Growth media

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 . Previous work from this laboratory had shown that estradiol (E(2)) regulates tumor suppressor proteins, p53 and retinoblastoma in breast cancer cells . In the present study, we have examined the phosphorylation of pRB in T47D breast cancer cells following treatments with R5020 and antiprogestins . In growth medium containing serum depleted of endogenous steroids by charcoal treatment, pRb appeared mainly in its hypophosphorylated form . Addition of 10 nM R5020 to the culture medium caused hyperphosphorylation of pRb within 24 h, but the hypophosphorylated form of pRb began to accumulate after 72 h . Upon prolonged R5020 treatment (72-96 h), pRb was detected exclusively in its hypophosphorylated form . While treatment of cells with R5020 caused a transient increase in the level of cyclin D1, E(2) addition caused a sustained increase in the level of cyclin D1 consistent with its role in stimulating pRb phosphorylation . Antagonists of both estrogen receptor (ER) and progesterone receptor (PR) blocked the E(2) and R5020-induced pRb phosphorylation, respectively . These results suggest that R5020 induces pRb phosphorylation via a transient increased expression of cyclin D1, whereas E(2) treatment results in sustained expression of cyclin D1 and increased pRb phosphorylation . Furthermore, R5020 effects on pRb phosphorylation appear PR-mediated as no cross-antagonism of pRb phosphorylation was observed: the R5020 effects were blocked by RU486 and ZK98299, but not by the pure ER antagonist, ICI 182, 780 (ICI).

J Clin Microbiol, 2001 Jun, 39(6), 2115 - 21
Utility of inoculum counting (Walshe and English criteria) in clinical diagnosis of onychomycosis caused by nondermatophytic filamentous fungi; Gupta AK et al.; Opportunistic onychomycosis caused by nondermatophytic molds may differ in treatment from tinea unguium . Confirmed diagnosis of opportunistic onychomycosis classically requires more than one laboratory analysis to show consistency of fungal outgrowth . Walshe and English in 1966 proposed to extract sufficient diagnostic information from a single patient consultation by counting the number of nail fragments positive for inoculum of the suspected fungus . Twenty fragments were plated per patient, and each case in which five or more fragments grew the same mold was considered an infection by that mold, provided that compatible filaments were also seen invading the nail tissue by direct microscopy . This widely used and often recommended method has never been validated . Therefore, the validity of substituting any technique based on inoculum counting for conventional follow-up study in the diagnosis of opportunistic onychomycosis was investigated . Sampling of 473 patients was performed repeatedly . Nail specimens were examined by direct microscopy, and 15 pieces were plated on standard growth media . After 3 weeks, outgrowing dermatophytes were recorded, and pieces growing any nondermatophyte mold were counted . Patients returned on two to eight additional occasions over a 1- to 3-year period for similar examinations . Onychomycosis was etiologically classified based on long-term study . Opportunistic onychomycosis was definitively established for 86 patients . Counts of nondermatophyte molds in initial examinations were analyzed to determine if they successfully predicted both true cases of opportunistic onychomycosis and cases of insignificant mold contamination . There was a strong positive statistical association between mold colony counts and true opportunistic onychomycosis . Logistic regression analysis, however, determined that even the highest counts predicted true cases of opportunistic onychomycosis only 89.7% of the time . The counting criterion suggested by Walshe and English was correct only 23.2% of the time . Acremonium infections were especially likely to be correctly predicted by inoculum counting . Inoculum counting could be used to indicate a need for repeat studies in cases of false-negative results from laboratory direct microscopy . Inoculum counting cannot serve as a valid substitute for follow-up study in the diagnosis of opportunistic onychomycosis . It may, nonetheless, provide useful information both to the physician and to the laboratory, and it may be especially valuable when the patient does not present for follow-up sampling.

Curr Microbiol, 2001 Jul, 43(1), 7 - 10
Guanidine hydrochloride inhibits Hsp104 activity in vivo: a possible explanation for its effect in curing yeast prions; Jung G et al.; The presence of millimolar concentrations of guanidine hydrochloride (Gdn-HCl) in growth media causes efficient loss of the normally stable {PSI+} element from yeast cells . Although it has become common practice to include 5 mm Gdn-HCl in growth media to cure {PSI+} and other prions of yeast, the biochemical mechanism by which it cures is unknown . We find that 5 mm Gdn-HCl significantly reduces Hsp104-mediated basal and acquired thermotolerance . Gdn-HCl also reduced the ability of Hsp104 to restore activity of thermally denatured luciferase in vivo . The abundance of Hsp104 was not reduced in cells grown in the presence of Gdn-HCl, ruling out negative effects on expression or stability of Hsp104 . We therefore conclude that Gdn-HCl inhibits Hsp104 activity in vivo . Since replication of yeast prions is dependent on Hsp104, our results suggest that Gdn-HCl cures prions by inhibiting Hsp104 activity.

Appl Environ Microbiol, 2001 Jun, 67(6), 2819 - 22
Incorporation of {(15)N} ammonia by the cellulolytic ruminal bacteria Fibrobacter succinogenes BL2, Ruminococcus albus SY3, and Ruminococcus flavefaciens 17; Atasoglu C et al.; The origin of cell nitrogen and amino acid nitrogen during growth of ruminal cellulolytic bacteria in different growth media was investigated by using (15)NH(3) . At high concentrations of peptides (Trypticase, 10 g/liter) and amino acids (15.5 g/liter), significant amounts of cell nitrogen of Fibrobacter succinogenes BL2 (51%), Ruminococcus flavefaciens 17 (43%), and Ruminococcus albus SY3 (46%) were derived from non-NH(3)-N . With peptides at 1 g/liter, a mean of 80% of cell nitrogen was from NH(3) . More cell nitrogen was formed from NH(3) during growth on cellobiose compared with growth on cellulose in all media . Phenylalanine was essential for F . succinogenes, and its (15)N enrichment declined more than that of other amino acids in all species when amino acids were added to the medium.

Microbiol Res, 2001, 156(1), 59 - 63
HCf-6, a novel class II hydrophobin from Cladosporium fulvum; Nielsen PS et al.; C . fulvum, a fungal tomato pathogen, has previously been shown to express a complex family of hydrophobin genes including four class I hydrophobins and one class II hydrophobin . Here we describe a gene for HCf-6, a sixth member of the hydrophobin family and the second class II gene . The protein is predicted to consist of a signal sequence, an N-terminus rich in glycine and asparagine and a C-terminal hydrophobic domain which bears the hall-marks of hydrophobins . In contrast to the previously described class II hydrophobin HCf-5, HCf-6 is expressed in mycelium growing in pure culture and mRNA levels do not increase during sporulation . It is down-regulated by carbon starvation but not by depletion of nitrogen in the growth medium.

Biochemistry, 2001 May 29, 40(21), 6275 - 83
High-level bacterial expression and 15N-alanine-labeling of bovine trypsin . Application to the study of trypsin-inhibitor complexes and trypsinogen activation by NMR spectroscopy; Peterson FC et al.; We describe here the high-level expression of bovine trypsinogen in E . coli, its refolding and activation to beta-trypsin, and the selective incorporation of (15)N-labeled alanine through supplementation of the growth medium . Using this procedure, we expressed (15)N-labeled S195A trypsinogens, both on a wild-type and on a D189S background, in amounts suitable for NMR spectroscopy . 2D {(1)H-(15)N}-HSQC NMR was used to follow conformational changes upon activation of trypsinogen and formation of noncovalent complexes between S195A or S195A/D189S trypsin and protein proteinase inhibitors of different structural families and different sizes, as well as to examine the effects of introduction of the D189S mutation . Spectra of good quality were obtained for both trypsins alone and in complexes of increasing size with the proteinase inhibitors BPTI (total molecular mass 31 kDa), SBTI (total molecular mass 44 kDa), and the serpin alpha(1)-proteinase inhibitor Pittsburgh (alpha(1)PI Pittsburgh) (total molecular mass 69 kDa) . Assignments of alanines 55 and 56, close to the active site histidine, and of alanine 195, present in the S195A variant used for most of the studies, were made by mutagenesis . These three alanines, together with two others, probably close to the S1 specificity pocket, were very sensitive to complex formation . In contrast, the remaining 10 alanines were invariant in chemical shift in all 3 of the noncovalent complexes formed, reflecting the conservation of structure in complexes with BPTI and SBTI known from X-ray crystal structures, but also indicating that there is no change in backbone conformation for the noncovalent complex with alpha(1)PI, for which there is no crystal structure . This was true both for S195A and for S195A/D189S trypsins . This high-level expression and labeling approach will be of great use for solution NMR studies on trypsin-serpin complexes, as well as for structural and mechanistic studies on trypsin variants.

J Exp Clin Cancer Res, 2001 Mar, 20(1), 103 - 9
A study of chromosome aneuploidy in hereditary breast cancer patients and their healthy blood relatives; Roy SK et al.; Chromosomal abnormalities that may predispose a group of individuals to develop certain neoplasms have been reported in lymphocytes . We evaluated cytogenetic abnormalities in 21 histopathologically confirmed primary breast cancer patients (BCPs), 52 healthy blood relatives (HBRs), belonging to 19 hereditary breast cancer families (HBFs) and 25 females as control . Phytohemagglutinin stimulated peripheral blood lymphocyte (PBL) cultures were used to study the chromosomal abnormalities in BCPs and their HBRs . Short term culture of the tumor tissue was also carried out in defined growth medium . Suitable metaphases (11 to 55) from tumors and a minimum of 100 metaphases from PBL were karyotyped for the cytogenetic analysis . Heterogeneous population of cells with random and nonrandom chromosomal abnormalities was noticed in tumors . In control groups 2-5% of metaphases showed numerical abnormalities, whereas this phenomenon was observed in 3-18% of metaphases in HBRs and 3-23% of metaphases in BCPs . In tumor tissue, 47.05% of BCPs showed numerical abnormalities in more than 16 metaphases . In lymphocytes, this event was observed in 33.33% of BCPs and 13.14% of HBRs . In controls 1.28%, in BCPs 52.04% (tumor) and 13.42% (lymphocytes), and in HBRs 9.03% of metaphases were found aneuploid . Statistically it was highly significant (Fisher's exact test, P<0.00001) . In lymphocytes of BCPs, chromosomes 1, 6, 8, 9, 15, 17, 18, 20, and X and in HBRs, chromosomes 8, 15, 17, 18, and X were frequently involved . It can be inferred from the findings that the above mentioned chromosomes may have an important role in early stage of breast carcinogenesis in BCFs . Moreover, presence of similar abnormalities in HBR indicates inherited pattern of this genetic error among them.

Biometals, 2001 Mar, 14(1), 23 - 31
Hexavalent chromium stimulation of riboflavin synthesis in flavinogenic yeast; Fedorovych D et al.; Flavinogenic yeast overproduce riboflavin (RF) in iron-deprived media . In optimal growth media supplemented with Fe, hexavalent chromium 'Cr (VI)' treatment led to elevated RF synthesis in all cases of 37 flavinogenic strains studied . The level of RF production exceeded the rate observed at iron-deficient conditions . At sublethal Cr concentrations the RF oversynthesis over time correlated well with the growth-inhibitory adaptational period as manifested by the prolonged lag phase . The consecutive logarithmic biomass growth was accompanied by a drop in RF biosynthesis . Cr (VI)-induced RF overproduction was not a result of cellular iron level decrease . The treatment of yeast with Cr (VI) led to the stimulation of GTP-cyclohydrolase and RF-synthase activities, the key enzymes of the RF biosynthesis pathway.

Arch Biochem Biophys, 2001 Apr 15, 388(2), 185 - 97
Human soluble guanylate cyclase: functional expression, purification and structural characterization; Kosarikov DN et al.; Soluble guanylate cyclase is an enzyme that catalyzes formation of cGMP from GTP and is a member of the nucleotide cyclase family of enzymes . sGC is a receptor for endogenous and exogenous nitric oxide and is activated several-fold upon its binding, constituting a core enzyme in the nitric oxide signal transduction pathway . cGMP generated by sGC is an important second messenger that regulates activity of several enzymes triggering such important physiologic reactions as vasodilation, smooth muscle relaxation and platelet aggregation . We report here the functional expression of the human isoform of soluble guanylate cyclase in HighFive insect cells using a baculovirus expression system . Highly active recombinant protein was obtained without heme reconstitution or supplementation of the cell growth medium and the level of protein expression was found to be heavily affected by the composition of the growth medium . We have successfully purified highly active sGC (sp act up to 940 nmol/min/mg) from adherent cultures using a three-column, 1-day procedure . The UV-Vis spectrum of the isolated protein shows a Soret band at 431 nm, consistent with a histidine-ligated, 5-coordinate heme as previously reported . Far UV CD spectroscopy, intrinsic tryptophan fluorescence, fluorescence of the hydrophobic dye bis-ANS, size-exclusion chromatography, and small angle X-ray scattering (SAXS) were used to characterize the structural properties of the purified sGC . We used two hierarchical neural network methods to predict the secondary structure of sGC and found it to be consistent with the observed CD spectrum of sGC.

Arch Biochem Biophys, 2001 Jan 15, 385(2), 250 - 8
Development and evaluation of a boronate inhibitor of gamma-glutamyl transpeptidase; London RE et al.; Gamma-glutamyl transpeptidase (gamma-GT) plays a central role in the metabolism of glutathione and is also a marker for neoplasia and cell transformation . We have investigated the compound L-2-amino-4-boronobutanoic acid (ABBA) as a structural analog of the putative ternary complex formed by the enzyme, L-serine, and borate, proposed to function as a transition state analog inhibitor . ABBA was found to be a potent inhibitor of the enzyme, with Ki = 17 nM using typical assay conditions (pH 8, gamma-glutamyl-p-nitroanilide substrate, 20 mM glycyl-glycine acceptor) . ABBA is a stable amino acid analog with pK values determined from 13C and 11B NMR to be 2.3, 11.0 (amino titration), and 7.9 (boronate titration) . The structural similarity to glutamate suggested that it might function as a glutamate analog for some glutamate-dependent enzymes or receptors . Transamination of pyruvate by ABBA to yield alanine in the presence of glutamic pyruvic transaminase was demonstrated by 13C NMR . The 2-keto-4-boronobutanoic acid transamination product is apparently fairly labile to hydrolysis, leading to formation of 2-ketobutanoic acid plus borate . The latter is also subsequently transaminated to yield 2-aminobutanoic acid . Both of these metabolites were observed in the 13C NMR spectrum . However, the corresponding transamination of oxaloacetate by ABBA in the presence of glutamic oxaloacetic transaminase was not observed . Effects of ABBA on the growth of cultured rat liver cell lines ARL-15C1 (nontumorigenic, low gamma-GT activity) and ARL-16T2 (tumorigenic, high gamma-GT activity) were also investigated, both in standard Williams Media as well as in a low cysteine growth medium . A high concentration (1 mM) of ABBA inhibited the growth of both cell lines in both media, with the degree of inhibition greater in the low cysteine medium . Alternatively, growth inhibition by 10 microM ABBA could be observed only in the low cysteine media . In general, there were no significant differences between the two cell lines in terms of sensitivity to ABBA.

Glycobiology, 2001 Apr, 11(4), 283 - 95
Analysis of Skp1 glycosylation and nuclear enrichment in Dictyostelium; Sassi S et al.; Skp1 is a subunit of SCF-E3 ubiquitin ligases and other protein complexes in the nucleus and cytoplasm of yeast and mammalian cells . In Dictyostelium, Skp1 is partially modified by an unusual pentasaccharide O-linked to hydroxyproline143 . This modification was found to be susceptible to known prolyl hydroxylase inhibitors based on M(r)-shift analysis using SDS-polyacrylamide gel electrophoresis/Western blotting . In addition, Dictyostelium Skp1 consists of 2 genetic isoforms, Skp1A and Skp1B, which differ by a single amino acid and appear to be expressed throughout the life cycle based on reverse-transcription polymerase chain reactions . The significance of these structural variations was examined by expressing myc-tagged Skp1s and mutants that lacked the glycosylation site . Gel-based M(r)-shift studies showed that Skp1A and Skp1B are both nearly completely glycosylated during growth and early development, and mass spectrometry of glycopeptides showed that they were glycosylated similarly . Skp1 expressed later in prespore cells was not glycosylated, unlike bulk Skp1 persisting from earlier in development, but became glycosylated after return to growth medium . Skp1A and Skp1B were each concentrated in the nucleus and regions of the cytoplasm, based on immunofluorescence localization . However, when Skp1 glycosylation was blocked by mutation, prolyl hydroxylase inhibitors, or expression in prespore cells, nuclear concentration of Skp1 was not detected . Furthermore, nuclear concentration occurred in a mutant that attached only the core disaccharide to Skp1 . Overall, there was no evidence for differential Skp1 isoform expression, glycosylation variants in the bulk Skp1 pool, or regulation of nuclear localization . However, these studies uncovered evidence that the glycosylation pathway is developmentally regulated and can function posttranslationally, and that core glycosylation is required for Skp1's nuclear concentration.

Can J Microbiol, 2001 Apr, 47(4), 290 - 3
Sensitivity of Saccharomyces cerevisiae to tannic acid is due to iron deprivation; Wauters T et al.; Tannic acid inhibited the growth of the yeast Saccharomyces cerevisiae . Growth medium supplementation with more nitrogen or metal ions showed that only iron ions could restore the maximal growth rate of S . cerevisiae . Tannic acid resistant mutants were previously isolated by screening for tannic acid resistance and were all cytoplasmic petite mutants . While the wild type was very sensitive to iron deprivation conditions when grown in aerobic conditions, the mutants, whether grown aerobically or anaerobically, showed the same growth rate under iron-limited conditions as under iron-repleted conditions . Also, the wild type grown anaerobically was not affected by iron-limited conditions . Cytoplasmic petite mutants obtained by ethidium bromide mutagenesis behaved like the other mutants . During iron limitation, the wild type showed a reduced oxygen uptake rate . Maximal growth rate of the wild type in iron-limited conditions could be restored by the addition to the media of unsaturated fatty acids and sterol . Iron deprivation caused by tannic acid may thus affect the synthesis of a functional respiratory chain as well as the synthesis of unsaturated fatty acids and (or) sterol.

Sheng Li Xue Bao, 2001 Feb, 53(1), 23 - 6
{Effect of aldosterone on the secretion of endothelin by ventricular fibroblasts}; Gong SZ et al.; Using cell culture, radioimmunoassay for endothelin and RT-PCR, the effect of aldosterone on the endothelin secretion of ventricular fibroblasts was studied . The results showed that aldosterone (1 x 10(-7) mol/L) promoted the expression of ppET-1 mRNA, which began to increase in 2 hours and attained the highest level in 4 hours, thereafter decreased; aldosterone increased the endothelin level in ventricular fibroblasts and fibroblast conditioned growth medium (FCGM) as well, which was blocked by spironolactone (1 x 10(-6) mol/L), an aldosterone receptor antagonist . The results suggest that aldosterone can increase endothelin secretion by ventricular fibroblasts, which can be inhibited by its receptor antagonist spironolactone.

Sheng Li Xue Bao, 2001 Feb, 53(1), 18 - 22
{Role of conditioned growth medium for ventricular fibroblasts in promoting fibroblast collagen synthesis and proliferation}; Gong SZ et al.; Ventricular fibroblasts were cultured using conditioned growth medium for ventricular fibroblasts (FCGM) . The rate of the total collagen synthesis of ventricular fibroblasts was measured by assaying the incorporation rate of {3H}-proline, whereas the proliferation of ventricular fibroblasts was assessed by determining the incorporation rate of {3H}-TdR and the expression of c-fos genes . FCGM significantly increased the {3H}-proline incorporation rate and {3H}-TdR incorporation rate of fibroblasts in a dose-dependent manner . Furthermore, FCGM promoted the c-fos gene expression of fibroblasts, which attained its maximum in 1 h . BQ123, an ETA receptor antagonist, partially blocked the above effects of FCGM, but AT1 receptor antagonist CV11974 and alpha-adrenergic receptor antagonist regitin did not . It is suggested that the ventricular fibroblast has an autorine function in promotion of collagen synthesis and proliferation of fibroblasts by secreting endothelin and other bioactive substances.

