate was desaturated to cis, cis 9,12 hexadecadienoate (9(Z)12(Z)C16:2), endogenous oleate to linoleate (9(Z)12(Z) octadecadienoate), and cis 10-nonadecenoate (provided as a supplement in the growth medium) to 10(Z)13(Z)C19:2 . The rule, Delta(x+3) where x=9 is the double bond location in the substrate, best describes the consistent placement of the second double bond in the above monounsaturated substrates for the oleoyl-PC desaturase of higher plants. J Pharmacol Toxicol Methods, 2000 Nov-Dec, 44(3), 533 - 42 Phenotypic stability of chick cardiomyocytes in serum-free media . Preservation of muscarinic receptor expression; Eatman D et al.; Chick cardiomyocytes cultured in fetal bovine serum (FBS)-supplemented media are phenotypically unstable, becoming noncontractile and unresponsive to stimuli after several days . We report a culturing protocol that preserves the differentiated cardiomyocyte phenotype for at least 9 days in culture . Cardiomyocytes isolated from 11-day chicken embryos, and cultured in either Dulbecco's Modified Earle's Medium (DMEM)/Ham's F12 medium with N-2 supplement or Medium 199 (M199) with 10% FBS continued to beat spontaneously for 4-5 days; only cells cultured in N-2-supplemented medium exhibited spontaneous beating beyond 5 days . Immunostaining for alpha-actinin after 9 days in culture revealed that myofibrils persisted in N-2-supplemented cells, while no myofibrils were observed in the FBS-supplemented cells . For cells in FBS-supplemented media, {3H}thymidine incorporation rates were 7.5 and 3 times greater than that of cells in N-2-supplemented media at Days 4 and 9 in culture, respectively . The effect of growth media on the binding parameters of the muscarinic antagonist, {3H}N-methyl-scopolamine (NMS), was also compared . While B(max) decreased 34% between Days 4 and 9 for cells maintained in N-2-supplemented media, a 77% decrease was observed for cells cultured in FBS-supplemented media . The phenotypic stability of this preparation makes it feasible for the first time to use these cells in experiments that require more than 4 days to complete. Arch Gerontol Geriatr, 2001 Jun, 32(3), 185 - 197 Metabolism and aging in the filamentous fungus Podospora anserina; Osiewacz HD et al.; In Podospora anserina, lifespan is under the control of environmental and genetic factors . Both suggest an important impact of metabolism on lifespan and aging . Environmental changes of temperature, of the carbon source in the growth medium, or the addition of specific inhibitors to the growth medium are some of the investigated factors . Genetic approaches underscore the significance of metabolism . In particular, the mitochondrial electron transport plays a major role . As a by-product of a cytochrome oxidase (COX) dependent energy transduction, reactive oxygen species (ROS) are generated and lead to damage of cellular biomolecules . Damaged mitochondria, compromised at complex IV (COX) of the respiratory chain, signal to the nucleus and induce a nuclear gene, PaAox, encoding an alternative oxidase (AOX) . This pathway resembles the retrograde response that, at least in yeast, is induced by dysfunctional mitochondria . ROS generation is lowered when electrons are transferred via an alternative pathway utilizing the AOX . As a consequence, lifespan of the corresponding strains is increased . Cellular copper levels were found to play a significant role not only in the generation of ROS but also have an impact on the cytoplasmic and the mitochondrial superoxide dismutase (SOD) . In addition, copper is involved in the control of mitochondrial DNA rearrangements and affects the ability of the system to remodel damaged mitochondria . All these different components and pathways are part of the complex molecular network involved in lifespan control of this aging model. Analyst, 2001 May, 126(5), 641 - 6 A microbiological six-plate method for the identification of certain antibiotic groups in incurred kidney and muscle samples; Myllyniemi AL et al.; A microbiological method was developed for group level identification of antibiotics in incurred kidney and muscle samples from cattle and pigs . The method was composed of six test bacterium-plate growth medium combinations and the result was recorded as a profile of growth inhibition zones . The sample profiles were compared to two sets of references: one constructed with standard antibiotic solution profiles, and the other with these combined with profiles of microbiologically and chemically identified residues from incurred samples . The algorithm employed in profile comparison located the minimal sum of absolute pairwise differences over the tests, with the addition of a number of experimentally observed intra-test criteria . Chemical identification and quantitation of incurred residues was based on liquid chromatography . The method identified penicillin G as a penicillinase sensitive penicillin, enrofloxacin and ciprofloxacin belonging to fluoroquinolone group, and oxytetracycline belonging to tetracycline group . Each of these residues was microbiologically identified below the Maximum Residue Limit (MRL) for kidney tissue . Combining sample profiles with the standard reference data set did not enhance the resolution . Microbiological and chemical identification test results were in good agreement . The results of this study show that a microbiological identification method is a useful tool in preliminary