Proc Natl Acad Sci U S A, 2001 May 22, 98(11), 6412 - 6 Epub 2001 May 15.
Trypanosoma brucei CTP synthetase: a target for the treatment of African sleeping sickness; Hofer A et al.; The drugs in clinical use against African sleeping sickness are toxic, costly, or inefficient . We show that Trypanosoma brucei, which causes this disease, has very low levels of CTP, which are due to a limited capacity for de novo synthesis and the lack of salvage pathways . The CTP synthetase inhibitors 6-diazo-5-oxo-l-norleucine (DON) and alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) reduced the parasite CTP levels even further and inhibited trypanosome proliferation in vitro and in T . brucei-infected mice . In mammalian cells, DON mainly inhibits de novo purine biosynthesis, a pathway lacking in trypanosomes . We could rescue DON-treated human and mouse fibroblasts by the addition of the purine base hypoxanthine to the growth medium . For treatment of sleeping sickness, we propose the use of CTP synthetase inhibitors alone or in combination with appropriate nucleosides or bases.

Biochem Biophys Res Commun, 2001 Apr 13, 282(4), 1019 - 25
Chilling-sensitive, post-transcriptional regulation of a plant fatty acid desaturase expressed in yeast; Dyer JM et al.; Plants respond to chilling exposure by increasing the relative proportion of polyunsaturated fatty acids in their lipids . However, unlike the response in many other organisms, plant fatty acid desaturase genes are typically not upregulated during this process . We expressed the Brassica napus FAD3 gene, which encodes an enzyme for synthesis of linolenic acid, in Saccharomyces cerevisiae and observed a temperature-dependent increase in linolenic acid production at cooler growth temperatures . Untransformed yeast cells, however, responded to cooler temperatures primarily by shortening fatty acid chains, even when polyunsaturated fatty acids were supplied in the growth media . Measurement of the steady-state levels of Fad3 protein in transformed yeast revealed an 8.5-fold increase in steady-state amount of desaturase enzyme when cells were cultivated at cooler temperatures . The increase was not due to changes in transcriptional activity, since Northern hybridization revealed no appreciable changes in abundance of FAD3 transcripts at cooler temperatures . Taken together, the results suggest that the increase in linolenic acid content in cells containing Fad3 was not due to enhanced physiological demand for polyunsaturated fatty acids by yeast, but rather a cold-inducible, post-transcriptional increase in steady-state amount of plant desaturase enzyme . Implications for plant adaptation to chilling are discussed .

Environ Sci Technol, 2001 Jan 1, 35(1), 76 - 83
Cerro Negro bitumen degradation by a consortium of marine benthic microorganisms; Potter TL et al.; Cerro Negro bitumen, separated from an Orimulsion sample, was incubated for up to 120 days with sediments collected at a petroleum-impacted site in Tampa Bay, Florida . Biodegradation conditions were optimized by increasing bitumen surface area, continuous agitation on a shaker apparatus, use of a complete growth medium, and maintenance at 37 degrees C . Aerobic degradation conditions were promoted by maintaining sediment contact with the laboratory atmosphere . Bitumen recovered in solvent extracts when compared to autoclaved controls decreased by up to 40% during the first 56 days . There was no detectable change after this . Molasses addition and use of a culture enriched from the sediments did not change the extent or rate of decrease in bitumen recovery . Chemical fractionation of bitumen control and degraded bitumen showed that aromatic and aliphatic fractions were depleted by approximately equals 50% . Accumulation of polars was observed; however, the apparent increase was relatively small when compared to the mass loss of the other fractions . Selected biomarker ratios were not affected by incubation indicating their utility for fingerprinting the source bitumen in environmental samples . PAH distribution in the aromatic fraction favored the higher alkyl-homologues with the relative degree of alkylation increasing as the mass of bitumen recovered decreased with degradation . The study showed that up to 40% of the bitumen was bioaccessible and that bioremediation may be a treatment option for sediments contaminated with bitumen by an Orimulsion spill.

J Microbiol Methods, 2001 Jul, 45(3), 207 - 12
Proteases from actinomycetes interfere in solid media plate assays of hyaluronidase activity; de Azeredo LA et al.; Four hundred and fifteen actinomycete strains were screened for hyaluronidase activity in two plate assays media . In the first one, using hyaluronic acid as substrate and bovine serum albumin (BSA) to help precipitation of the nondegraded substrate, only strain 594 and hyaluronidase control were positive . In the second assay, plates with hyaluronic acid, but not BSA, gave the same results . For plates containing only BSA, proteinase activity was detected in strain 594 . When hyaluronic acid was treated with pronase, the only clear zones, in the second assay without BSA, were those around hyaluronidase controls . Protease activity, commonly found in actinomycetes, was detected only in strain 594, among the 415 studied, when tested in hyaluronidase assay using hyaluronate plus BSA . This may be due to the composition of the growth medium, since media with different composition gave different results for protease activity in each of the 15 strains analyzed . These data suggest that proteases can affect an accurate detection of hyaluronidase in media containing proteins, not only from hyaluronate preparations, but also from other medium ingredients . Thus, for a correct interpretation of the method, they must be excluded . Commercial Hyaluronidase used as controls must be also tested for the presence of protease contamination.

J Surg Res, 2001 May 15, 97(2), 131 - 7
Antitumor and antiangiogenic effects of somatostatin receptor-targeted in situ radiation with (111)In-DTPA-JIC 2DL; Gulec SA et al.; INTRODUCTION: Expression of somatostatin receptor subtype 2 (sst 2) in angiogenic tumor vessels appears to be homogeneous, while tumor cell expression of this receptor is often heterogeneous . We have developed a novel in vitro three-dimensional tumor angiogenesis model to study the antitumor and the antiangiogenic effects of radiolabeled somatostatin analogs . We hypothesized that targeted in situ radiation with an Auger electron-emitting radiolabeled somatostatin analog would produce receptor-specific cytotoxicity in sst 2-expressing cells . MATERIALS and METHODS: IMR-32 human neuroblastoma (sst 2-positive) and MDA MB-231 human breast cancer (sst 2-negative) xenografts were created in nude mice from monolayer cell cultures . Fragments of these tumors were embedded in three-dimensional fibrin gels supplemented with endothelial growth media and incubated for a period of 14 days . Tumor fragments were treated with 50 microCi/ml of (111)In-JIC 2DL, a sst 2-preferring somatostatin analog, or medium on Day 1 . Initial angiogenic activity was determined at 48 h and the mean angiogenic score and tumoricidal responses were assessed on Day 14 . RESULTS and CONCLUSION: Tumoricidal effects of (111)In-JIC 2DL were seen only in sst 2-positive IMR-32 tumors . However, the angiogenic response was inhibited in both IMR-32 and MDA MB-231 tumors independent of the tumor cells' sst 2 status . Somatostatin receptor-mediated in situ radiation therapy has profound cytotoxic effects on angiogenic blood vessels and sst 2-expressing tumor cells .

Bioresour Technol, 2001 Jul, 78(3), 273 - 5
Two-stage biohumus production from inedible potato biomass; Manukovsky NS et al.; The feasibility of a two-stage bioconversion of inedible potato biomass into biohumus by oyster mushroom followed by worms was tested . As a raw material for biohumus production the inedible potato biomass in certain properties ranked below wheat straw . The most feasible method to convert the potato wastes into biohumus was to mix them with wheat straw at the mass ratio of 1:3 and then treat with mushrooms followed by worms . This gave a good yield of mushrooms . The biohumus produced from the mixture was suitable for use as a plant growth medium.

Nutr Cancer, 2000, 38(1), 123 - 30
Molecular mechanisms of cell cycle block by methionine restriction in human prostate cancer cells; Lu S et al.; Previous studies have shown that dietary or pharmacological methionine restriction inhibits growth of human prostate cancer cells in vitro or as xenografts in mice . We undertook the present studies to clarify the molecular mechanisms by which methionine restriction inhibits prostate cancer cell growth . We found that PC-3 and DU 145 cells stopped proliferating within six days in growth medium containing homocysteine in place of methionine . In contrast, proliferation of LNCaP cells was only partially inhibited by the absence of methionine . Using flow cytometry, we found that methionine restriction caused PC-3 cells to arrest in all phases of the cell cycle, but predominantly in the G2/M phase, whereas LNCaP cells accumulated exclusively in the G1 phase . Methionine restriction led to accumulation of the cyclin-dependent kinase inhibitors p21 and p27, as determined by Western blot analysis, and inhibited the enzymatic activities of the cyclin-dependent kinases CDK2 and cdc2, as determined by an in vitro kinase assay: However, methionine restriction had little or no effect on CDK2 or cdc2 protein levels . Methionine restriction also induced PC-3 cells to undergo apoptosis, as indicated by the appearance of a typical nucleosomal ladder on gel electrophoresis of genomic DNA . We conclude that methionine restriction has potential as a novel treatment strategy for prostate cancer.

J Control Release, 2001 May 18, 73(1), 49 - 57
LDL-induced association of anionic liposomes with cells and delivery of contents . II . Interaction of liposomes with cells in serum-containing medium; Amin K et al.; In the present study we have investigated cell binding and drug delivery potency of various anionic liposomal formulations in a serum-supplemented growth medium, in order to understand the role of the LDL receptor in targeted drug delivery mediated by anionic liposomes . The cell lines used were CV1-P and CHO wild type, which both express the LDL receptor, and CHOldlA7, which lacks the LDL receptor . Cellular association of encapsulated methotrexate and fluorescein labeled phosphatidylethanolamine in the lipid bilayer was measured . Potency of two liposome-dependent drugs (N-phosphonacetyl-L-aspartic acid and fluoroorotic acid) was also measured by growth inhibition . Association of ePG liposomal aqueous contents with cells grown in serum-supplemented growth medium was up to 30-fold higher with CV1-P or CHO wild type cells than with CHOldlA7 . Increased association was not paralleled by a corresponding increase in potency of liposome-dependent drugs . The serum-dependent association of fluid, anionic (ePG) liposomes with cells expressing the LDL receptor is caused by an interaction of ePG liposomes with LDL . The failure of this association to increase drug delivery seems to be caused by the downregulation of LDL receptor expression when cells are continuously exposed to LDL.

Mutat Res, 2001 May 9, 476(1-2), 99 - 107
Mutations induced by 5-formyl-2'-deoxyuridine in Escherichia coli include base substitutions that can arise from mispairs of 5-formyluracil with guanine, cytosine and thymine; Anensen H et al.; 5-Formyluracil (5-foU) is a major oxidation product of thymine formed in yields comparable to that of 8-oxoguanine in DNA by ionizing radiation . Whereas the mutagenic effects of 8-oxoguanine are well understood, the investigation of the biological implications of 5-foU has so far been limited . Here we demonstrate that 5-formyl-2'-deoxyuridine (5-fodUrd) supplied to the growth medium of Escherichia coli induces several base substitutions at different frequencies at position 461 in the lacZ gene in the following order: A.T-->G.C>G.C-->A.T>G.C-->T.A>>A.T-->T.A>A.T-->C.G . No induction of G.C-->C.G transversions was observed . It is inferred that 5-fodUrd will be incorporated into the DNA during cell growth, forming mispairs with guanine, cytosine and thymine during replication . It, thus, appears that cell growth in the presence of 5-fodUrd may represent a good model for elucidating the cellular effects of 5-foU residues in DNA.

Dev Biol, 2001 May 15, 233(2), 347 - 64
Study of the murine allantois by allantoic explants; Downs KM et al.; The murine allantois will become the umbilical artery and vein of the chorioallantoic placenta . In previous studies, growth and differentiation of the allantois had been elucidated in whole embryos . In this study, the extent to which explanted allantoises grow and differentiate outside of the conceptus was investigated . The explant model was then used to elucidate cell and growth factor requirements in allantoic development . Early headfold-stage murine allantoises were explanted directly onto tissue culture plastic or suspended in test tubes . Explanted allantoises vascularized with distal-to-proximal polarity, they exhibited many of the same signaling factors used by the vitelline and cardiovascular systems, and they contained at least three cell types whose identity, gene expression profiles, topographical associations, and behavior resembled those of intact allantoises . DiI labeling further revealed that isolated allantoises grew and vascularized in the absence of significant cell mingling, thereby supporting a model of mesodermal differentiation in the allantois that is position- and possibly age-dependent . Manipulation of allantoic explants by varying growth media demonstrated that the allantoic endothelial cell lineage, like that of other embryonic vasculatures, is responsive to VEGF(164) . Although VEGF(164) was required for both survival and proliferation of allantoic angioblasts, it was not sufficient to induce appropriate epithelialization of these cells . Rather, other VEGF isoforms and/or the outer sheath of mesothelium, whose maintenance did not appear to be dependent upon endothelium, may also play important roles . On the basis of these findings, we propose murine allantoic explants as a new tool for shedding light not only on allantoic development, but for elucidating universal mechanisms of blood vessel formation, including vascular supporting cells, either in the intact organism or in existing in vitro systems .

J Biol Chem, 2001 Jul 6, 276(27), 25262 - 72 Epub 2001 May 02.
Lack of mitochondrial anionic phospholipids causes an inhibition of translation of protein components of the electron transport chain . A yeast genetic model system for the study of anionic phospholipid function in mitochondria; Ostrander DB et al.; Reduction of mitochondrial cardiolipin (CL) levels has been postulated to compromise directly the function of several essential enzymes and processes of the mitochondria . There is limited genetic evidence for the critical roles with which CL and its precursor phosphatidylglycerol (PG) have been associated . A null allele of the PGS1 gene from Saccharomyces cerevisiae, which encodes the enzyme responsible for the synthesis of the CL precursor PG phosphate, was created in a yeast strain in which PGS1 expression is exogenously regulated by doxycycline . The addition of increasing concentrations of doxycycline to the growth medium causes a proportional decrease to undetectable levels of PGS1 transcript, PG phosphate synthase activity, and PG plus CL . The doubling time of this strain with increasing doxycycline increases to senescence in non-fermentable carbon sources or at high temperatures, conditions that do not support growth of the pgs1Delta strain . Doxycycline addition also causes mitochondrial abnormalities as observed by fluorescence microscopy . Products of four mitochondrial encoded genes (COX1, COX2, COX3, and COB) and one nuclear encoded gene (COX4) associated with the mitochondrial inner membrane are not present when PGS1 expression is fully repressed . No translation of these proteins can be detected in cells lacking the PGS1 gene product, although transcription and splicing appear unaffected . Protein import of other nuclear encoded proteins remains unaffected . The remaining proteins encoded by mitochondrial DNA are expressed and translated normally . Thus, the molecular basis for the lack of mitochondrial function in pgs1Delta cells is the failure to translate gene products essential to the electron transport chain.

Int Microbiol, 2000 Dec, 3(4), 231 - 4
Extracellular cyanobacterial substances inhibit microbial growth; Heyduck-Soller B et al.; Cyanobacteria are able to produce extracellular substances with different biological activities and behaviors . The marine cyanobacteria Anabaena sp . strain Hi 26 and Oscillatoria subtilissima strain Bo 62 cause significant color changes in their growth media, while viscosity of the medium is influenced by Rivularia sp . strain Bo 85 and Oscillatoria limnetica strain Flo 1 . Sterile-filtered media, pregrown with the organisms mentioned above, were used to study the influence of changes in media bioactivity induced by "excreted substances" on the growth and/or morphological development of five related filamentous cyanobacterial species and on selected heterotrophic microorganisms . Cell lysis, empty sheaths, different lengths of filaments, or even single cells and a decrease in chlorophyll a and protein content were the characteristic changes obtained by such a "cyanobacterial assay." The use of a "precultured" medium, as demonstrated in an "agar diffusion assay," affects in varying degrees the growth of gram-positive and gram-negative heterotrophic bacteria, as well as of the yeast Saccharomyces cerevisiae.

Appl Microbiol Biotechnol, 2001 Mar, 55(2), 164 - 9
Enhanced anthocyanin production by repeated-batch culture of strawberry cells with medium shift; Zhang W et al.; Repeated-batch cultures of strawberry cells (Fragaria ananassa cv . Shikinari) subjected to four medium-shift procedures (constant LS medium, constant B5 medium, alternation between LS and B5 starting from LS and alternation between LS and B5 starting from B5) were investigated for the enhanced anthocyanin productivity . To determine the optimum period for repeated batch cultures, two medium-shift periods of 9 and 14 days were studied, which represent the end of the exponential growth phase and the stationary phase . By comparison with the corresponding batch cultures, higher anthocyanin productivity was achieved for all the repeated-batch cultures at a 9-day medium-shift period . The average anthocyanin productivity was enhanced 1.7- and 1.76-fold by repeated-batch cultures in constant LS and constant B5 medium at a 9-day shift period for 45 days, respectively . No further improvement was observed when the medium was alternated between LS (the growth medium) and B5 (the production medium) . Anthocyanin production was unstable at a 14-day shift period regardless of the medium-shift procedures . The results show that it is feasible to improve anthocyanin production by a repeated-batch culture of strawberry cells.

FEMS Microbiol Lett, 2001 Apr 20, 198(1), 73 - 8
Characterization of FruR as a putative activator of the fructose operon of Spiroplasma citri; Gaurivaud P et al.; The role of fruR, the first gene of the Spiroplasma citri fructose operon, was investigated . In vivo transcription of the fructose operon is greatly enhanced by the presence of fructose in the growth medium while glucose has no effect . When fruR is not expressed, transcription of the fructose operon is not stimulated by fructose, and fructose fermentation is decreased, indicating that FruR is an activator of the fructose operon . The promoter of the fructose operon was localized by primer extension, and a direct T-rich repeat was found to overlap the -35 box . This repeat could be the binding site of FruR . The presence of fructose in the culture medium also decreases the toxicity of methyl alpha-glucoside, however FruR is not involved in this regulation . This is the first description of transcription regulation of a mollicute operon.

Appl Environ Microbiol, 2001 May, 67(5), 2051 - 5
Effect of culture conditions on ergosterol as an indicator of biomass in the aquatic hyphomycetes; Charcosset JY et al.; Ergosterol is a membrane component specific to fungi that can be used to estimate fungal biomass using appropriate factors of conversion . Our objectives were to determine the limits of use of ergosterol content as a measure of biomass for aquatic hyphomycetes, and to evaluate a previously established ergosterol-to-biomass conversion factor . We varied inoculum quality, growth medium, and degree of shaking of four aquatic hyphomycete species . In cultures inoculated with homogenized mycelium, we found a significant effect of shaking condition and culture age on ergosterol content . In liquid cultures with defined medium, ergosterol content reached 10 to 11 microg/mg of mycelium (dry mass) and varied by factors of 2.2 during exponential growth and 1.3 during stationary phase . The increase in ergosterol content during exponential phase could be attributed, at least in part, to rapid depletion of glucose . Oxygen availability to internal hyphae within the mycelial mass is also responsible for the differences found between culture conditions . Ergosterol concentration ranged from 0.8 to 1.6 microg/mg in static cultures inoculated with agar plugs . Ergosterol content varied by a factor of 4 in two media of different richnesses . For different combinations of these parameters, strong (r(2) = 0.83 to 0.98) and highly significant (P << 0.001) linear relationships between ergosterol and mycelial dry mass (up to 110 mg) were observed . Overall, the ergosterol content varied by a factor of 14 (0.8 to 11 mg/g) . These results suggest that care must be taken when the ergosterol content is used to compare data generated in different field environments.

Plant J, 2001 Mar, 25(6), 595 - 604
A potato tuber-expressed mRNA with homology to steroid dehydrogenases affects gibberellin levels and plant development; Bachem CW et al.; Using cDNA-AFLP RNA fingerprinting throughout potato tuber development, we have isolated a transcript-derived fragment (TDF511) with strong homology to plant steroid dehydrogenases . During in vitro tuberization, the abundance profile of the TDF shows close correlation to the process of tuber formation . However, when tuberization is inhibited by the addition of gibberellins (GAs) to the growth medium, the appearance of TDF511 in the fingerprint is delayed, then steadily increases in intensity during later stages of development . TDF511 was used to isolate the corresponding cDNA (CB12) . The DNA and deduced amino-acid sequences of the cDNA show high homology to a fruit-ripening gene from tomato, a series of steroid dehydrogenases, and the maize Ts2 gene . A section of the cDNA was cloned in antisense orientation behind a 35S CaMV promoter and transformed into potato . Transgenic plants expressing the antisense gene showed significantly earlier emergence, an increase in height, and longer tuber shape . In vitro tuberization experiments reveal extended stolon lengths in comparison to the controls . The analysis of endogenous GA levels showed that the transgenic antisense plants have elevated levels of biologically active GAs and their respective precursors . We propose that this gene plays a role in the metabolism of plant-growth substances important for tuber life cycle and plant development.

J Parasitol, 2001 Apr, 87(2), 399 - 405
Effect of jasplakinolide on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens; Makioka A et al.; The effect of jasplakinolide . an actin-polymerizing and filament-stabilizing drug, on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens was examined . Jasplakinolide inhibited the growth of E . histolytica strain HM-1:IMSS and E . invadens strain IP-1 in a concentration-dependent manner, the latter being more resistant to the drug . The inhibitory effect of jasplakinolide on the growth of E . histolytica trophozoites was reversed by removal of the drug after exposure to 1 microM for 1 day . Encystation of E . invadens as induced in vitro was also inhibited by jasplakinolide . Trophozoites exposed to jasplakinolide in encystation medium for 1 day did not encyst after removal of the drug, whereas those exposed to the drug in growth medium for 7 days did encyst without the drug . The process of cyst maturation was unaffected by jasplakinolide . Large round structures were formed in trophozoites of both amoebae grown with jasplakinolide; these were identified as F-actin aggregates by staining with fluorescent phalloidin . Accumulation in trophozoites of both amoebae of actin aggregates was observed after culture in jasplakinolide . Also, E . invadens cysts formed from trophozoites treated with jasplakinolide contained the actin aggregate . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis revealed that the jasplakinolide treatment led to an increase in the proportion of F-actin associated with formation of the aggregate . The results suggest that aggregates are formed from the cortical flow of F-actin filaments, and that these filaments would normally be depolymerized but are artificially stabilized by jasplakinolide binding.

Water Res, 2001 Apr, 35(6), 1592 - 8
Evaluation of the fern Azolla for growth, nitrogen and phosphorus removal from wastewater; Forni C et al.; Water fern (Azolla filiculoides Lam.) has been assessed for nitrogen and phosphorus removal in outdoor experiments comparing sewage water (S) from an experimental aquaculture plant, well water (W) and mineral growth medium Hoagland (H) . The experiments were undertaken during the spring and the summer . The yield of fern biomass and nitrogenase activity was higher in H than in W and S waters . The enzyme activity had a decreasing trend with significant differences (p < 0.05) in the three waters . Peroxidase (POD) activity in April decreased with significative differences in W and S waters (p < 0.05) . The electrical conductivity and the concentrations of NO3- in the three waters decreased significantly (p < 0.05) . The highest removal of nitrate from the media was obtained in July . In S water, NO2- concentration decreased, while it increased in W water . PO(4)3- concentration was very low in W and S waters and decreased in H medium . The results obtained confirm the ability of the fern to grow in sewage water.

Mikrobiologiia, 2000 Sep-Oct, 69(5), 642 - 6
{Effect on microelements on biosynthes of secondary metabolites in the fungus Penicillium citrinum Thom VKM F-1079}; Kozlovskii AG et al.; Penicillium citrinum VKM F-1079 was found to produce clavine ergot alkaloids and citrinin, a secondary O-heterocyclic metabolite . Citrinin was produced in the idiophase, whereas the production of ergot alkaloids paralleled fungal growth . The addition of manganese ions to the growth medium stimulated the biosynthesis of both citrinin and ergot alkaloids . Zinc ions stimulated only citrinin synthesis . The presence of these microelements in the growth medium influenced the proportion between the ergot alkaloids synthesized . Copper, manganese, and iron ions affected but little fungal growth and alkaloid production . The effect of microelements on the main kinetic parameters of growth and alkaloid production was studied.

Biotechniques, 2001 Apr, 30(4), 782 - 4, 786, 788 passim
Specific inhibitors prevent proteolytic degradation of recombinant proteins expressed in High Five cells; Martensen PM et al.; The insect cell line BTI-TN-5B1-4 (High Five) is frequently used to express recombinant proteins in large amounts using the baculovirus expression system . However, extensive proteolytic degradation of recombinant proteins is often encountered . Furthermore, we have observed that recombinant proteins migrate in SDS-PAGE in agreement with poly-ubiquitinated forms of the protein, suggesting a ubiquitin/proteasome degradation pathway . Here, we describe a systematic study unraveling the effect of adding proteasome inhibitors or specific protease inhibitors to the growth medium of High Five insect cells infected with recombinant baculovirus . Furthermore, protease inhibitors were added to the lysis buffer to establish the most efficient way to inhibit proteolytic activity after lysis of baculovirus-infected cells expressing recombinant proteins . We conclude that a combination of adding protease inhibitors to the growth medium and to the lysis buffer minimizes the proteolytic activity in High Five cells . The most efficient protease inhibitors were E-64 in the growth medium together with Leupeptin in the lysis buffer at concentrations higher than with available cocktails of inhibitors . The optimal treatment of High Five cells is different from the optimal treatment of Sf9 cells . For proteins susceptible to ubiquitinylation, a treatment of insect cell cultures with the proteasome inhibitor MG132 (LLL) leads to a considerable reduction of the yield of production of recombinant protein.

Calcif Tissue Int, 2001 Feb, 68(2), 87 - 94
In vitro and in vivo induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene; Cheng SL et al.; It has been well established that bone morphogenetic protein-2 (BMP-2) can induce bone formation both in vivo and in vitro, although high concentrations (up to milligrams) of BMP-2 have been required to achieve this effect in vivo . Further, clinical applications are usually limited to a single dose at the time of implantation . In an attempt to prolong the transforming effect of BMP-2 we used a recombinant adenoviral vector carrying the human BMP-2 gene (Adv-BMP2) to transduce marrow-derived mesenchymal stem cells (MSC) of skeletally mature male New Zealand white rabbits . The pluripotential MSC were incubated with Adv-BMP2 overnight followed by culture in growth medium for 1 week . Assays on tissue cultures demonstrated that these Adv-BMP2 transduced MSC produced BMP-2 protein, differentiated into an osteoprogenitor line, and induced bone formation in vitro . These MSC had increased alkaline phosphatase activity, increased expression of type I collagen, osteopontin, and osteocalcin mRNA, and induced matrix mineralization compared with both non-transduced cells and cells transduced with a control adenoviral construct . To analyze the osteogenic potential in vivo, Adv-BMP2-transduced MSC were autologously implanted into the intertransverse process space between L5 and L6 of the donor rabbits . The production of new bone was demonstrated by radiographic examination 4 weeks later in areas implanted with cells transduced with Adv-BMP2, whereas no bone was evident at sites implanted with cells transduced with the control adenoviral construct . Histological examination further confirmed the presence of new bone formation . These accumulated data indicate that it is possible to successfully transduce mesenchymal stem cells with a recombinant adenoviral vector carrying the gene for BMP-2 such that these cells will produce BMP-2, differentiate into an osteoprogenitor line, and induce bone formation both in vitro and in vivo . Moreover, incubation of the Adv-BMP2-transduced cells for an additional 7 days in culture before transplantation enhances the success rate in bone formation (three out of three) as compared with our previous report (one out of five, Calcif Tissue Int 63:357-360, 1998).

Chemosphere, 2001 Apr, 43(3), 265 - 72
Biosynthesis and release of methylarsenic compounds during the growth of freshwater algae; Hasegawa H et al.; Arsenic transformations by freshwater algae have been studied under laboratory conditions . By the use of a new analytical method, we identified methylarsenic(III) species in the growth medium of green-alga Closterium aciculare incubated under axenic conditions . The arsenate concentration in the experimental medium began to decrease just after inoculation, and the levels of arsenite and methylarsenicals increased with the growth of C . aciculare . Initially, most of the arsenate was converted into arsenite, which peaked in concentration during the exponential phase . Methylarsenicals accumulated rapidly in the stationary phase . DMAA(V) production was enhanced when the ratio of phosphate to arsenate decreased in the culture medium . The levels of DMAA(V) increased continuously toward the end of the experiment . On the other hand, methylarsenic(III) species remained relatively steady during the stationary phase . Methylarsenic(III) species accounted for 0-35% of methylarsenicals . These results suggest that arsenite and methylarsenicals (containing methylarsenic(III) species) are supplied by phytoplankton, and serve as evidence of the origin of methylarsenic(III) species in natural waters.

Plant Physiol, 2001 Apr, 125(4), 1679 - 87
Iron stress-induced changes in root epidermal cell fate are regulated independently from physiological responses to low iron availability; Schikora A et al.; Iron-overaccumulating mutants were investigated with respect to changes in epidermal cell patterning and root reductase activity in response to iron starvation . In all mutants under investigation, ferric chelate reductase activity was up-regulated both in the presence and absence of iron in the growth medium . The induction of transfer cells in the rhizodermis appeared to be iron regulated in the pea (Pisum sativum L . cv Dippes Gelbe Viktoria and cv Sparkle) mutants bronze and degenerated leaflets, but not in roots of the tomato (Lycopersicon esculentum Mill . cv Bonner Beste) mutant chloronerva, suggesting that in chloronerva iron cannot be recognized by putative sensor proteins . Experiments with split-root plants supports the hypothesis that Fe(III) chelate reductase is regulated by a shoot-borne signal molecule, communicating the iron status of the shoot to the roots . In contrast, the formation of transfer cells was dependent on the local concentration of iron, implying that this shoot signal does not affect their formation . Different repression curves of the two responses imply that the induction of transfer cells occurs after the enhancement of electron transfer across the plasma membrane rather than being causally linked . Similar to transfer cells, the formation of extra root hairs in the Arabidopsis mutant man1 was regulated by the iron concentration of the growth medium and was unaffected by interorgan signaling.

Mol Biol Cell, 2001 Apr, 12(4), 997 - 1007
Roles of phosphatidylethanolamine and of its several biosynthetic pathways in Saccharomyces cerevisiae; Birner R et al.; Three different pathways lead to the synthesis of phosphatidylethanolamine (PtdEtn) in yeast, one of which is localized to the inner mitochondrial membrane . To study the contribution of each of these pathways, we constructed a series of deletion mutants in which different combinations of the pathways are blocked . Analysis of their growth phenotypes revealed that a minimal level of PtdEtn is essential for growth . On fermentable carbon sources such as glucose, endogenous ethanolaminephosphate provided by sphingolipid catabolism is sufficient to allow synthesis of the essential amount of PtdEtn through the cytidyldiphosphate (CDP)-ethanolamine pathway . On nonfermentable carbon sources, however, a higher level of PtdEtn is required for growth, and the amounts of PtdEtn produced through the CDP-ethanolamine pathway and by extramitochondrial phosphatidylserine decarboxylase 2 are not sufficient to maintain growth unless the action of the former pathway is enhanced by supplementing the growth medium with ethanolamine . Thus, in the absence of such supplementation, production of PtdEtn by mitochondrial phosphatidylserine decarboxylase 1 becomes essential . In psd1Delta strains or cho1Delta strains (defective in phosphatidylserine synthesis), which contain decreased amounts of PtdEtn, the growth rate on nonfermentable carbon sources correlates with the content of PtdEtn in mitochondria, suggesting that import of PtdEtn into this organelle becomes growth limiting . Although morphological and biochemical analysis revealed no obvious defects of PtdEtn-depleted mitochondria, the mutants exhibited an enhanced formation of respiration-deficient cells . Synthesis of glycosylphosphatidylinositol-anchored proteins is also impaired in PtdEtn-depleted cells, as demonstrated by delayed maturation of Gas1p . Carboxypeptidase Y and invertase, on the other hand, were processed with wild-type kinetics . Thus, PtdEtn depletion does not affect protein secretion in general, suggesting that high levels of nonbilayer-forming lipids such as PtdEtn are not essential for membrane vesicle fusion processes in vivo.

J Dairy Res, 2001 Feb, 68(1), 109 - 16
Influence of growth conditions on heat-stable phospholipase activity in Pseudomonas; Koka RA et al.; Many psychrotrophic bacteria contaminating raw milk produce phospholipase that withstands pasteurization and UHT treatments . This enzyme acts on the milk fat globule membrane and exposes triacylglycerides to the action of lipase . Phospholipase production by various isolates of Pseudomonas was investigated . The isolates were cultured aerobically at 8 degrees C in nutrient broth, McKellar's minimal salts medium, Chrisope's medium, and skim milk . Each strain produced phospholipase during the 50 h incubation . Enzyme production varied significantly (P < 0.001) with strain and growth medium . Strains varied significantly (P < 0.001) in their enzyme production in each medium and during the incubation time as well . Strain, incubation time, and the growth medium significantly influenced (P < 0.001) heat stability of the enzyme activity . Pasteurization reduced the activity, but did not eliminate it in skim milk.

Ann Biomed Eng, 2001 Feb, 29(2), 153 - 63
Compressive deformation and damage of muscle cell subpopulations in a model system; Bouten CV et al.; To study the effects of compressive straining on muscle cell deformation and damage an in vitro model system was developed . Myoblasts were seeded in agarose constructs and cultured in growth medium for 4 days . Subsequently, the cells were allowed to fuse into multinucleated myotubes for 8 days in differentiation medium, resulting in a population of spherical myoblasts (50%), spherical myotubes (35%), and elongated myotubes (15%) with an overall viability of 90% . To evaluate cell deformation upon construct compression half-core shaped constructs were compressed up to 40% strain and the resulting cell shape was assessed from confocal scans through the central plane of spherical cells . The ratio of cell diameters measured parallel and perpendicular to the axis of compression was used as an index of deformation (DI) . The average DI of myoblasts decreased with strain level (0.99+/-0.03, 0.70+/-0.04, and 0.56+/-0.10 at 0%, 20%, and 40% strain), whereas for myotubes DI decreased up to 20% strain and then remained fairly constant (0.99+/-0.06, 0.55+/-0.06, 0.50+/-0.11) . The discrepancy in DI between spherical myoblasts and myotubes at 20% strain was explained by the relative sensitivity of the cell membrane to buckling, which is more pronounced in the myotubes . Sustained compression up to 24 h at 20% strain resulted in a significant increase in cell damage with time as compared to unstrained controls . Despite differences in membrane buckling no difference in damage between myoblasts and spherical myotubes was observed over time, whereas the elongated myotubes were more susceptible to damage.

Yeast, 2001 Apr, 18(6), 511 - 21
The activity of plasma membrane H(+)-ATPase is strongly stimulated during Saccharomyces cerevisiae adaptation to growth under high copper stress, accompanying intracellular acidification; Fernandes AR et al.; For the adaptation of cells of Saccharomyces cerevisiae, a period of latency is necessary before exponential growth is resumed in a medium supplemented with a highly inhibitory concentration of copper . In this work, we have examined some physiological responses occurring during this period of adaptation . The results revealed that plasma membrane H(+)-ATPase (PM-ATPase) activity is strongly stimulated (up to 24-fold) during copper-induced latency in growth medium with glucose, reaching maximal levels when the cells were about to start inhibited exponential growth . This in vivo activation of the ATPase activity by copper was accompanied by the stimulation of the H(+)-pumping activity of the enzyme in vivo and was essentially due to the increase of the apparent V(max) for MgATP . Although the exact molecular basis of the reported plasma membrane ATPase activation was not clarified, no increase in the mRNA levels from the encoding genes PMA1 and PMA2 was apparently detected during copper-induced latency . The physiological response reported here may allow the cells to cope with copper-induced lipid peroxidation and consequent decrease in plasma membrane lipid ordering and increase in the non-specific permeability to protons . The consequences of these copper deleterious effects were revealed by the decrease of the intracellular pH (pH(i)) of the yeast population, from approximately pH(i) 6 to pH(i) 5, during copper-induced latency in growth medium at pH 4.3 . The time-dependent patterns of plasma membrane ATPase activation and of the decrease of pH(i) during the period of adaptation to growth with copper correlate, suggesting that the regulation of this membrane enzyme activity may be triggered by intracellular acidification . Consistent with this idea, when exponential growth under copper stress was resumed and the pH(i) of the yeast population recovered up to physiological values, plasma membrane ATPase activity simultaneously decreased from the highly stimulated level attained during the adaptation period of latency .

Protein Expr Purif, 2001 Apr, 21(3), 500 - 9
Heterologous expression of soluble, active proteins in Escherichia coli: the human estrogen receptor hormone-binding domain as paradigm; Nygaard FB et al.; The human estrogen receptor ligand-binding domain (hER-E/F), including the distal F domain, has been expressed to high levels in a soluble, active form in Escherichia coli in order to facilitate biophysical studies . The ability of a series of vectors incorporating strong transcriptional and translational signals to provide an efficient expression system for hER-E/F was investigated . High-level expression was obtained from all of the vectors used in the study . Although the majority of hER-E/F protein was produced in insoluble form under standard bacterial culture conditions, hER-E/F could be produced in soluble, biologically active form by altering the sequence of the expressed protein and by varying the host culture conditions . Several parameters, including the presence of a His tag, growth temperature, and addition of ethanol and 17beta-estradiol to the growth medium were shown to have a positive effect on production of soluble hER-E/F . An optimized expression system capable of producing from 25 to 35 mg of biologically active hER-E/F in 1 liter of cell culture was designed, and a simple, rapid purification protocol for hER-E/F produced in this system was developed . Characterization of purified hER-E/F by Edman degradation and mass spectrometry verified the identity of the protein . The K(D) for 17beta-estradiol binding to purified hER-E/F was determined to be 0.6 +/- 0.1 nM . The parameters controlling soluble, heterologous protein production observed in this study may be generally applicable to the expression of other heterologous proteins in E . coli .

J Biol Chem, 2001 May 25, 276(21), 18161 - 8 Epub 2001 Mar 09.
A defect in coenzyme Q biosynthesis is responsible for the respiratory deficiency in Saccharomyces cerevisiae abc1 mutants; Do TQ et al.; Ubiquinone (coenzyme Q or Q) is an essential component of the mitochondrial respiratory chain in eukaryotic cells . There are eight complementation groups of Q-deficient Saccharomyces cerevisiae mutants designated coq1-coq8 . Here we report that COQ8 is ABC1 (for Activity of bc(1) complex), which was originally isolated as a multicopy suppressor of a cytochrome b mRNA translation defect (Bousquet, I., Dujardin, G., and Slonimski, P . P . (1991) EMBO J . 10, 2023-2031) . Previous studies of abc1 mutants suggested that the mitochondrial respiratory complexes were thermosensitive and function inefficiently . Although initial characterization of the abc1 mutants revealed characteristics of Q-deficient mutants, levels of Q were reported to be similar to wild type . The suggested function of Abc1p was that it acts as a chaperone-like protein essential for the proper conformation and functioning of the bc(1) and its neighboring complexes (Brasseur, G., Tron, P., Dujardin, G., Slonimski, P . P . (1997) Eur . J . Biochem . 246, 103-111) . Studies presented here indicate that abc1/coq8 null mutants are defective in Q biosynthesis and accumulate 3-hexaprenyl-4-hydroxybenzoic acid as the predominant intermediate . As observed in other yeast coq mutants, supplementation of growth media with Q(6) rescues the abc1/coq8 null mutants for growth on nonfermentable carbon sources . Such supplementation also partially restores succinate-cytochrome c reductase activity in the abc1/coq8 null mutants . Abc1/Coq8p localizes to the mitochondria, and is proteolytically processed upon import . The findings presented here indicate that the previously reported thermosensitivity of the respiratory complexes of abc1/coq8 mutants results from the lack of Q and a general deficiency in respiration, rather than a specific phenotype due to dysfunction of the Abc1 polypeptide . These results indicate that ABC1/COQ8 is essential for Q-biosynthesis and that the critical defect of abc1/coq8 mutants is a lack of Q.