characterisation of antibiotic residues in animal tissues. Indoor Air, 2001 Jun, 11(2), 99 - 110 Review of methods to collect settled dust and isolate culturable microorganisms; Macher JM; Examination of settled dust is often included in investigations of indoor environments to identify the types and concentrations of particles to which building occupants may be exposed . Fungi and bacteria are among the many components in dust that have been studied . Isolation by culture is an established method that is used widely to quantify and identify microorganisms in environmental samples . However, no standard procedures for culturing fungi or bacteria from dust have been adopted widely to ensure the validity of comparing findings from different studies . This paper reviews methods various researchers have used to study surface particles and to isolate culturable microorganisms from dust . Factors that were found to differ included the method of sample collection, the ways dust was prepared for inoculation onto growth media, and the culture media chosen for specific categories of agents . The need for reference methods in environmental microbiology for use in the assessment of indoor environmental quality is discussed. Phytochemistry, 2001 Jun, 57(3), 377 - 83 Biotransformation of squamulosone by Curvularia lunata ATCC 12017; Collins DO et al.; Squamulosone (aromadendr-1(10)-en-9-one), isolated in large quantity from the plant Hyptis verticillata Jacq . (Labiatae), was incubated with the fungus Curvularia lunata ATCC 12017 in two different growth media . Six metabolites were isolated from each medium . with five of the products being common to both fermentations . All seven metabolites are novel . The insecticidal activity of these aromadendranes was evaluated against the sweet potato weevil Cylas formicarius elegantus. J Steroid Biochem Mol Biol, 2001 Jan-Mar, 76(1-5), 105 - 17 Hormonal regulation of tumor suppressor proteins in breast cancer cells; Moudgil VK et al.; This laboratory is studying hormonal regulation of tumor suppressor proteins, p53 and retinoblastoma (pRB) . Estrogen receptor and progesterone receptor positive human breast cancer cell lines, T47D and MCF-7, were utilized for determining influence of hormonal and antihormonal agents on the level of expression of p53, state of phosphorylation of pRB, and rate of cell proliferation . The expression of p53 in T47D cells grown for 4-5 days in culture medium containing charcoal-treated (stripped) fetal bovine serum declined gradually to 10% of the level seen in control (whole serum, non charcoal-treated) groups . Supplementation of culture medium containing stripped serum with 0.1-1 nM estradiol (E(2)) restored p53 to its level seen in the control within 6-24 h . Under above conditions, treatment of cells with R5020 or RU486 reduced (15-30%) the level of p53 . Incubation of cells in E(2)-containing growth medium caused cell proliferation and hyperphosphorylation of pRB; the latter effect was seen maximally between 24-72 h . The E(2)-induced hyperphosphorylation of pRB and increase in the level of p53 were sensitive to the presence of ICI and 4-hydroxy tamoxifen (OHT) . T47D and MCF-7 cells were also transiently transfected with a P1CAT reporter plasmid containing c-Myc responsive element and the levels of chloramphenicol acetyltransferase (CAT) activity were observed in response to various treatments . E(2) and OHT caused P1CAT induction as seen by increased CAT activity: E(2) caused an endogenous increase in the expression of an ICI-sensitive c-Myc form . These data suggest that estrogen upregulates p53 expression while progesterone downregulates this process . Further, E(2) regulates p53 level and pRB activity in a coordinated manner. Regul Pept, 2001 Jun 15, 99(2-3), 169 - 74 Overexpression of parathyroid hormone-related protein promotes cell growth in the rat intestinal cell line IEC-6; Ye Y et al.; The rat intestinal cell line, IEC-6, was used as a model to study effects of parathyroid hormone-related protein (PTHrP) on crypt cell growth . Studies showed that addition of PTHrP analogs (1-34), (67-86), or (107-139) to growth medium did not affect proliferation of cells grown in either high (10% Nu-Serum) or low serum (1% Nu-Serum) . However, studies on clonal lines of IEC-6 cells stably transfected with PTHrP cDNA and overexpressing PTHrP showed that increased PTHrP production enhanced cell growth and 3H-thymidine incorporation in high, but not low, serum . Additional studies examined the role of the nuclear localization sequence (NLS) of PTHrP in mediating the growth effect . In three clonal IEC-6 lines transfected with PTHrP cDNA bearing a mutated NLS, the ability of PTHrP to stimulate 3H-thymidine incorporation and cell growth was lost . The results suggest that endogenously produced PTHrP can promote proliferation of IEC-6 cells and that the integrity of the NLS of PTHrP is required for its growth effects. Toxicon, 2001 Sep, 39(9), 1411 - 20 Investigations into the inhibitory effects of microcystins on plant growth, and the toxicity of plant tissues following exposure; McElhiney J et al.; The cyanobacterial toxins microcystins are known to affect a number of processes in plant tissues, and their presence in water used for irrigation may have considerable impact on the growth and development of crop plants . In this study, two plant bioassays were employed to investigate the phytotoxic effects of microcystins . A