J Biol Chem, 2001 Apr 20, 276(16), 12702 - 11 Epub 2001 Jan 18.
The effect of the erg26-1 mutation on the regulation of lipid metabolism in Saccharomyces cerevisiae; Baudry K et al.; A temperature-sensitive Saccharomyces cerevisiae mutant harboring a lesion in the ERG26 gene has been isolated . ERG26 encodes 4alpha-carboxysterol-C3 dehydrogenase, one of three enzymatic activities required for the conversion of 4,4-dimethylzymosterol to zymosterol . Gas chromatography/mass spectrometry analyses of sterols in this mutant, designated erg26-1, revealed the aberrant accumulation of a 4-methyl-4-carboxy zymosterol intermediate, as well as a novel 4-carboxysterol . Neutral lipid radiolabeling studies showed that erg26-1 cells also harbored defects in the rate of biosynthesis and steady-state levels of mono-, di-, and triglycerides . Phospholipid radiolabeling studies showed defects in the rate of biosynthesis of both phosphatidic acid and phosphatidylinositol . Biochemical studies revealed that microsomes isolated from erg26-1 cells contained greatly reduced 4alpha-carboxysterol-C3 dehydrogenase activity when compared with microsomes from wild type cells . Previous studies have shown that loss of function mutations in either of the fatty acid elongase genes SUR4/ELO3 or FEN1/GNS1/ELO2 can "bypass" the essentiality of certain ERG genes (Ladeveze, V., Marcireau, C., Delourme, D., and Karst, F . (1993) Lipids 28, 907-912; Silve, S., Leplatois, P., Josse, A., Dupuy, P . H., Lanau, C., Kaghad, M., Dhers, C., Picard, C., Rahier, A., Taton, M., Le Fur, G., Caput, D., Ferrara, P., and Loison, G . (1996) Mol . Cell . Biol . 16, 2719-2727) . Studies presented here have shown that this sphingolipid-dependent "bypass" mechanism did not suppress the essential requirement for zymosterol biosynthesis . However, studies aimed at understanding the underlying physiology behind the temperature-sensitive growth defect of erg26-1 cells showed that the addition of several antifungal compounds to the growth media of erg26-1 cells could suppress the temperature-sensitive growth defect . Fluorescence microscopic analysis showed that GFP-Erg26p and GFP-Erg27p fusion proteins were localized to the endoplasmic reticulum . Two-hybrid analysis indicated that Erg25p, Erg26p, and Erg27p, which are required for the biosynthesis of zymosterol, form a complex within the cell.

J Biol Chem, 2001 May 25, 276(21), 18031 - 7 Epub 2001 Feb 23.
Expression analysis of the nrdHIEF operon from Escherichia coli . Conditions that trigger the transcript level in vivo; Monje-Casas F et al.; Escherichia coli has two aerobic ribonucleotide reductases encoded by the nrdAB and nrdHIEF operons . While NrdAB is active during aerobiosis, NrdEF is considered a cryptic enzyme with no obvious function . Here, we present evidence that nrdHIEF expression might be important under certain circumstances . Basal transcript levels were dramatically enhanced (25-75-fold), depending on the growth-phase and the growth-medium composition . Likewise, a large increase of >100-fold in nrdHIEF mRNA was observed in bacteria lacking Trx1 and Grx1, the two main NrdAB reductants . Moreover, nrdHIEF expression was triggered in response to oxidative stress, particularly in mutants missing hydroperoxidase I and alkyl-hydroperoxide reductase activities (69.7-fold) and in cells treated with oxidants (up to 23.4-fold over the enhanced transcript level possessed by cells grown on minimal medium) . The mechanism(s) that triggers nrdHIEF expression remains unknown, but our findings exclude putative global regulators like RpoS, Fis, cAMP, OxyR, SoxR/S, or RecA . What we have learned about nrdHIEF expression indicates strong differences between its regulation and that of the nrdAB operon and of genes coding for components of both thioredoxin/glutaredoxin pathways . We propose that E . coli might optimize the responses to different stimuli by co-evolving the expression levels for its multiple reductases and electron donors.

Eur J Biochem, 2001 Apr, 268(7), 1940 - 52
FeMo cofactor biosynthesis in a nifE- mutant of Rhodobacter capsulatus; Siemann S et al.; In all diazotrophic micro-organisms investigated so far, mutations in nifE, one of the genes involved in the biosynthesis of the FeMo cofactor (FeMoco), resulted in the accumulation of cofactorless inactive dinitrogenase . In this study, we have found that strains of the phototrophic non-sulfur purple bacterium Rhodobacter capsulatus with mutations in nifE, as well as in the operon harbouring the nifE gene, were capable of reducing acetylene and growing diazotrophically, although at distinctly lower rates than the wild-type strain . The diminished rates of substrate reduction were found to correlate with the decreased amounts of the dinitrogenase component (MoFe protein) expressed in R . capsulatus . The in vivo activity, as measured by the routine acetylene-reduction assay, was strictly Mo-dependent . Maximal activity was achieved under diazotrophic growth conditions and by supplementing the growth medium with molybdate (final concentration 20-50 microM) . Moreover, in these strains a high proportion of ethane was produced from acetylene ( approximately 10% of ethylene) in vivo . However, in in vitro measurements with cell-free extracts as well as purified dinitrogenase, ethane production was always found to be less than 1% . The isolation and partial purification of the MoFe protein from the nifE mutant strain by Q-Sepharose chromatography and subsequent analysis by EPR spectroscopy and inductively coupled plasma MS revealed that FeMoco is actually incorporated into the protein (1.7 molecules of FeMoco per tetramer) . On the basis of the results presented here, the role of NifNE in the biosynthetic pathway of the FeMoco demands reconsideration . It is shown for the first time that NifNE is not essential for biosynthesis of the cofactor, although its presence guarantees formation of a higher content of intact FeMoco-containing MoFe protein molecules . The implications of our findings for the biosynthesis of the FeMoco will be discussed.

Toxicol Lett, 2001 Feb 3, 119(1), 71 - 8
5-Formyluracil and its nucleoside derivatives confer toxicity and mutagenicity to mammalian cells by interfering with normal RNA and DNA metabolism; Klungland A et al.; Oxidation of the methyl group of thymine yields 5-(hydroxymethyl)uracil (5-hmU) and 5-formyluracil (5-foU) as major products . Whereas 5-hmU appears to have normal base pairing properties, the biological effects of 5-foU are rather poorly characterised . Here, we show that the colony forming ability of Chinese hamster fibroblast (CHF) cells is greatly reduced by addition of 5-foU, 5-formyluridine (5-foUrd) and 5-formyl-2'-deoxyuridine (5-fodUrd) to the growth medium . There are no toxic effects of 5-fodUrd on cells defective in thymidine kinase or thymidylate synthetase, suggesting that the toxicity may be caused by 5-fodUrd phosphorylation and subsequent inhibition of thymidylate synthetase . Whereas 5-fodUrd was the most effective 5-foU derivative causing cell growth inhibition, the corresponding ribonucleoside 5-foUrd was more effective in inhibiting {3H}uridine incorporation in non-dividing rat nerve cells in culture, suggesting that 5-foUrd exerts its toxicity through interference with RNA rather than DNA synthesis . Addition of 5-foU and 5-fodUrd was also found to promote mutagenicity at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus of CHF cells; 5-fodUrd being three orders of magnitude more potent than 5-foU . In contrast, neither 5-hmU nor 5-(hydroxymethyl)-2'-deoxyuridine induced HPRT mutations . The mutation induction indicates that 5-foU will be incorporated into DNA and has base pairing properties different from that of thymine . These results suggest that 5-foU residues, originating from incorporation of oxidised bases, nucleosides or nucleotides or by oxidation of DNA, may contribute significantly to the damaging effects of oxygen radical species in mammalian cells.

Br J Radiol, 2000 Oct, 73(874), 1034 - 41
Effects of radiographic contrast media on proliferation and apoptosis of human vascular endothelial cells; Zhang H et al.; The aim of the study was to determine the effects of radiographic contrast media (RCM) on proliferation and apoptosis of human vascular endothelial cells . Human umbilical vein endothelial cells (HUVECs) were exposed for either 1 min or 15 min to RCM (diatrizoate, ioxaglate, iopromide, iotrolan) at an iodine concentration of 250 mgl ml-1 . Controls were complete growth medium (CGM) and saturated mannitol (osmotic control) . {3H}thymidine incorporation was used to determine cell proliferation 24 h after exposure . Apoptosis was determined at 1 h and 6 h by terminal uridine nick end labelling (TUNEL), time lapse video microscopy (TLVM) and DNA electrophoresis . Mean proliferation rates (%) (+/- SEM) (p-values compared with the CGM control) at 1 min and 15 min, respectively, were: diatrizoate: 31.9 (10.6), 5.8 (1.5) (p < 0.001); ioxaglate: 48.4 (10.9), 20.4 (4.5) (p < 0.001); iopromide: 63.4 (8.7), 58.2 (10.2) (p < 0.05); iotrolan: 84.7 (7.3), 72.8 (12.4) (p = ns); saturated mannitol 50.5 (9.6), 45.9 (10.0) (p < 0.001) . Mean apoptotic indices (%) (+/- SEM) at 1 h and 6 h following 1 min exposure, respectively, were: CGM: 0.25 (0.13), 0.23 (0.08); diatrizoate: 2.18 (0.19), 2.69 (0.34) (p < 0.001); ioxaglate: 1.90 (0.23), 1.69 (0.02) (p < 0.05); iopromide: 0.59 (0.04), 0.33 (0.02) (p = ns); iotrolan: 0.30 (0.07), 0.27 (0.1) (p = ns); saturated mannitol 2.11 (0.24), 1.4 (0.1) (p < 0.05) . After 15 min exposure, apoptosis rates at both 1 h and 6 h, respectively, were: iotrolan: 0.29 (0.17), 0.51 (0.16) (p = ns); diatrizoate: 3.19 (0.81), 11.66 (1.75) (p < 0.001); ioxaglate: 1.88 (0.14), 2.87 (0.20) (p < 0.05); iopromide: 1.06 (0.11), 1.52 (0.15) (p < 0.05); saturated mannitol 1.62 (0.09), 4.63 (0.74) (p < 0.05) . TLVM and DNA electrophoresis confirmed the occurence of apoptosis after exposure to RCM . In conclusion, saturated mannitol and all tested RCM, with the exception of iotrolan, (diatrizoate > ioxaglate > iopromide) reduced proliferation and increased apoptosis of HUVECs . The effects were more pronounced with ionic RCM and seem to depend on osmolality as well as the chemical structure of these agents . Endothelial injury and apoptosis may be responsible for some of the side effects associated with intravascular use of RCM.

Electrophoresis, 2000 Nov, 21(17), 3823 - 32
Identification of nolR-regulated proteins in Sinorhizobium meliloti using proteome analysis; Chen H et al.; Extractable proteins from Sinorhizobium meliloti strains AK631 and EK698 (a Tn5-induced noIR-deficient mutant of AK631), grown in tryptone agar (TA) medium with or without the addition of the plant signal luteolin, were separated by two-dimensional gel electrophoresis and compared . Analysis of silver-stained gels showed that the noIR mutant had 189 proteins that were significantly altered in their levels (101 protein spots up- and 88 downregulated) . Coomassie-stained preparative two-dimensional (2-D) gels or polyvinylidene difluoride (PVDF) membranes blotted from preparative gels showed that at least 52 of the altered proteins could be reproducibly detected and isolated from the noIR mutant . These 52 altered protein spots were classified into five groups based on an assessment of protein abundance by computer analysis and the effect of the presence or absence of luteolin addition to the growth medium . N-terminal microsequencing of 38 proteins revealed that the most striking feature of the consequence of the noIR mutation is the number and broad spectrum of cellular functions that are affected by the loss of the NoIR function . These include proteins involved in the tricarboxylic acid (TCA) cycle, heat shock and cold shock proteins, protein synthesis, a translation elongation factor, oxidative stress and cell growth and maintenance functions . We propose that the NoIR repressor is a global regulatory protein which responds to environmental factors to fine-tune intracellular metabolism.

Water Res, 2001 Apr, 35(5), 1209 - 18
Environmental and nutritional factors affecting geosmin synthesis by Anabaena sp; Saadoun IM et al.; A cyanobacterium isolated from a source-water reservoir during a spring odor and taste episode and identified as Anabaena sp . consistently produced geosmin during laboratory culture on modified BG-11 liquid medium . Maximal geosmin/biomass occurred at 20 degrees C and a light intensity of 17 microE/m2/s; geosmin/chla values directly correlated with increasing light intensity (r2 = 0.95, P < 0.01) . It was concluded that at 20 degrees C, increasing light intensity favors less chla synthesis and higher geosmin synthesis; at 17 microE/m2/s, increasing temperature stimulates chla production (to 25 degrees C) while repressing geosmin synthesis (above 20 degrees C) . Nutritional factors promoting biomass, chla, and geosmin synthesis by Anabaena sp . were also investigated . For cultures grown at 17 microE/m2/s and 20 degrees C for 20 days, both ammonium-N and nitrate-N generally enhanced the growth of Anabaena sp . Nitrate-N promoted more chla production (r2 = 0.99) than ammonium-N . Geosmin synthesis was directly correlated with ammonium-N concentrations (r2 = 0.89), with low nitrate-N (123.5 micrograms/l) favoring maximal geosmin production (2.8 micrograms/l) . Increasing nitrate-N concentrations promoted a three-fold increase in chla content with geosmin synthesis decreased by two-fold . Geosmin/mg biomass was directly related to ammonium-N concentration; high nitrate-N levels suppressed geosmin production . No geosmin was detected at or below 118 micrograms phosphate-phosphorus/l . Geosmin, dry weight biomass, and chla production were correlated with increasing phosphorus (P) concentration (r2 = 0.76, 0.96 and 0.98, respectively) . No geosmin was detected when copper was present in growth media at or above 6.92 micrograms Cu2+/l (CuSO4.5H2O) . Dry weight biomass and chla production were negatively correlated with Cu2+ ion concentrations.

Bioresour Technol, 2001 May, 78(1), 103 - 6
Composted grape marc as growing medium for hypostases (Hypostases phyllostagya); Baran A et al.; The use of composted grape marc (CGM) as a plant growth medium was investigated with Hypostases (Hypostases phyllostagya) . Seven media were prepared using CGM mixed, in different ratios, with native peat and perlite . The following mixtures were used: 100% CGM, 75% CGM + 25% peat, 50% CGM + 50% perlite, 25% CGM + 75% peat, 50% CGM + 25%) peat + 25% perlite, 25%, CGM + 50%, peat + 25% perlite and 100% peat . The experiment was arranged in a randomized plot design with four replicates under greenhouse conditions . After a growing period of three months, some horticultural parameters were measured . Besides, some physical and chemical properties of the growing medium were determined . The mixtures of 50% CGM + 50% peat, 25% CGM + 75% peat and 100% peat were found to be most suitable based on the horticultural parameters . This was confirmed through the physical characteristics . Up to 50% composted grape marc can be used in mixtures with peat on account of its low cost and high nutrient content.

Pac Symp Biocomput . 2001;:471-82.
Nutrient-related analysis of pathway/genome databases; Romero PR et al.; We present an algorithm that solves two related problems in the analysis of metabolic networks stored within a pathway/genome database . (1) The Forward Propagation Problem: given a set of nutrients that are inputs to the metabolic network, what compounds will be produced by the metabolic network? (2) The Backtracking Problem: given the results of a forward propagation, and given a set of essential compounds that are not produced as a result of the forward propagation, what precursors must be supplied to produce those essential compounds? A program based on this algorithm is applied to the EcoCyc database, which is a pathway/genome database for E . coli that consists of annotated genomes and the metabolic reactions and pathways associated with the known gene products . The inputs to the program are a description of the metabolic network of an organism (EcoCyc), a set of nutrients corresponding to a known minimal growth medium, and a list of essential compounds to be produced . The program "fires" the microorganism's metabolism contained in the database and predicts all synthesized and nonsynthesized essential compounds, along with the missing precursors required to produce the latter . When applied to the EcoCyc database, the program identifies a number of missing precursors that indicate incomplete regions of the database . Thus the program results can be used to evaluate existing pathway databases like EcoCyc.

Phytochemistry, 2001 Mar, 56(5), 417 - 21
Biotransformation of cedrol by Curvularia lunata ATCC 12017; Collins DO et al.; Bioconversion of cedrol (1) with the Curvularia lunata was investigated in two different growth media . Five products were obtained in potato dextrose broth, whereas nine compounds were produced in a medium containing beef extract . Only three of the metabolites: 3beta-hydroxycedrol (2), 3alpha-hydroxycedrol (3) and 12-hydroxycedrol (4) were common to both media . They were also obtained as the major products in each case . Three new derivatives have also been identified.

J Urol, 2001 Apr, 165(4), 1319 - 24
Insulin-like growth factor binding protein-3 mediates 1 alpha,25-dihydroxyvitamin d(3) growth inhibition in the LNCaP prostate cancer cell line through p21/WAF1; Boyle BJ et al.; PURPOSE: We determined that insulin-like growth factor binding protein 3 (IGFBP-3) induction by 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) is a necessary component of 1,25-(OH)(2)D(3) mediated growth inhibition of the LNCaP human prostate cancer cell line . In addition, induction of the cyclin dependent kinase inhibitory protein p21/WAF/CIP1 by 1,25-(OH)(2)D(3) is mediated by IGFBP-3 . MATERIALS AND METHODS: Induction of IGFBP-3 by 1,25-(OH)(2)D(3) was determined by enzyme-linked immunosorbent assay for IGFBP-3 protein and by Northern blot analysis for IGFBP-3 messenger (m) RNA . Growth assays for LNCaP cells were determined by measuring DNA content . The contribution of IGFBP-3 toward 1,25-(OH)(2)D(3) mediated growth inhibition was determined by adding either antisense oligonucleotides or immuno-neutralizing antibodies to culture media of growth assays . Regulation of p21/WAF/CIP1 was determined by Western blot analysis . RESULTS: Adding 1,25-(OH)(2)D(3) to LNCaP prostate cancer cells demonstrated that 1,25-(OH)(2)D(3) significantly up-regulated IGFBP-3 at the mRNA and protein levels in these cells approximately 3-fold over control levels . Also, adding IGFBP-3 protein to LNCaP cell growth medium inhibited LNCaP cell growth . Interestingly adding IGFBP-3 antisense oligonucleotides or antibodies directed toward IGFBP-3 abolished the growth inhibitory actions of 1,25-(OH)(2)D(3), indicating that this effect is IGFBP-3 dependent . Furthermore, to connect the mechanisms of IGFBP-3 and 1,25-(OH)(2)D(3) mediated growth inhibition we demonstrated that IGFBP-3 up-regulates the expression of p21/WAF1 protein to approximately 2-fold over the control level . Adding an IGFBP-3 immuno-neutralizing antibody completely prevented the 1,25-(OH)(2)D(3) induced up-regulation of p21/WAF1 . CONCLUSIONS: 1,25-(OH)(2)D(3) up-regulates IGFBP-3 in the LNCaP cell line at the mRNA and protein levels . The growth inhibitory action of 1,25-(OH)(2)D(3) on LNCaP cells depends on active IGFBP-3, as evidenced by the loss of growth inhibition induced by IGFBP-3 antisense oligonucleotide and immuno-neutralization experiments . A possible connection between IGFBP-3 and 1,25-(OH)(2)D(3) lies in the cyclin dependent kinase inhibitory protein p21/WAF1 since IGFBP-3 and 1,25-(OH)(2)D(3) each up-regulate this protein and both inhibit LNCaP cell growth . Therefore, we hypothesize that the mechanism of action by which IGFBP-3 and 1,25-(OH)(2)D(3) induce growth inhibition is the induction of p21/WAF1 because IGFPB-3 immuno-neutralizing antibodies completely abrogate the 1,25-(OH)(2)D(3) mediated up-regulation of p21/WAF1 and growth inhibition.

Enzyme Microb Technol, 2001 Mar 8, 28(4-5), 415 - 419
The potential of positively-charged cellulose sponge for malolactic fermentation of wine, using Oenococcus oeni; Maicas S et al.; Malolactic fermentation (MLF) is a secondary bioconversion developed in some wines involving malic acid decarboxylation . The induction of MLF in wine by cultures of free and immobilized Oenococcus oeni cells was investigated . This work reports on the effect of surface charges in the immobilization material, a recently described fibrous sponge, as well as the pH and the composition of the media where cells are suspended . A chemical treatment provided positive charge to the sponges (DE or DEAE) and gave the highest cell loadings and subsequent resistance to removal . Preculture media to grow the malolactic bacteria before the immobilization procedure were also evaluated . We have established favorable conditions for growth (Medium of Preculture), suspension solution (Tartrate-Phosphate Buffer), suspension pH (3.5-5.5) and immobilization matrix (DE or DEAE cellulose sponge) to induce MLF in red wine . The use of a semi-continuous system permitted a high-efficiency malic acid conversion by 2 x 10(9) cfu sponge(-)(1) in at least four subsequent batch fermentations.

Environ Res, 2001 Mar, 85(3), 246 - 55
Preliminary description of antigenic components characteristic of Stachybotrys chartarum; Raunio P et al.; The objective of this study was to characterize preliminarily immunogenic components characteristic of Stachybotrys chartarum to be used later for the development of a detection method for the fungus in environmental samples . The procedure for S.chartarum extract preparation was first optimized related to the age of the culture, culture type, and growth medium, and the antigenic composition of S . chartarum cultured in two different media was then characterized by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting method . Cross-reactivity of S . chartarum antigenic components with 10 other fungal species was identified by the inhibition immunoblotting method . The 10-day-old S . chartarum culture extract cultured in malt extract broth revealed a wider selection of proteins and antigenic components than the 30-day-old culture extract or the culture medium extracts . When cultured in cellulose broth, S . chartarum produced a higher number of proteins and antigenic components than in malt extract broth . The most dominant immunogenic components of S . chartarum cultured in cellulose broth were those of 65, 50, 37, and 27 kDa . The components of 65 and 50 kDa proved to be the most characteristic of this fungus according to the inhibition immunoblotting analyses . Several of the S . chartarum components were identified as glycoproteins . Carbohydrate moieties of the S . chartarum components also possessed an antibody binding activity.

Fresenius J Anal Chem, 2000 Feb, 366(3), 273 - 82
Determination of trace elements in marine plankton by inductively coupled plasma mass spectrometry (ICP-MS); Arslan Z et al.; A method has been developed for the determination of 23 elements in marine plankton in which inductively coupled plasma (ICP) source mass spectrometry (MS) was used to quantify the elements in the solution after digestion in a mixture of hydrofluoric and nitric acids in sealed PTFE vessels in a microwave field . The procedure was validated by the analysis of a standard reference soil (SRM 2709 San Joaquin Soil) and a standard reference fresh water plankton (CRM 414) . The method was applied to the analysis of several marine plankton samples grown under controlled conditions including several whose growth media had been enriched with selenium . Matrix induced signal suppressions and instrumental drift were corrected by internal standardization . The suitabilities of germanium, indium, rhodium, scandium and yttrium as internal standard elements were evaluated . Neither scandium nor yttrium could be used due to the presence of these elements in the samples, germanium was used for the determination of As, Co, Cu, Fe, Ni, Se, Si and Zn, indium was used for Al, Ba, Ca, Eu, Sr, and Tl, and rhodium was used for Cd, Cr, Hg, Mg, Pb, Sb, Sn, and V . For Al, Ca, Cr, Cu, Fe, Mg, Mn, Ni, Si, Sr, V, and Zn internal standardization did not completely compensate for the suppressive effect of the heavier elements and the solutions were diluted . However, for As, Ba, Cd, Co, Eu, Hg, Pb, Sb, Se, Sn and T1, it was possible to obtain accurate results despite the 35-40% suppression in the signals . Isobaric overlap was only a problem in the cases of 42Ca and 78Se; 44Ca and 77Se, respectively, were used . Memory effects were only observed with Hg for which a nitric acid-sodium chloride solution was the most effective wash-out solution . The marine plankton samples were able to tolerate a higher concentration of Hg as the selenium concentration increased.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Sep-Oct, (5), 82 - 4
{The role of oleic acid in the process of induced destruction of Escherichia coli M-17}; Akaizin ES; The influence of oleic acid on the process of the destruction of E . coli, induced by the transfer of the bacteria from the growth medium into solution without nutrient substances and their treatment with oleic acid at a temperature of 45 degrees C, was studied . Oleic acid at a concentration of 200 nmol/ml accelerated the destruction of E . coli in comparison with the control organisms, but did not lead to the significant increase of the final degree of the destruction of biomass . The dynamics of the inclusion of labeled oleic acid into E . coli cell fractions after the induction of autolysis was analyzed . The radioactive label was most actively included into the lipid (chloroform) fraction of bacterial biopolymers . Simultaneously with the destruction of some cells in the population the inclusion of labeled oleic acid into intact cells was found to occur.

FEBS Lett, 2001 Feb 16, 490(3), 171 - 8
Regulation of MyoD function in the dividing myoblast; Wei Q et al.; Proliferating myoblasts express MyoD, yet no phenotypic markers are activated as long as mitogen levels are sufficient to keep the cells dividing . Depending upon mitogen levels, a decision is made in G1 that commits the myoblast to either continue to divide or to exit from the cell cycle and activate terminal differentiation . Ectopic expression of MyoD under the control of the RSV or CMV promoters causes 10T1/2 cells to rapidly exit the cell cycle and differentiate as single myocytes, even in growth medium, whereas expression of MyoD under the weaker SV40 promoter is compatible with proliferation . Co-expression of MyoD and cyclin D1, but not cyclins A, B, E or D3, blocks transactivation of a MyoD responsive reporter . Similarly, transfection of myoblasts with the cyclin-dependent kinase (cdk) inhibitors p16 and p21 supports some muscle-specific gene expression even in growth medium . Taken altogether, these results suggest cell cycle progression negatively regulates myocyte differentiation, possibly through a mechanism involving the D1 responsive cdks . We review evidence coupling growth status, the cell cycle and myogenesis . We describe a novel mitogen-sensitive mechanism that involves the cyclin D1-dependent direct interaction between the G1 cdks and MyoD in the dividing myoblast, which regulates MyoD function in a mitogen-sensitive manner.

J Bacteriol, 2001 Mar, 183(6), 2006 - 12
SecG function and phospholipid metabolism in Escherichia coli; Flower AM; SecG is an auxiliary protein in the Sec-dependent protein export pathway of Escherichia coli . Although the precise function of SecG is unknown, it stimulates translocation activity and has been postulated to enhance the membrane insertion-deinsertion cycle of SecA . Deletion of secG was initially reported to result in a severe export defect and cold sensitivity . Later results demonstrated that both of these phenotypes were strain dependent, and it was proposed that an additional mutation was required for manifestation of the cold-sensitive phenotype . The results presented here demonstrate that the cold-sensitive secG deletion strain also contains a mutation in glpR that causes constitutive expression of the glp regulon . Introduction of both the glpR mutation and the secG deletion into a wild-type strain background produced a cold-sensitive phenotype, confirming the hypothesis that a second mutation (glpR) contributes to the cold-sensitive phenotype of secG deletion strains . It was speculated that the glpR mutation causes an intracellular depletion of glycerol-3-phosphate due to constitutive synthesis of GlpD and subsequent channeling of glycerol-3-phosphate into metabolic pathways . In support of this hypothesis, it was demonstrated that addition of glycerol-3-phosphate to the growth medium ameliorated the cold sensitivity, as did introduction of a glpD mutation . This depletion of glycerol-3-phosphate is predicted to limit phospholipid biosynthesis, causing an imbalance in the levels of membrane phospholipids . It is hypothesized that this state of phospholipid imbalance imparts a dependence on SecG for proper function or stabilization of the translocation apparatus.

J Environ Pathol Toxicol Oncol, 2001, 20(1), 1 - 7
Stress-inducible DNA repair in Saccharomyces cerevisiae; Verma NC et al.; Saccharomyces cerevisiae shows altered radiation response under various stress conditions, such as nutrition depletion, nitrogen starvation, osmotic shock, heat shock, and mild chemical treatments . In general, the cells show higher levels of UV or gamma radiation resistance under the stress . However, not all the stress conditions affect the repair system in the same manner . For example, depletion of nitrogen supply in the growth medium has been shown to enhance the repair of gamma ray-induced DNA damage without significantly affecting the UV response of the cells . On the other hand, a mild treatment with alkali or hydrogen peroxide improves the response to UV light but not to gamma radiation . It has further been shown that the effect of these stresses are not additive, e.g., the alkali and hydrogen peroxide treatments given simultaneously show the same effect as either of them alone . Low levels of gamma and UV radiation exposures are also treated as stress in the present context . Studies show that irradiation of low-dose gamma rays results in enhanced excision repair of UV-induced pyrimidine dimers . However, in all the wild-type strains tested, none showed any effect on gamma rays response . The exposure to low doses of UV light did not show any effect on either the gamma rays or the UV response . It is suggested that the stress-induced enhancement of DNA repair can be of two types: 1) A general response to stress, which prepares the organism to survive in adverse circumstances (some of the proteins produced during this response also take part in the DNA repair), and 2) a particular response involving DNA damage, such as that caused by gamma irradiation . In this case, the DNA damage may act as a signal for enhancement of the DNA repair.

Mol Gen Genet, 2001 Jan, 264(5), 604 - 12
Molecular control of copper homeostasis in filamentous fungi: increased expression of a metallothionein gene during aging of Podospora anserina; Averbeck NB et al.; The lifespan of the ascomycete Podospora anserina was previously demonstrated to be significantly increased in a copper-uptake mutant, suggesting that copper is a potential stressor involved in degenerative processes . In order to determine whether changes in copper stress occur in the cells during normal aging of cultures, we cloned and characterized a gene coding for a component of the molecular machinery involved in the control of copper homeostasis . This gene, PaMt1, is a single-copy gene that encodes a metallothionein of 26 amino acids . The coding sequence of PaMt1 is interrupted by a single intron . The deduced amino acid sequence shows a high degree of sequence identity to metallothioneins of the filamentous ascomycete Neurospora crassa and the basidiomycete Agaricus bisporus, and to the N-terminal portion of mammalian metallothioneins . Levels of PaMt1 transcript increase in response to elevated amounts of copper in the growth medium and during aging of wild-type cultures . In contrast, in the long-lived mutant grisea, transcript levels first increase but then decrease again . The ability of wild-type cultures to respond to exogenous copper stress via the induction of PaMt1 transcription is not affected as they grow older.

Tsitologiia, 2000, 42(11), 1033 - 6
{Ultrastructure of bacteroid-containing tissue of Pisum sativum L . peas having various regulatory mechanisms of nodulation}; Bolobolova EU et al.; The infected root nodule cells of Pisum sativum cvs . Torsdag, Rondo and its supernodulating mutant nod3 have been investigated by transmission electron microscopy and morphometrically . Torsdag and nod3 developed effective nodules, when grown with or without nitrates in the growth medium . The nodules developed by Rondo were ineffective in the presence of nitrates, and otherwise effective . An obvious similarity in the fine structure of bacteroid tissue of root nodules has been observed in Torgsdag (Nod5) and the supernodulating mutant nod3, both forms being nitrate-tolerant, but nodulation being controlled by different genetic systems . The statistical processing results showed significant differences in the respective morphometric parameters of nodule cells between the plants grown according to either scheme: with and without nitrates . Combined nitrogen is likely to affect the ratio of symbionts in the infected nodule cells of cultivars with nitrate-tolerant nodulation.

Plant Mol Biol, 2000 Dec, 44(6), 759 - 75
Methods of double-stranded RNA-mediated gene inactivation in Arabidopsis and their use to define an essential gene in methionine biosynthesis; Levin JZ et al.; Controlled down-regulation of endogenous plant gene expression is a useful tool, but antisense and sense silencing lack predictability . Recent studies show that expression of both antisense and sense RNA together is an effective means of inactivating reporter and viral genes in plants . We created transgenic plants expressing antisense and sense RNA together in a single 'double-stranded RNA' (dsRNA) transcript . This approach shows great promise as a highly effective means for reducing gene function . With this approach, we demonstrated that the Arabidopsis cystathionine beta-lyase gene, which encodes a methionine biosynthetic enzyme, is essential for viability . Inactivation of this gene was rescued by the addition of methionine to the growth medium . Compared to antisense and sense constructs, the dsRNA construct showed a much more consistent and complete suppression of gene activity . Additionally, expression of a transcript with a spacer sequence containing an unrelated gene between antisense and sense luciferase gene fragments led to stronger inactivation of a second luciferase transgene than did constructs with a minimal spacing between sense and antisense fragments . However, the gene in the spacer region was neither functionally expressed nor functional in silencing a second, unlinked homologous transgene.

Environ Microbiol, 2000 Jun, 2(3), 310 - 8
Cultivatable microbial biodiversity: gnawing at the Gordian knot; Tindall BJ et al.; Rapid and inexpensive sorting of bacterial isolates may be achieved using Fourier transform infrared spectroscopy (FT-IR), a method that has hitherto been applied to identification and classification . The comprehensive characterization of environmental samples requires the isolation of large numbers of isolates using different growth media and growth conditions . In such cases, sorting the isolates is critical before isolates are subjected to more detailed studies . Using FT-IR, isolates are grown under standardized conditions, and 100 strains can be tested within less than 8 h . Chemotaxonomic and molecular characterization of members of clusters emerging from FT-IR analysis either at a level of spectral distance values below 20-30 (analysis of region 600-800 cm(-1), average linkage algorithm) or at spectral heterogeneity values below 75 (regions 1,200-900, 3,000-2,798 and 901-698, scaling to first region, Ward's algorithm) reveals great similarities in fatty acids and 16S rDNA sequences . As judged from riboprinting analyses and fatty acid analyses, FT-IR analysis is able to unravel intraspecific subclustering . The example used in this study of 100 isolates from a mat system, Lake Fryxell, Dry Valleys, Antarctica, selected from a larger number of isolates, picked mainly on the basis of colony pigmentation and form, reveals the utility of the method for identifying the number of putative species quickly . The method described is able to select strains rapidly that represent clusters at the specific and intraspecific level for subsequent characterization.

Environ Microbiol, 2000 Jun, 2(3), 266 - 73
N2-dependent growth and nitrogenase activity in the metal-metabolizing bacteria, Geobacter and Magnetospirillum species; Bazylinski DA et al.; Cells of Geobacter metallireducens, Magnetospirillum strain AMB-1, Magnetospirillum magnetotacticum and Magnetospirillum gryphiswaldense showed N2-dependent growth, the first anaerobically with Fe(III) as the electron acceptor, and the latter three species microaerobically in semi-solid oxygen gradient cultures . Cells of the Magnetospirillum species grown with N2 under microaerobic conditions were magnetotactic and therefore produced magnetosomes . Cells of Geobacter metallireducens reduced acetylene to ethylene (11.5+/-5.9 nmol C2H4 produced min(-1) mg(-1) cell protein) while growing with Fe(III) as the electron acceptor in anaerobic growth medium lacking a fixed nitrogen source . Cells of the Magnetospirillum species, grown in a semi-solid oxygen gradient medium, also reduced acetylene at comparable rates . Uncut chromosomal and fragments from endonuclease-digested chromosomal DNA from these species, as well as Geobacter sulphurreducens organisms, hybridized with a nifHDK probe from Rhodospirillum rubrum, indicating the presence of these nitrogenase structural genes in these organisms . The evidence presented here shows that members of the metal-metabolizing genera, Geobacter and Magnetospirillum, fix atmospheric dinitrogen.

J Basic Microbiol, 2000, 40(5-6), 295 - 301
Evidence for high affinity nickel transporter genes in heavy metal resistant Streptomyces spec; Amoroso MJ et al.; We have isolated 25 new strains of streptomycetes from soil samples of a polluted site at the former uranium mine, Wismut, in eastern Thuringia, Germany . The strains grew on medium containing 1 mM NiCl2 and thus were resistant to the heavy metal ion . Seven of the strains were further characterized . All of these strains were resistant to heavy metals in various degrees with up to 10 mM resistance against NiCl2 supplied with the liquid minimal growth medium . The high level of resistance prompted us to look for high affinity nickel transporter genes thought to provide a means to eliminate the excess nickel ions form the cells . Degenerate oligonucleotide primers derived from sequences of P-type ATPase transporter genes of Gram negative bacteria identified a fragment which shows deduced amino acid sequence similarities to known high affinity nickel transporters . Investigation of two genes obtained from the isolates Streptomyces spec . E8 and F4 showed high sequence divergence . This was unexpected since a transmissible plasmid had been thought to convey heavy metal resistance.

J Mol Med, 2000, 78(10), 569 - 74
Elevated p21 mRNA level in skeletal muscle of DMD patients and mdx mice indicates either an exhausted satellite cell pool or a higher p21 expression in dystrophin-deficient cells per se; Endesfelder S et al.; Abnormalities in proliferation and differentiation of the dystrophin-deficient muscle are a controversial aspect of the pathogenesis of Duchenne muscular dystrophy (DMD) . Analyses of molecules involved in cell cycle modulation do not exist in this context . Cells withdrawn from the cell cycle permanently express p21 . The fact that p2 1, in contrast to other cell cycle proteins, is not diminished when myotubes are reexposed to growth media, allocates this cyclin-dependent kinase inhibitor a special function . Here we report for the first time statistically increased p21 mRNA levels in dystrophin-deficient muscle tissue . Only 42% of conventional RT-PCRs from six muscle samples of human controls yielded positive results but almost all skeletal muscle biopsy samples (87%) from DMD patients (n=5) . For p21 mRNA quantification in murine muscle samples we were able to use the exact real-time TaqMan PCR method due to generally higher p21 mRNA levels than in human muscles . In addition, contamination with fibroblasts can be excluded for the murine samples because they do not demonstrate fibrosis at the age of 350 days but start to lose their regenerative capacity . In accord with the results in humans, we observed p21 mRNA levels in mdx mice that were approx . four times as high as those in control mice . Elevated p21 mRNA level may indicate a shift in cell composition towards differentiated p21 expressing cells as a result of an exhausted pool of undifferentiated, non-p21-expressing satellite cells due to previous cycles of de- and regeneration . Alternatively, dystrophin-deficient cells per se may express higher p21 levels for unknown reasons . Although we cannot distinguish between these possibilities, the eventual transfec tion of a patient's own satellite cells with p21 antisense oligonucleotides may enable the dystrophic process to be influenced.

Int J Artif Organs, 2000 Dec, 23(12), 817 - 23
Optimized growth conditions for tissue engineering of human cardiovascular structures; Hoerstrup SP et al.; Optimized in vitro formation of strong tissue is a prerequisite for tissue engineering of cardiovascular structures, such as heart valves and blood vessels . This study evaluates different growth media additives as to cell proliferation, extracellular matrix formation, and mechanical characteristics . Biodegradable polymers were seeded with human vascular myofibroblasts . Group A was cultured with standard medium, groups B, C, and D were in addition supplemented with ascorbate, fibroblast growth factor (bFGF), and both respectively . Analysis included histology, electron microsocopy, mechanical testing, and biochemical assays for cell proliferation (DNA) and extracellular matrix (collagen) . DNA content increased in all groups, showing significantly more cells in group C and D after 14d . Collagen increased in all groups, except for C . Morphology showed viable, layered cellular tissue, with collagen fibrils after 2w, most pronounced in B and D . Mechanical properties decreased initially, stabilizing after 2w . In conclusion, standard nutrient media were efficient for seeded human vascular cells cultured on biodegradable meshes . Supplementation with bFGF+ascorbate resulted in enhanced early cell proliferation and structurally more mature tissue formation.