plant tissue culture assay revealed that the growth and chlorophyll content of Solanum tuberosum L . cultures was inhibited at microcystin-LR concentrations of 0.005 and 0.05 microg x cm(-3), respectively . A previously developed bioassay was also employed to determine the effects of three commonly occurring microcystin variants on the growth of Synapis alba L . seedlings . Microcystins-LR, -RR, and -LF inhibited the growth of seedlings, with GI50 values of 1.9, 1.6 and 7.7 microg x ml(-1), respectively . The growth of Phaseolus vulgaris was also examined in the presence of microcystin-LR . The toxin was found to have little effect on growth for up to 18 days, but impaired the development of the roots of exposed plants, causing them to take up approximately 30% less growth medium than those grown in the absence of toxin . Microcystin was also detected in the tissues of exposed plants using a commercially available ELISA kit, suggesting that the uptake of these toxins by edible plants may have significant implications for human health. Exp Eye Res, 2001 Jun, 72(6), 661 - 6 Glutathione transport in human retinal pigment epithelial (HRPE) cells: apical localization of sodium-dependent gsh transport; Kannan R et al.; The study was undertaken to identify and localize GSH transport in non-transformed cultured human retinal pigmented epithelial cells (HRPE) . In confluent monolayers exhibiting high transepithelial resistance (TER 700-1000 Omega cm(-2)), apical and basolateral GSH uptake were determined after introducing(35)S-GSH (+ 1 m M GSH) to the apical side or basal side in NaCl (Na+ -containing) or choline chloride (Na+ -free) buffers . Cells in growth medium or in incubation buffers were pretreated with acivicin to inhibit gamma-glutamyltranspeptidase (GGT) . GSH efflux was measured after labelling the intracellular GSH pool by incubation overnight with 35 S-cysteine and quantitating the release of labelled GSH into the medium . Uptake of GSH was found at both the apical and basolateral membranes of HRPE cells . Inhibition of gamma-glutamyltranspeptidase (GGT) with acivicin did not alter mean GSH uptake (nmol per million cells per 30 min) significantly at the apical (1.63 +/- 0.32 vs 1.45 +/- 0.30; with and without acivicin respectively) or the basolateral (1.17 +/- 0.21 vs 1.44 +/- 0.38) membranes . Transport was verified to be in the form of intact GSH by HPLC . Uptake was unaffected by the removal of Na+ at the basolateral membrane while apical uptake exhibited partial but significant (approximately 40%) Na+ -dependency . Net GSH efflux (nmol per million cells per min) to the apical side of HRPE cells was higher than to the basolateral side in the presence of sodium . Transepithelial flux in the basolateral to apical direction was approximately 17-fold higher than the apical to basolateral direction resulting in a net flux of GSH to the apical side . In conclusion, HRPE cells exhibit GSH transport by Na+ -dependent and Na+ -independent mechanisms . The Na+ -dependent GSH transporter is localized to the apical membrane of HRPE cells . Kidney Int, 2001 Jun, 59(6), 2182 - 91 Hypertonic activation of the renal betaine/GABA transporter is microtubule dependent; Basham JC et al.; BACKGROUND: Epithelial cells in the renal inner medulla accumulate osmolytes such as betaine to maintain normal cell volume during prolonged extracellular hypertonic stress . Betaine accumulation is the result of activation of transcription of the BGT1 transporter gene followed by increased betaine transport . METHODS: We studied the possible role of microtubules in this adaptive mechanism using renal cells in culture . RESULTS.: In cultured renal cell lines {Madin-Darby canine kidney (MDCK) and mouse inner medullary collecting duct (mIMCD-3)}, up-regulation of BGT1 activity was maximal after 24 to 30 hours in growth medium made hypertonic (510 mOsm/kg) by the addition of sucrose or NaCl . Up-regulation was reversed within 24 to 36 hours after returning cells to isotonic medium . Both cycloheximide (20 micromol/L) and nocodazole (20 micromol/L) blocked the hypertonic up-regulation of BGT1 . Nocodazole was partially effective even when added 16 to 20 hours after the switch to hypertonic medium . Recovery from nocodazole action was rapid, and there was full activation of BGT1 transport within three to six hours after nocodazole removal, suggesting rapid trafficking to the cell surface once microtubules repolymerized . Hypertonic activation of BGT1 transport was detected in an isolated membrane fraction and was blocked by cycloheximide but not by nocodazole . Confocal microscopy confirmed the increased abundance of BGT1 proteins in the plasma membrane of hypertonic cells and showed that BGT1 remained intracellular during nocodazole treatment . CONCLUSIONS: Hypertonic activation of BGT1 in renal cells requires de novo protein synthesis and microtubule-dependent trafficking of additional transporters to the cell surface . The apparent resistance of membrane BGT1 to nocodazole blockade is likely due to the presence in the membrane fraction of an increased intracellular pool of active BGT1 transporters. J Steroid Biochem Mol Biol, 2001 May, 77(2-3), 135 - 41 Differential regulation of retinoblastoma protein by hormonal and antihormonal agents in T47D breast cancer cells; Alban P et al.; Phosphorylation of the tumor suppressor protein, retinoblastoma (pRb), regulates the progression of the cell cycle . Prev