Inorg Chem, 2000 Jun 12, 39(12), 2631 - 4
New calcium germanium nitrides: Ca2GeN2, Ca4GeN4, and Ca5Ge2N6; Clarke SJ et al.; We report three new calcium germanium nitrides synthesized as crystals from the elements in sealed niobium tubes at 760 degrees C using liquid sodium as a growth medium . Black Ca2GeN2 is isostructural with the previously reported strontium analogue . It is tetragonal P4(2)/mbc (no . 135) with a = 11.2004(8) A, c = 5.0482(6) A, and Z = 8 . It contains GeN2(4-) units which have 18 valence electrons, and consequently are bent, like the isoelectronic molecule SO2 . In contrast, clear, orange Ca4GeN4 with fully oxidized germanium contains isolated GeN4(8-) tetrahedra and is monoclinic P2(1)/c (no . 14) with a = 9.2823(8) A, b = 6.0429(5) A, c = 11.1612(9) A, beta = 116.498(6) degrees, and Z = 4 . Clear, colorless Ca5Ge2N6, also with fully oxidized germanium, contains infinite chains, 1 infinity{GeN2N2/2(5-)}, of corner-sharing tetrahedra similar to those found in pyroxenes . However, the precise structure of this latter phase has not yet been determined because of twinning problems.

Biotechniques, 2001 Jan, 30(1), 94 - 8, 100
cDNA library screening using the SOS recruitment system; Huang W et al.; The SOS recruitment system (SRS), a recently developed method for detecting protein-protein interactions, provides an attractive alternative to identify biologically important protein interactions . In SRS, the protein-protein interactions take place in the cytoplasm instead of the nucleus, as is the case in the conventional two-hybrid system . Although the SRS has overcome some of the disadvantages of the conventional two-hybrid system, it still has several problems and limitations . Here, we describe a new protocol for SRS library screening . A new combination of growth media to avoid the tedious step of replica plating greatly increases the number of independent colonies in a single library screening . Furthermore, we designed a pair of ras-specific primers and a one-step simple PCR to rule out the most abundant false positive, the mammalian ras cDNA, in SRS library screening.

Biosci Biotechnol Biochem, 2000 Nov, 64(11), 2508 - 11
Regulation of virulence factors of enterohemorrhagic Escherichia coli O157:H7 by self-produced extracellular factors; Kanamaru K et al.; Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes serious diarrhea and hemolytic uremic syndrome in humans . The expressions of EspD and intimin by O157:H7 have now been shown to be down-regulated by medium conditioned by O157:H7 grown at stationary phase . Preparation of conditioned medium showing the effect on the amount of EspD was not dependent on temperature or growth medium, but was dependent on growth phase . Inhibition of EspD and intimin expression was also induced by medium conditioned by E . coli K-12 strains and homoserine lactone, a signal molecule of the quorum-sensing system in gram-negative bacteria . These results suggest the possibility that the quorum-sensing system mediated by self-produced extracellular factors plays an important role in control of colonization of EHEC O157:H7.

Radiat Res, 2001 Mar, 155(3), 387 - 96
Induction of radioresistance by a nitric oxide-mediated bystander effect; Matsumoto H et al.; To elucidate whether nitric oxide secreted from irradiated cells affects cellular radiosensitivity, we examined the accumulation of inducible nitric oxide synthase, TP53 and HSP72, the concentration of nitrite in the medium of cells after X irradiation, and cellular radiosensitivity using two human glioblastoma cell lines, A-172, which has a wild-type TP53 gene, and a transfectant of A-172 cells, A-172/mp53, bearing a mutated TP53 gene . Accumulation of inducible nitric oxide synthase was caused by X irradiation of the mutant TP53 cells but not of the wild-type TP53 cells . Accumulation of TP53 and HSP72 in the wild-type TP53 cells was observed by cocultivation with irradiated mutant TP53 cells, and the accumulation was abolished by the addition of an inhibitor for inducible nitric oxide synthase, aminoguanidine, to the medium . Likewise, accumulation of these proteins was observed in the wild-type TP53 cells after exposure to conditioned medium from irradiated mutant TP53 cells, and the accumulation was abolished by the addition of a specific nitric oxide scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, to the medium . The radiosensitivity of wild-type TP53 cells was reduced when the cells were cultured in conditioned medium from irradiated mutant TP53 cells compared to conventional fresh growth medium . Collectively, these findings indicate the potential importance of an intercellular signal transduction pathway initiated by nitric oxide in the cellular response to ionizing radiation.

Helicobacter, 2000 Dec, 5(4), 240 - 7
pH-dependent protein profiles of Helicobacter pylori analyzed by two-dimensional gels; Slonczewski JL et al.; BACKGROUND: Helicobacter pylori survives transient exposure to extreme acid prior to adherence and growth on the gastric epithelium at neutral pH . MATERIALS AND METHODS: The effect of pH stress on protein profiles of H . pylori was observed using two-dimensional gel electrophoresis (2-D gels) . H . pylori 26695 was grown microaerobically in tryptone-yeast extract broth, 3% fetal bovine serum . Growth in acid alkalinized the medium, whereas growth in base caused acidification . For 2-D gel analysis of protein profiles, cultures were grown in media buffered at pH 5.7 and at pH 7.5 . RESULTS: Under all pH conditions, the most abundant proteins observed were the urease structural subunit UreB and the chaperonin GroEL . Growth in acid significantly increased the abundance of UreB . Thus, urease expression is not completely constitutive, as reported previously, but shows regulation by pH . Another protein observed only at low pH was identified as mammalian apolipoprotein A-I, possibly taken up by H . pylori from bovine serum in the growth medium . This finding, if confirmed, suggests that uptake of high-density lipoprotein from the human host may facilitate acquisition of cholesterol, required for formation of the unique cholesteryl glucosides in the membrane of H . pylori . In growth above pH 7, three stress proteins were induced: GroES (HspA), GroEL (HspB), and the antioxidant AhpC homolog TsaA . In addition, N-terminal sequence analysis identified five additional proteins that had not previously been reported on 2-D gels of H . pylori (FMN, SodB, TrxB, TsaA, and Tsr) . CONCLUSIONS: In summary, our 2-D gel study reveals expression of several proteins dependent on growth pH.

Altern Lab Anim, 2001 Jan-Feb, 29(1), 9 - 13
Indirect cytotoxicity of dental materials: a study with Transwell inserts and the neutral red uptake assay; Babich H et al.; A modification of the Transwell insert methodology was evaluated by using the neutral red uptake (NRU) assay in a cytotoxicity test . The Transwell insert methodology was developed to assess the biocompatibility of solid materials used in dentistry and, when initially designed, used the release of radiochromium ((51)Cr) in the cytotoxicity assay . Another aim of this study was to evaluate different exposure regimes with which to assess cytotoxicity . The exposure regimes included: a 1-hour exposure in buffer followed by a 24-hour incubation in growth medium; a 2-hour exposure in buffer followed by a 24-hour incubation in growth medium; a 24-hour exposure in serum-limited medium; and a 24-hour exposure in a serum-sufficient medium . The bioindicator target was the Smulow-Glickman (S-G) human gingival cell line and the biomaterials were dental restoratives . The Transwell insert methodology with the NRU cytotoxicity assay as the cytotoxicity endpoint was effective in differentiating the potencies of the dental restoratives; a 2-hour exposure in buffer and a 24-hour exposure in serum-limited medium were the exposure regimes that most clearly differentiated the test agents according to their potencies . The sequence of cytotoxicity of the dental restoratives to the S-G cells was Vitremer > Ketac-Molar Aplicap > Flow-It.

Radiat Res, 2001 Feb, 155(2), 320 - 7
Hypertonic treatment inhibits radiation-induced nuclear translocation of the Ku proteins G22p1 (Ku70) and Xrcc5 (Ku80) in rat fibroblasts; Endoh D et al.; The effects of X irradiation and hypertonic treatment with 0.5 M NaCl on the subcellular localization of the Ku proteins G22p1 (also known as Ku70) and Xrcc5 (also known as Ku80) in rat fibroblasts with normal radiosensitivity were examined using confocal laser microscopy and immunoblotting . Although these proteins were observed mainly in the nuclei of human fibroblasts, approximately 80% of the intensities of immunofluorescence from both G22p1 and Xrcc5 was observed in the cytoplasm of rat fibroblasts . When the rat cells were X-irradiated with 4 Gy, the intensities of the fluorescence derived from G22p1 and Xrcc5 in the nuclei increased from 20% to 50% of the total cellular fluorescence intensity at 20 min postirradiation . No significant differences were observed between the total intensities of the cellular fluorescence from the proteins in unirradiated and irradiated rat fibroblasts . The results showed that the proteins were translocated from the cytoplasm to the nucleus in the rat cells after X irradiation . The nuclear translocation of the proteins from the cytoplasm was inhibited by hypertonic treatment of the cells with 0.5 M NaCl for 20 min, which inhibits the fast repair process of potentially lethal damage (PLD) . When the rat cells were treated with 0.5 M NaCl immediately after X irradiation, the repair of DNA DSBs was inhibited . The surviving fraction was approximately 60% of that of irradiated cells that were not treated with 0.5 M NaCl . The surviving fraction increased with incubation time in the growth medium before treatment with NaCl . The proportions of the intensities of fluorescence from G22p1 in the nuclei of X-irradiated cells also increased from 20% to 50% with increasing interval between X irradiation and treatment with NaCl . These results suggest that nuclear translocation of G22p1 and Xrcc5 is important for the fast repair process of PLD in rat cells.

J Biotechnol, 2001 Feb 23, 85(3), 305 - 14
Phenyl propenoic side chain degradation of ferulic acid by Pycnoporus cinnabarinus - elucidation of metabolic pathways using {5-2H}-ferulic acid; Krings U et al.; The white-rot fungus Pycnoporous cinnabarinus (DMS-1184) was submerged cultured for 22 days under controlled conditions in a bioreactor . After 6, 9, and 15 days of culture the growth medium was supplemented with {5-2H}-labelled ferulic acid (I) . The major phenolic compounds identified labelled were four lignans, the methyl esters of ferulic (I) and vanillic acid (VIII), (E)-coniferyl aldehyde (II), (E)-coniferyl alcohol (III), vanillic acid (VIII), vanillin (IX) and vanillyl alcohol (X) . The detection of considerable amounts of labelled 4-hydroxy-3-methoxyacetophenone (VII) in the late growth phase suggested the increasing formation and decarboxylation of free 4-hydroxy-3-methoxybenzoylacetic acid (VI) and, thus, a beta-oxidation-like degradation of ferulic acid (I) or its methyl ester to vanillic acid (VIII) . 4-Hydroxy-3-methoxybenzoylacetic acid methyl ester (VI) and 3-hydroxy-(4-hydroxy-3-methoxyphenyl)-propanoic acid methyl ester (V) were synthesised and then identified as metabolites in the culture medium . The fungal degradation of the phenyl propenoic side chain of ferulic acid (I), a principal key step of lignin decomposition, appeared to proceed analogous to fatty acids.

Br J Clin Pharmacol, 2001 Jan, 51(1), 99 - 102
Effect of alpha1-acid glycoprotein on the intracellular accumulation of the HIV protease inhibitors saquinavir, ritonavir and indinavir in vitro; Jones K et al.; AIMS: Since alpha1-acid glycoprotein (AGP) levels may be raised during HIV infection, we have examined in vitro the effect of increasing the concentration of AGP on the intracellular accumulation of the HIV protease inhibitors saquinavir (SQV), ritonavir (RTV) and indinavir (IDV) . METHODS: U937 cells (5 x 10(6) cells in 5 ml RPMI growth medium) were incubated at 37 degrees C for 18 h with {14C}-SQV (0.1 microCi), {3H}-RTV and {3H}-IDV (0.135 microCi) to a final concentration of 1 microM in the presence of 0, 0.5 and 2.0 mg x ml(-1) AGP . Following extraction in 60% methanol the intracellular drug concentration was determined by liquid scintillation counting . RESULTS: Binding to AGP (2.0 mg x ml(-1)) reduced the mean intracellular concentration of SQV from 31.5 microM to 7.4 microM (P < 0.0001; 95% CI 19.4-28.8) . RTV concentration was also reduced (8.8 microM to 1.6 microM; P < 0.0001; 95% CI 5.4-9.0) as was the concentration of IDV (3.0 microM to 1.5 microM; P < 0.0001; 95% CI 1.1-1.9) . CONCLUSIONS: Reduced intracellular protease inhibitor concentrations in the presence of increasing concentrations of AGP will certainly impact on the antiviral activity in vitro . However, since protease inhibitors are high clearance drugs, free drug concentration will likely remain unaffected in the presence of elevated AGP during chronic oral dosing although there will be an increase in total plasma drug concentration.

Plant Sci, 2001 Feb 5, 160(3), 563 - 570
Carbon isotopic ratios of atmospheric CO(2) affect the delta(13)C values of heterotrophic growth in Nicotiana tabacum; Le Roux-Swarthout D et al.; Heterotrophic Nicotiana tabacum (L . CV . Wisconsin 38) plants are enriched in 13C relative to the carbon sources in their growth medium . We examined whether carboxylation via phosphoenolpyruvate carboxylase contributes to the enrichment . Achlorophyllous plants were produced using an inhibitor of carotenoid synthesis and were grown on sucrose with known delta(13)C values . Groups of plants were exposed to air with different delta(13)C values as well as to CO(2)-free air . The delta(13)C values of heterotrophic plants were greater than the sucrose source in all treatments and this enrichment increased as 13CO(2)/12CO(2) ratios increased in the source air . Rubisco activity was ruled out as a cause for the enrichment observed as 13CO(2)/12CO(2) ratios increased because the delta(13)C values of heterotrophic plants were similar when exposed to high 13CO(2) while grown in the light or dark . Neither was enrichment due to the adsorption of 13CO(2) in the high 13CO(2) treatment because dead plants did not exhibit this effect when subjected to the same atmospheric treatments . Carboxylation by PEP carboxylase is a likely mechanism causing the 13C-enriched values of living white tissues relative to their organic carbon sources . These results experimentally support suggestions that the anaplerotic activity of PEP is responsible for the 13C-enrichment commonly observed where heterotrophic inputs to growth are large such as in very young leaves.

Brain Res Mol Brain Res, 2001 Jan 31, 86(1-2), 1 - 12
Characterization of a transformed rat retinal ganglion cell line; Krishnamoorthy RR et al.; The purpose of the present study was to establish a rat retinal ganglion cell line by transformation of rat retinal cells . For this investigation, retinal cells were isolated from postnatal day 1 (PN1) rats and transformed with the psi2 E1A virus . In order to isolate retinal ganglion cells (RGC), single cell clones were chosen at random from the transformed cells . Expression of Thy-1 (a marker for RGC), glial fibrillary acidic protein (GFAP, a positive marker for Muller cells), HPC-1/syntaxin (a marker for amacrine cells), 8A1 (a marker for horizontal and ganglion cells) and neurotrophins was studied using reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunocytochemistry . One of the retinal cell clones, designated RGC-5, was positive for Thy-1, Brn-3C, Neuritin, NMDA receptor, GABA-B receptor, and synaptophysin expression and negative for GFAP, HPC-1, and 8A1, suggesting that it represented a putative RGC clone . The results of RT-PCR analysis were confirmed by immunocytochemistry for Thy-1 and GFAP . Upon further characterization by immunoblotting, the RGC-5 clone was positive for Thy-1, negative for GFAP, 8A1 and syntaxin . RGC 5 cells were also positive for the expression of neurotrophins and their cognate receptors . To establish the physiological relevance of RGC-5, the effects of serum/trophic factor deprivation and glutamate toxicity were analyzed to determine if these cells would undergo apoptosis . The protective effects of neurotrophins on RGC-5 after serum deprivation was also investigated . Apoptosis was studied by terminal deoxynucleotidyl transferase-mediated fluoresceinated dUTP nick end labeling (TUNEL) . Serum deprivation resulted in apoptosis and supplementation with both BDNF and NT-4 in the growth media, protected the RGC-5 cells from undergoing apoptosis . On differentiation with succinyl concanavalin A (sConA), RGC-5 cells became sensitive to glutamate toxicity, which could be reversed by inclusion of ciplizone (MK801) . In conclusion, a transformed rat retinal cell line, RGC-5, has certain characteristics of retinal ganglion cells based on Thy-1 and Brn-3C expression and its sensitivity to glutamate excitotoxicity and neurotrophin withdrawal . These cells may be valuable in understanding of retinal ganglion cell biology and physiology including in vitro manipulations in experimental models of glaucoma.

Biochem Biophys Res Commun, 2001 Feb 9, 280(5), 1224 - 8
Insulin-transferrin-selenous acid in growth medium alters the expression of PKC isoforms in mesangial cells; Kumar A et al.; Glomerular mesangial cells (MCs) have been used as an in vitro model for glomerular disease . The culture conditions used for these cells vary and include the use of insulin or insulin-transferrin-selenous acid (ITS) in the growth medium . We studied the effect of ITS in the growth medium containing either normal or high glucose on the expression of protein kinase C (PKC) isoforms in MCs . In the presence of ITS in the medium, MCs expressed lower levels of both PKC isoforms in their cytosol in comparison to MCs grown in medium without ITS . Upon stimulation with PMA, both isoforms were translocated to the particulate (nucleus/cytoskeleton) compartment in MCs grown in presence of ITS . However, in the absence of ITS in the growth medium, both PKC isoforms were primarily translocated to the membrane compartment upon PMA stimulation . These results indicate that insulin in the growth medium may activate MCs resulting in translocation of PKC from the cytosol to other subcellular compartments . This effect is even evident in MCs grown in normal glucose concentration . Our data indicate that the use of ITS in growth medium and eventual interpretation from such experiments involving primary mesangial cells grown in culture needs careful evaluation.

Protein Expr Purif, 2001 Feb, 21(1), 201 - 9
One-step purification of recombinant yeast 6-phosphofructo-2-kinase after the identification of contaminants by MALDI-TOF MS; Dihazi H et al.; His-tagged yeast 6-phosphofructo-2-kinase was overexpressed in the yeast strain DFY658 under the control of the Gal1 promoter . Here we describe a simple and fast purification protocol for the recombinant enzyme under native conditions using a HiTrap affinity column loaded with CuSO(4) . The use of MALDI-TOF MS after in-gel-digestion enabled us to identify a critical contamination of the end product as yeast alcohol dehydrogenase1 (Adh1p) . After identification this contaminant could be efficiently removed by carrying out the washing steps at 25 degrees C instead of at 4 degrees C . To reduce the cellular proteolytic activities a low phosphate concentration in the growth medium was applied . This simple modification of the yeast cell growth conditions increased significantly the yield of the recombinant protein .

Anal Biochem, 2001 Feb 15, 289(2), 217 - 30
Generation of a mammalian cell line stably expressing a tetracycline-regulated epitope-tagged human androgen receptor: implications for steroid hormone receptor research; Wang Q et al.; The androgen receptor (AR) is hormone-activated transcription factor that regulates the expression of genes involved in differentiation, development, and maintenance of male reproductive functions . To establish a useful model system for studying molecular mechanisms of AR action, we generated a HeLa-derived cell line (termed E19) that stably expresses human AR . Because overexpression of AR in cultured cells can be cytotoxic, we placed AR expression under the control of a tetracycline-regulated promoter . The stably expressed AR also contains an N-terminal FLAG-epitope tag (f:AR) that provides an advantageous method for immunopurification . We show that f:AR expression in E19 cells can be precisely modulated by varying the concentration of tetracycline or its chemical derivative doxycycline in the growth media . The functional activity of E19-expressed f:AR is demonstrated in vivo by its ability to activate transiently transfected AR reporter genes in an androgen-dependent manner, and in vitro by its ability to specifically bind AR-response elements using DNA-mobility shift assays . We further show that f:AR in androgen-stimulated E19 cells is markedly phosphorylated and coimmunopurifies with the transcriptional coactivator CREB-binding protein (CBP) . The implications of these findings on steroid receptor research and the identification of receptor coregulatory factors will be discussed .

Anal Biochem, 2001 Feb 1, 289(1), 68 - 76
Marine corrinoid-binding proteins for the direct determination of vitamin B12 by radioassay; Sahni MK et al.; Under conditions of vitamin B12 deficiency the marine phytoplankton Thalassiosira pseudonana secretes into the growth medium a protein (Gm protein) that binds B12 specifically with an affinity constant of 2 x 10(11) M(-1) . When Gm protein was used as a B12-specific binder in radioassays for the direct determination of the vitamin, the detection limit of the technique approached 5 pg B12/mL . In its natural state, Gm protein is oligomeric having molecular weight of over 400 kDa . Modification of the solution environment of this protein produces unique changes in its tertiary and quaternary structure . The amino acid and submit composition of the protein is reported .

J Bacteriol, 2001 Mar, 183(5), 1600 - 9
Modulation of gonococcal piliation by regulatable transcription of pilE; Long CD et al.; The gonococcal pilus, a member of the type IV family of pili, is composed of numerous monomers of the pilin protein and plays an important role in the initiation of disease by providing the primary attachment of the bacterial cell to human mucosal tissues . Piliation also correlates with efficient DNA transformation . To investigate the relationships between these pilus-related functions, the piliation state, and the availability of pilin, we constructed a derivative of MS11-C9 (DeltapilE1) in which the lacIOP regulatory sequences control pilE transcription . In this strain, MS11-C9.10, the steady-state levels of pilin mRNA and protein directly correlate with the concentration of IPTG (isopropyl-beta-D-thiogalactopyranoside) in the growth medium and can reach near-wild-type levels of expression . Transmission electron microscopy (TEM) demonstrated that the number of pili per cell correlated with the steady-state expression levels: at a low level of transcription, single long pili were observed; at a moderate expression level, many singular and bundled pili were expressed; and upon full gene expression, increased lateral association between pili was observed . Analysis of pilus assembly by TEM and epithelial cell adherence over a time course of induction demonstrated that pili were expressed as early as 1 h postinduction . Analysis at different steady-state levels of transcription demonstrated that DNA transformation efficiency and adherence of MS11-C9.10 to transformed and primary epithelial cells also correlated with the level of piliation . These data show that modulation of the level of pilE transcription, without a change in pilE sequence, can alter the number of pili expressed per cell, pilus bundling, DNA transformation competence, and epithelial cell adherence of the gonococcus.

Invest Ophthalmol Vis Sci, 2001 Feb, 42(2), 488 - 96
Proliferation of CECs requires dual signaling through both MAPK/ERK and PI 3-K/Akt pathways; Zubilewicz A et al.; PURPOSE: To analyze the intracellular signaling involved in the proliferation of choroidal endothelial cells (CECs) in vitro . METHODS: Bovine CECs were cultured in endothelial growth medium (EGM) containing 2% fetal calf serum (FCS), 10 microg/ml bovine brain extract (BBE), and 10 ng/ml epidermal growth factor (EGF) in fibronectin-coated plates . Cells were treated with various specific pharmacologic inhibitors of the mitogen-activated protein kinase (MAPK) and of the phosphatidylinositol 3-kinase (PI 3-K) pathways to analyze signaling involved in CEC proliferation . Activation of the MAPK and PI 3-K was detected by Western blot analysis, using specific antiphosphosignaling protein antibodies . RESULTS: FCS, EGF, and BBE were all necessary to induce optimal CEC proliferation . Individually, these three components were not mitogenic . EGM-stimulated CEC proliferation involved the activation of the Raf/mitogen extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90(RSK) cascade . Inhibition of Ras resulted in a 92% reduction of CEC proliferation, whereas inhibition of ERK1/2 activity reduced it by only 46% . The PI 3-K/p70(S6K)/Akt pathway was also stimulated during CEC proliferation, and inhibition of PI 3-K activity resulted in a 94% reduction in CEC proliferation . Inhibition of PI 3-K/p70(S6K) activities also unexpectedly inhibited ERK activity, whereas the converse was not observed, suggesting that PI 3-K acted upstream from ERK and controlled this pathway for CEC proliferation . CONCLUSIONS: CEC proliferation involves both ERK and PI 3-K . That PI 3-K signaling is a key component in cell proliferation can be demonstrated by controlling ERK activity . These data on the molecular mechanism and signaling of CEC proliferation may have major implications for developing more selective methods for antiangiogenic and antitumoral therapy.

EMBO J, 2001 Feb 1, 20(3), 411 - 21
t-SNARE dephosphorylation promotes SNARE assembly and exocytosis in yeast; Marash M et al.; The role of protein phosphorylation in secretion is not well understood . Here we show that yeast lacking the Snc1,2 v-SNAREs, or bearing a temperature-sensitive mutation in the Sso2 t-SNARE, are rescued at restrictive conditions by the addition of ceramide precursors and analogs to the growth medium . Rescue results from dephosphorylation of the Sso t-SNAREs by a ceramide-activated type 2A protein phosphatase (Sit4) involved in cell cycle control . Sso t-SNARE dephosphorylation correlated with its assembly into complexes with the Sec9 t-SNARE, both in vitro and in vivo, and with an increase in protein trafficking and secretion in cells . SNARE complexes isolated under these conditions contained only Sso and Sec9, suggesting that a t-t-SNARE fusion complex is sufficient to confer exocytosis . Mutation of a single PKA site (Ser79 to Ala79) in Sso1 resulted in a decrease in phosphorylation and was sufficient to confer growth to snc cells at restrictive conditions . Thus, modulation of t-SNARE phosphorylation regulates SNARE complex assembly and membrane fusion in vivo.

Arch Ophthalmol, 2001 Jan, 119(1), 81 - 8
The influence of zinc on caspase-3 and DNA breakdown in cultured human retinal pigment epithelial cells; Wood JP et al.; OBJECTIVES: To investigate the role of extracellular zinc on the death process of cultured human retinal pigment epithelial (RPE) cells . METHODS: Confluent cells on borosilicate glass coverslips were treated with substances in serum-free growth medium for various times and were analyzed for death by means of changes in morphologic features, numbers of attached cells, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) procedure . Some cultures were also exposed to experimental ischemia (defined as a lack of oxygen, glucose, and serum) . Electrophoresis and Western blotting and enzyme assays were used to investigate changes in expression of the protease enzyme, caspase-3 . RESULTS: Experimental ischemia caused death of RPE cells . Zinc sulfate had no effect on these cells at low concentrations (100 pmol/L to 10 nmol/L), but protected them at higher concentrations (< or = 10 micromol/L) and appeared to exacerbate cell death at still greater concentrations . Moreover, zinc compounds (>10 micromol/L) also induced death of cells in control cultures that could be blocked by zinc chelators and partially by the caspase-3 inhibitor, DEVD-FMK . Zinc also increased the amount of the active form of caspase-3 in RPE cells . CONCLUSIONS: Zinc salts protect RPE cells from experimental ischemia-induced death at low concentrations (100 pmol/L-10 nmol/L) . However, at higher concentrations, zinc causes cell death and alters the cellular level of caspase-3 . These observations are consistent with the death process being apoptosis . CLINICAL RELEVANCE: Zinc supplements are taken by many individuals . Low doses of zinc can protect RPE cells against ischemic-type insults as may occur in certain ocular complaints . Furthermore, high concentrations of zinc can damage RPE cells . Because zinc ions are known to be taken up by RPE cells from the choroidal circulation, the actual therapeutic dose taken by patients is critical.

Cancer Chemother Pharmacol, 2000, 45(4), 335 - 44
Evidence for a role of chloroethylaziridine in the cytotoxicity of cyclophosphamide; Flowers JL et al.; A number of investigators have observed that the use of 4-hydroperoxycyclophosphamide (4-HC) in multiwell plate cytotoxicity assays can be associated with toxicity to cells in wells that contain no drug . Previous reports have implicated diffusion of 4-HC decomposition products, and acrolein in particular, as the active species . PURPOSE: The purpose of this study was to elucidate the species responsible for the airborne cytotoxicity of 4-HC, and to devise ways to minimize such effects in chemosensitivity assays . METHODS: To this end, analogues of 4-HC were synthesized to identify the contributions of individual cyclophosphamide metabolites to cytotoxicity . The analogues were then tested for activity against three human breast tumor cell lines (including a line resistant to 4-HC), and one non-small-cell lung carcinoma line . Cytotoxicity was evaluated by assays that quantitate cellular metabolism and nucleic acid content . RESULTS: Didechloro-4-hydroperoxycyclophosphamide, a compound that generates acrolein and a nontoxic analogue of phosphoramide mustard, gave no cross-well toxicity . In contrast, a significant neighboring well effect was observed with phenylketophosphamide, a compound that generates phosphoramide mustard but not acrolein . Addition of authentic chloroethylaziridine reproduced the airborne toxicity patterns generated by 4-HC and phenylketophosphamide . Increasing the buffering capacity of the growth medium and sealing the microtiter plates prevented airborne cytotoxicity . CONCLUSION: Since it is unlikely that phosphoramide mustard is volatile, these findings implicate chloroethylaziridine rather than acrolein as the volatile metabolite of 4-HC that is responsible for airborne cytotoxicity . The fact that chloroethylaziridine is generated in amounts sufficient to volatilize, diffuse across wells and cause cytotoxicity indicates that it is an important component in the overall cytotoxicity of 4-HC in vitro . Furthermore, these findings suggest that chloroethylaziridine may also contribute to the toxicity of cyclophosphamide in vivo.

J Biol Chem, 2001 Mar 30, 276(13), 10126 - 33 Epub 2001 Jan 03.
Regulation of the Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate phosphatase by zinc; Han GS et al.; The DPP1 gene, encoding diacylglycerol pyrophosphate (DGPP) phosphatase from Saccharomyces cerevisiae, has recently been identified as a zinc-regulated gene, and it contains a putative zinc-responsive element (UAS(ZRE)) in its promoter . In this work we examined the hypothesis that expression of DGPP phosphatase was regulated by zinc availability . The deprivation of zinc from the growth medium resulted in a time- and dose-dependent induction of beta-galactosidase activity driven by a P(DPP1)-lacZ reporter gene . This regulation was dependent on the UAS(ZRE) in the DPP1 promoter and was mediated by the Zap1p transcriptional activator . Induction of the DGPP phosphatase protein and activity by zinc deprivation was demonstrated by immunoblot analysis and measurement of the dephosphorylation of DGPP . The regulation pattern of DGPP phosphatase in mutants defective in plasma membrane (Zrt1p and Zrt2p) and vacuolar membrane (Zrt3p) zinc transporters indicated that enzyme expression was sensitive to the cytoplasmic levels of zinc . DGPP phosphatase activity was inhibited by zinc by a mechanism that involved formation of DGPP-zinc complexes . Studies with well characterized subcellular fractions and by indirect immunofluorescence microscopy revealed that the DGPP phosphatase enzyme was localized to the vacuolar membrane.

Clin Chem Lab Med, 2000 Nov, 38(11), 1191 - 3
The effect of human organ preservation and albumin flush solution on in vitro cell metabolic activity; Riga AT et al.; In liver transplantation, the organ during the recipient's operation is traditionally flushed with 4.5% of human albumin solution to wash away the potassium-rich University of Wisconsin (UW) solution . It has been argued whether albumin could be useful at this stage . We used a new simple non-toxic assay to determine cell viability in vitro . Alamar Blue incorporates a redox indicator which changes colour from blue (oxidised form) to magenta (reduced form) in response to metabolic activity . Cultured human hepatocyte and HUVEC cell lines were exposed for 3, 6, 12 or 24 hours to plain medium, UW solution, human albumin 4.5% solution, UW-containing effluents before and after preservation as well as albumin flushes from different transplantation cases . After addition of Alamar Blue the optical density was measured at 570 nm and the background measured at 600 nm was subtracted . The studies showed a significantly lower metabolic rate of the cells exposed to albumin and albumin-containing flushes at all time periods, even after a short exposure such as 3 hours (p < 0.001) . On the other hand, there was no significant difference of growth and metabolic activity rate between cells exposed to UW solution, different UW-containing flushes and medium for up to 12 hours . In conclusion, human albumin is a very poor solution for cell maintenance . In contrast, UW solution has comparable results with the full growth medium for up to 12 hours of exposure.

J Appl Microbiol, 2001 Jan, 90(1), 27 - 33
Comparison of selective procedures for isolation and enumeration of Legionella species from hot water systems; Leoni E et al.; Various sample pre-treatment techniques and different growth media for the isolation of Legionellae from hot water supplies in public buildings were compared . A total of 102 hot water samples from taps and showers was examined . The highest recovery frequency was obtained with the heat pre-treatment method and using the selective medium GVPC . However, the results differed according to the concentration of legionellas . In the case of low plate counts (< or =5000 cfu l(-1)), the heat pre-treatment technique gave a significantly higher percentage of positive samples compared with other techniques (P < 0.05) . With increasing concentration, the differences between the procedures decreased until they became statistically not significant for concentrations above 50 000 cfu l(-1) . The direct inoculum method allowed a significantly higher detection of concentrations (P < 0.001) compared with heat and acid decontamination methods, which brought about a 67-68% reduction in detectable Legionellae . Heat decontamination techniques show greater sensitivity and specificity . However, they underestimate the number of legionellas . In environmental surveillance programmes, this underestimate must be taken into consideration when assessing the health risk.

Inhal Toxicol, 2001 Jan, 13(1), 55 - 68
Effect of growth medium on potential of Streptomyces Anulatus spores to induce inflammatory responses and cytotoxicity in RAW264.7 macrophages; Hirvonen MR et al.; Epidemiological studies have shown an association between microbial growth in buildings and increased risk of respiratory symptoms and disease related to inflammatory reactions in the inhabitants96 . The current study examined the affects of growth conditions of Streptomyces anulatus, isolated from indoor air of a moldy building, on the inflammatory potential of spores of this microbe . Spores were harvested from 15 growth media formulations, applied to RAW264.7 macrophages (10(5), 10(6), or 10(7) spores/million cells), and evaluated for the ability to stimulate production of inflammatory mediators and cytotoxicity in these cells 24 h after exposure . Streptomyces anulatus spores induced dose-dependent production of nitric oxide (NO) in macrophages, reaching a level from 4.2 microM to 39.2 microM depending on the composition of the growth medium of the microbe . Expression of inducible NO synthase (iNOS) was detected in macrophages after exposure to spores collected from all growth media . Production of reactive oxygen species (ROS) was significantly increased only by the highest dose of S . anulatus spores grown on glycerol-arginine agar . Furthermore production of cytokines was affected by growth medium; the highest dose-dependent levels of interleukin 6 (IL-6) ranged from 900 to 7800 pg/ml, and the levels of tumor necrosis factor alpha (TNFalpha) varied from 490 to 3200 pg/ml . The amount of dead macrophages after the exposure varied from 11% to 96%, depending also on the growth media of the microbe . Altogether, our results suggest that the growth medium of S . anulatus has a fundamental role in the ability of the spores to induce inflammatory responses and cytotoxicity in mammalian cells.

Cell Growth Differ, 2000 Dec, 11(12), 655 - 64
Inhibition of mitogen-activated protein kinase kinase selectively inhibits cell proliferation in human breast cancer cells displaying enhanced insulin-like growth factor I-mediated mitogen-activated protein kinase activation; Hermanto U et al.; Mitogen-activated protein (MAP) kinase mediates cell proliferation, cell differentiation, and cell survival by regulating signaling pathways activated by receptor protein tyrosine kinases (RPTKs), including the insulin-like growth factor 1 receptor (IGF-IR) . We analyzed the upstream signaling components of the MAP kinase pathway, including RPTKs, in human breast cancer cell lines and found that some of those components were overexpressed . Importantly, signaling molecules such as IGF-IR, insulin receptor, and insulin receptor substrate 1, leading to the MAP kinase pathway, were found to be concomitantly overexpressed within certain tumor lines, i.e., MCF-7 and T-47D . When compared with the nonmalignant and other breast tumor lines examined, MCF-7 and T-47D cells displayed a more rapid, robust, and sustained MAP kinase activation in response to insulin-like growth factor I (IGF-I) stimulation . By contrast, IGF-I treatment led to a sustained down-regulation of MAP kinase in those lines overexpressing ErbB2-related RPTKs . Interestingly, blocking the MAP kinase pathway with PD098059 had the greatest antiproliferative effect on MCF-7 and T-47D among the normal and tumor lines tested . Furthermore, addition of an IGF-IR blocking antibody to growth medium attenuated the ability of PD098059 to suppress the growth of MCF-7 and T-47D cells . Thus, our study suggests that concomitant overexpression of multiple signaling components of the IGF-IR pathway leads to the amplification of IGF-I-mediated MAP kinase signaling and resultant sensitization to PD098059 . The enhanced sensitivity to PD098059 implies an increased requirement for the MAP kinase pathway in those breast cancer cells, making this pathway a potential target in the treatment of selected breast malignancies.

Acta Orthop Scand, 2000 Dec, 71(6), 630 - 6
Reduced NO accumulation in arthrotic cartilage by exposure to methylene blue; Cohen N et al.; Nitric oxide (NO) appears to be a final common inflammation mediator of cartilage degradation . Halting the pathological formation of excessive NO, by suppressing the inducible NO synthase (iNOS) activity, may help to preserve cartilage integrity . We used fresh ex-vivo human articular cartilage explants from normal and arthrotic joints for assessment of NO levels, as determined by its nitrite degradation products and nitric oxide synthase expression . We measured matrix proteoglycan content, assessed by image analysis of alcian blue staining, and proteoglycan synthesis, assessed by sulfate incorporation into proteoglycans . The effect of methylene blue, a nitric oxide synthase inhibitor, on matrix preservation was evaluated . Cartilage discs in vitro, derived from normal appearing joints, secreted about one tenth as much NO compared to discs derived from arthrotic cartilage . Cartilage explants showed a time-dependent reduction in the amount of aggrecan within the cartilaginous matrix . Addition of methylene blue to the growth medium lowered nitric oxide accumulation and prevented matrix degradation in the cultured cartilage discs . The cartilage matrix preservation effect was mediated through downregulation of all three isoforms of NOS, i.e., the neuronal NOS, endothelial NOS and inducible NOS and upregulation of TGF beta receptor in the chondrocytes . Our findings indicate that inhibition of NOS activity preserves cartilage matrix in vitro.

Clin Diagn Lab Immunol, 2001 Jan, 8(1), 85 - 92
Genetic and biochemical characterization of glycerol uptake in mycoplasma mycoides subsp . mycoides SC: its impact on H(2)O(2) production and virulence; Vilei EM et al.; Highly virulent strains of Mycoplasma mycoides subsp . mycoides SC belonging to the African cluster contain an operon with the genes gtsA, gtsB, and gtsC, encoding membrane ATP binding cassette transporter proteins GtsA, GtsB, and GtsC, which are involved in glycerol transport . Strain Afade from the African cluster incorporated {U-(14)C}glycerol with a time-dependent increase . The less virulent strain L2 of the European cluster, which lacks gtsB and gtsC, failed to incorporate glycerol . Antibodies against GtsB noncompetitively inhibited glycerol uptake . L-alpha-Glycerophosphate was not transported by M . mycoides subsp . mycoides SC . It is postulated to be synthesized by phosphorylation of glycerol during transport and subsequently metabolized further to dihydroxyacetone phosphate accompanied by release of H(2)O(2) . Peroxide production in glycerol-containing growth medium was high for the African strain Afade but very low for the European strain L2 . Virtually no H(2)O(2) was produced by both strains without glycerol . Hence, the efficient glycerol uptake system found in the virulent strain of the African cluster leads to a strong release of peroxide, a potential virulence factor which is lacking in the less virulent European strains . M . mycoides subsp . mycoides SC might have adopted, as a strategy for virulence, a highly efficient uptake system for glycerol which allows the production of an active metabolic intermediate that damages host cells.

Biotechnol Bioeng, 2001 Feb 5, 72(3), 278 - 88
Accumulation of poly{(R)-3-hydroxyalkanoates} in Pseudomonas oleovorans during growth in batch and chemostat culture with different carbon sources; Durner R et al.; Pseudomonas oleovorans (ATCC 29347) was grown in batch and chemostat cultures with citrate, hexanoate, heptanoate, octanoate, and nonanoate as single carbon substrates . The growth medium for batch cultures was adjusted such that nitrogen (NH(4)(+)) limitation terminated the exponential-growth phase . During batch cultivation with octanoate or nonanoate the biomass continued to increase after depletion of ammonium due to the accumulation of medium-chain-length poly{(R)-3-hydroxyalkanoates} (mcl-PHAs) . Additionally, a significant rate of mcl-PHA accumulation was also observed in the exponential-growth phase of batch cultures . It is well known that the accumulation of reserve materials is strongly dependent on the ratio of nutrients (here of carbon, C, and of nitrogen, N) and that in a batch culture the ratio of C:N is continuously changing . Therefore, we have also investigated the effect of defined ratios of C:N under constant cultivation conditions, namely at a fixed dilution rate (D) in a chemostat fed with different medium C:N ratios . These experiments were performed at a constant D of 0.2 h(-1) . The concentration of the nitrogen source in the inflowing medium (N()) was kept constant, while its carbon concentration (C()) was increased stepwise, resulting in an increase of the medium carbon to nitrogen ratio (C()/N() ratio) . The culture parameters and the cell composition of steady-state cultures were determined as a function of the C()/N() ratio in the feed medium . Mcl-PHA accumulation was detected during growth with the fatty acids, and three distinct regimes of growth limitation were discovered: In addition to carbon limitation at low, and nitrogen limitation at high C()/N() ratios, an intermediate growth regime of simultaneous limitation by carbon and nitrogen was detected where both substrates were used to completion . The width of this dual-nutrient-limited growth regime was dependent on the change in the yield factors for carbon and nitrogen (Y(X/C), Y(X/N)) measured during single-nutrient-limited growth .

J Bacteriol, 2001 Jan, 183(2), 716 - 24
Key role for sulfur in peptide metabolism and in regulation of three hydrogenases in the hyperthermophilic archaeon Pyrococcus furiosus; Adams MW et al.; The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100 degrees C by the fermentation of peptides and carbohydrates . Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S(0)) . Growth rates were highest on media containing peptides and S(0), with or without maltose . Growth did not occur on the peptide medium without S(0) . S(0) had no effect on growth rates in the maltose medium in the absence of peptides . Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S(0) with or without maltose were the same, suggesting that S(0) is required for peptide utilization . The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P . furiosus were shown to be regulated under the five different growth conditions studied . The presence of S(0) in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude . The primary S(0)-reducing enzyme in this organism and the mechanism of the S(0) dependence of peptide metabolism are not known . This study provides the first evidence for a highly regulated fermentation-based metabolism in P . furiosus and a significant regulatory role for elemental sulfur or its metabolites.

J Magn Reson, 2001 Jan, 148(1), 142 - 6
Selectively labeling the heterologous protein in Escherichia coli for NMR studies: a strategy to speed up NMR spectroscopy; Almeida FC et al.; Nuclear magnetic resonance is an important tool for high-resolution structural studies of proteins . It demands high protein concentration and high purity; however, the expression of proteins at high levels often leads to protein aggregation and the protein purification step can correspond to a high percentage of the overall time in the structural determination process . In the present article we show that the step of sample optimization can be simplified by selective labeling the heterologous protein expressed in Escherichia coli by the use of rifampicin . Yeast thioredoxin and a coix transcription factor Opaque 2 leucine zipper (LZ) were used to show the effectiveness of the protocol . The (1)H/(15)N heteronuclear correlation two-dimensional NMR spectrum (HMQC) of the selective (15)N-labeled thioredoxin without any purification is remarkably similar to the spectrum of the purified protein . The method has high yields and a good (1)H/(15)N HMQC spectrum can be obtained with 50 ml of M9 growth medium . Opaque 2 LZ, a difficult protein due to the lower expression level and high hydrophobicity, was also probed . The (15)N-edited spectrum of Opaque 2 LZ showed only the resonances of the protein of heterologous expression (Opaque 2 LZ) while the (1)H spectrum shows several other resonances from other proteins of the cell lysate . The demand for a fast methodology for structural determination is increasing with the advent of genome/proteome projects . Selective labeling the heterologous protein can speed up NMR structural studies as well as NMR-based drug screening . This methodology is especially effective for difficult proteins such as hydrophobic transcription factors, membrane proteins, and others .

Int J Radiat Biol, 2000 Dec, 76(12), 1649 - 57
Induction of radioresistance to accelerated carbon-ion beams in recipient cells by nitric oxide excreted from irradiated donor cells of human glioblastoma; Matsumoto H et al.; PURPOSE: To investigate whether nitric oxide excreted from cells irradiated with accelerated carbon-ion beams modulates cellular radiosensitivity against irradiation in human glioblastoma A-172 and T98G cells . MATERIALS AND METHODS: Western-blot analysis of inducible nitric oxide synthase, hsp72 and p53, the concentration assay of nitrite in medium and cell survival assay after irradiation with accelerated carbon-ion beams were performed . RESULTS: The accumulation of inducible nitric oxide synthase was caused by accelerated carbon-ion beam irradiation of T98G cells but not of A-172 cells . The accumulation of hsp72 and p53 was observed in A-172 cells after exposure to the conditioned medium of the T98G cells irradiated with accelerated carbon-ion beams, and the accumulation was abolished by the addition of an inhibitor for inducible nitric oxide synthase to the medium . The radiosensitivity of A-172 cells was reduced in the conditioned medium of the T98G cells irradiated with accelerated carbon-ion beams compared with conventional fresh growth medium, and the reduction of radiosensitivity was abolished by the addition of an inducible nitric oxide synthase inhibitor to the conditioned medium . CONCLUSIONS: Nitric oxide excreted from the irradiated donor cells with accelerated carbon-ion beams could modulate the radiosensitivity of recipient cells . These findings indicate the importance of an intercellular signal transduction pathway initiated by nitric oxide in the cellular response to accelerated heavy ions.

Appl Microbiol Biotechnol, 2000 Nov, 54(5), 686 - 91
Differential expression of manganese peroxidase and laccase in white-rot fungi in the presence of manganese or aromatic compounds; Scheel T et al.; White-rot fungi (basidiomycetes) play an important role in the degradation of lignin which is, beside cellulose, the major compound of wood . This process is catalyzed by ligninolytic enzymes, which are able to cleave oxidatively aromatic rings in lignin structure . Manganese peroxidase and laccase of white-rot-fungi are the most important of these among the ligninolytic enzymes . In addition, they are able to degrade xenobiotic aromatic polymers, persisting as environmental pollutants . Manganese and aromatic compounds have often been discussed as being inducers, enhancers or mediators of these ligninolytic enzymes . It is known that supplementing the growth medium with either Mn2+, veratryl alcohol or coal-derived humic acids leads to significantly enhanced extracellular ligninolytic activities . Measuring the amount of expressed mRNA of the two enzymes by quantitative RT-PCR provided evidence that the expression of manganese peroxidase was induced in the three tested white-rot fungi, Clitocybula dusenii b11, Nematoloma frowardii b19, and a straw-degrading strain designated i63-2 . Laccase, on the other hand, was expressed in all three fungi with a significant basic activity even without inducer added . However, since the level of laccase mRNA was higher in cultures supplemented with any one of the tested inducers, we conclude that both manganese and the aromatic substances also increase the expression of laccase.

Life Sci, 2000 Nov 10, 67(25), 3075 - 85
Norepinephrine as a growth stimulating factor in bacteria--mechanistic studies; Kinney KS et al.; Catecholamines (norepinephrine, epinephrine, dopamine) enhance the growth of several species of gram-negative bacteria . Since catechol rings are known siderophores in bacteria, the administration of catecholamines may enhance growth by improving iron uptake in growth-limiting media, serving as auxiliary siderophores . We have tested the iron content in bacterial growth media which are known to support rapid growth and "slow growth" media . Additionally, we have examined the uptake of 3H-norepinephrine, to determine whether the catecholamine is actually taken into the bacteria or is merely adsorbed to the outside of the bacteria . Finally, we have been examining the supernatants produced by culturing bacteria with norepinephrine . These supernatants have been shown to have the capacity to enhance growth of naive cultures of bacteria, and are suggested to contain an "autoinducer of growth" . We have found that both fast-growth and slow-growth media contain similar concentrations of iron, and that these levels do not change in most supernatants from NE-supplemented bacterial cultures . Examination of culture supernatants from NE-supplemented bacteria under different temperature conditions reveals some interesting differences . First, culture supernatant from NE-treated Escherichia coli, cultured at 37 degrees C, when examined by HPLC, exhibits a change in the norepinephrine content over time which is not seen in supernatant from 21 degrees C cultures or other media treatments . Second, the 37 degrees C culture NE-supplemented E . coli supernatant was significantly more effective in enhancing growth of three bacterial species than any other culture method other than NE-supplementation itself (this includes supernatant from NE-supplemented cultures of the other two species as well as supernatants from unsupplemented cultures of all three species).

Yeast, 2001 Jan 15, 18(1), 81 - 8
Subtle alterations in growth medium composition can dramatically alter the percentage of unsaturated fatty acids in the yeast Saccharomyces cerevisiae; Chatterjee MT et al.; The essence of the scientific method is the production of reproducible results in repeated experiments . Cells of the yeast strain DBY747 normally contain 36% unsaturated fatty acids but suddenly, and initially inexplicably, lipid analysis revealed 72% unsaturated fatty acids in the same strain at the same growth temperature . A comparative lipid analysis of DBY747 grown in YEPD and in a number of different types and batches of Yeast Nitrogen Base media revealed two heretofore unreported phenomena . We provide mass spectroscopy and yeast bioassay evidence suggesting that the increase in lipid unsaturation can be attributed to the presence of the plasticizing agent dioctylphthalate in YNB and bactopeptone packaged in 'new' plastic containers first introduced by Difco some 3-4 years ago . We also demonstrate that L-methionine plays an important role in determining the percentage of unsaturated fatty acids in cells grown in laboratory-produced YNB . The results illustrate a novel aspect of methionine metabolism while at the same time highlighting the need for more stringent control to be exercised by the companies that formulate and package defined media .

Mol Microbiol, 2001 Jan, 39(1), 176 - 82
Requirement of nickel metabolism proteins HypA and HypB for full activity of both hydrogenase and urease in Helicobacter pylori; Olson JW et al.; The nickel-containing enzymes hydrogenase and urease require accessory proteins in order to incorporate properly the nickel atom(s) into the active sites . The Helicobacter pylori genome contains the full complement of both urease and hydrogenase accessory proteins . Two of these, the hydrogenase accessory proteins HypA (encoded by hypA) and HypB (encoded by hypB), are required for the full activity of both the hydrogenase and the urease enzymes in H . pylori . Under normal growth conditions, hydrogenase activity is abolished in strains in which either hypA (HypA:kan) or hypB (HypB:kan) have been interrupted by a kanamycin resistance cassette . Urease activity in these strains is 40 (HypA:kan)- and 200 (HypB:kan)-fold lower than for the wild-type (wt) strain 43504 . Nickel supplementation in the growth media restored urease activity to almost wt levels . Hydrogenase activity was restored to a lesser extent, as has been observed for hyp mutants in other (H(2)-oxidizing) bacteria . Expression levels of UreB (the urease large subunit) were not affected by inactivation of either hypA or hypB, as determined by immunoblotting . Urease activity was not affected by lesions in the genes for either the hydrogenase accessory proteins HypD or HypF or the hydrogenase large subunit structural gene, indicating that the urease deficiency was not caused by lack of hydrogenase activity . When crude extracts of wt, HypA:kan and HypB:kan were separated by anion exchange chromatography, the urease-containing fractions of the mutant strains contained about four (HypA:kan)- and five (HypB:kan)-fold less nickel than did the urease from wt, indicating that the lack of urease activity in these strains results from a nickel deficiency in the urease enzyme.

Radiat Res, 2001 Jan, 155(1 Pt 2), 239 - 247
Neoplastic transformation in C3H 10T(1/2) cells after exposure to 835.62 MHz FDMA and 847.74 MHz CDMA radiations; Roti Roti JL et al.; The effect of radiofrequency (RF) radiation in the cellular phone communication range (835.62 MHz frequency division multiple access, FDMA; 847.74 MHz code division multiple access, CDMA) on neoplastic transformation frequency was measured using the in vitro C3H 10T(1/2) cell transformation assay system . To determine if 835.62 MHz FDMA or 847.74 MHz CDMA radiations have any genotoxic effects that induce neoplastic transformation, C3H 10T(1/2) cells were exposed at 37 degrees C to either of the above radiations {each at a specific absorption rate (SAR) of 0.6 W/kg} or sham-exposed at the same time for 7 days . After the culture medium was changed, the cultures were transferred to incubators and refed with fresh growth medium every 7 days . After 42 days, the cells were fixed and stained with Giemsa, and transformed foci were scored . To determine if exposure to 835.62 MHz FDMA or 847.74 MHz CDMA radiation has any epigenetic effects that can promote neoplastic transformation, cells were first exposed to 4.5 Gy of X rays to induce the transformation process and then exposed to the above radiations (SAR = 0.6 W/kg) in temperature-controlled irradiators with weekly refeeding for 42 days . After both the 7-day RF exposure and the 42-day RF exposure after X irradiation, no statistically significant differences in the transformation frequencies were observed between incubator controls, the sham-exposed (maintained in irradiators without power to the antenna), and the 835.62 MHz FDMA or 847.74 MHz CDMA-exposed groups.

FEBS Lett, 2000 Dec 15, 486(3), 237 - 42
Enhanced release of soluble urokinase receptor by endothelial cells in contact with peripheral blood cells; Mustjoki S et al.; The urokinase receptor (uPAR) on the cell surface plays an important role in extracellular proteolysis, cell migration and adhesion . Soluble uPAR (suPAR) has been recently discovered in plasma, but its origin is unclear . Our results now demonstrate that both unstimulated blood mononuclear and endothelial cells can release suPAR and that the release is enhanced when either mononuclear cells or thrombocytes are cultured together with endothelial cells . Co-culture without cell-cell contacts fails to enhance suPAR release . We also found suPAR fragments, known to be potent inducers of chemotaxis, in co-culture growth medium samples . Taken together, our results suggest that normal plasma suPAR can be produced by endothelial and mononuclear cells and that suPAR release in cell-cell contacts may have a regulatory role in cell adhesion.

Int J Oncol, 2001 Jan, 18(1), 195 - 201
Integrin alpha 5 beta 1 suppresses apoptosis triggered by serum starvation but not phorbol ester in MCF-7 breast cancer cells that overexpress protein kinase C-alpha; Noti JD et al.; MCF-7 breast cancer cells grow as adherent cells, but following overexpression of protein kinase C-alpha these cells (MCF-7-PKC-alpha cells) become anchorage-independent and exhibit increased tumorigenicity in nude mice . MCF-7-PKC-alpha cells are also sensitized to apoptosis in response to phorbol ester but not serum starvation . Flourescence-activated cell sorting revealed that several integrin subunits were down-regulated in MCF-7-PKC-alpha cells, however, the fibronectin receptor alpha 5 beta 1 was upregulated . MCF-7-PKC-alpha cells growing under non-adherent conditions underwent cell death when antibodies to alpha 5 beta 1 were added to growth media lacking serum but not when serum was present . Addition of soluble fibronectin to cells incubated without serum suppressed apoptosis triggered by anti-alpha 5 beta 1 antibodies but not by phorbol esters . MCF-7-PKC-alpha cells also were shown to express more fibronectin on their cell surface than MCF-7V cells (MCF-7 cells transfected with pSV(2)M(2)6 vector only) . This study indicates that the survival of MCF-7-PKC-alpha cells under non-adherent conditions in the absence of serum results from the ligation of alpha 5 beta 1 with surface-bound fibronectin, which may account, in part, for the increased aggressiveness of these cells.

Yeast, 2000 Dec, 16(16), 1497 - 508
Isolation and characterization of Saccharomyces cerevisiae mutants with derepressed thiamine gene expression; Burrows RJ et al.; Using a THI4-lacZ reporter gene, mutant strains have been isolated that display constitutive expression of thiamine genes in the presence of normally repressing levels of exogenous thiamine . In total, eight strains were isolated in which this derepressed expression on thiamine (Det(-)) phenotype was the result of single gene mutations . The Det(-) mutations of three of these strains were partially dominant in a heterozygous diploid configuration, whereas the other five were recessive . The partially dominant mutants DET1, DET12 and DET13, and the recessive mutant det2, all showed derepressed THI4-lacZ expression levels comparable to those of a fully induced normal strain . Use of other promoter-lacZ gene fusions revealed that these four mutants were pleiotropic; expression levels of all thiamine-regulated genes tested were also derepressed . Genetic analysis of the four mutants suggested that det2 and DET13 were allelic, whereas the others were at different loci; these four mutations therefore represent three different genes . None of the mutations were allelic with THI80, mutations of which have previously been shown to confer derepression on thiamine-regulated genes . Also, intracellular thiamine levels were close to normal and none of the four mutants excreted thiamine into the growth medium . All mutant strains were found to be prototrophic for thiamine and none of those tested were compromised for thiamine uptake . It is possible that some may be alleles of, or interact with, the activator gene THI3 . Taken together, these results imply that DET1, det2, DET12 and DET13 represent new genes encoding negative regulators of thiamine-repressed genes .

Mol Cell Biol, 2001 Jan, 21(1), 16 - 25
The Sko1p repressor and Gcn4p activator antagonistically modulate stress-regulated transcription in Saccharomyces cerevisiae; Pascual-Ahuir A et al.; In the transcriptional response of Saccharomyces cerevisiae to stress, both activators and repressors are implicated . Here we demonstrate that the ion homeostasis determinant, HAL1, is regulated by two antagonistically operating bZIP transcription factors, the Sko1p repressor and the Gcn4p activator . A single CRE-like sequence (CRE(HAL1)) at position -222 to -215 with the palindromic core sequence TTACGTAA is essential for stress-induced expression of HAL1 . Down-regulation of HAL1 under normal growth conditions requires specific binding of Sko1p to CRE(HAL1) and the corepressor gene SSN6 . Release from this repression depends on the function of the high-osmolarity glycerol pathway . The Gcn4p transcriptional activator binds in vitro to the same CRE(HAL1) and is necessary for up-regulated HAL1 expression in vivo, indicating a dual control mechanism by a repressor-activator pair occupying the same promoter target sequence . gcn4 mutants display a strong sensitivity to elevated K(+) or Na(+) concentrations in the growth medium . In addition to reduced HAL1 expression, this sensitivity is explained by the fact that amino acid uptake is drastically impaired by high Na(+) and K(+) concentrations in wild-type yeast cells . The reduced amino acid biosynthesis of gcn4 mutants would result in amino acid deprivation . Together with the induction of HAL1 by amino acid starvation, these results suggest that salt stress and amino acid availability are physiologically interconnected.

Can J Gastroenterol, 2000 Nov, 14(10), 871 - 5
Antibiotic susceptibility and resistance testing: an overview; Smaill F; The results of in vitro antibiotic susceptibility testing can predict the clinical response to treatment and guide the selection of antibiotics . The minimum inhibitory concentration (MIC) of an organism is the lowest concentration of an antibiotic that will inhibit its growth . Bacteria are classified as sensitive, intermediate or resistant based on breakpoint MIC values that are arbitrarily defined and reflect the achievable levels of the antibiotic, the distribution of MICs for the organism and their correlation with clinical outcome . Broth dilution, agar dilution and gradient diffusion (the 'E test'), where twofold serial dilutions of antibiotic are incorporated into tubes of broth, agar plates or on a paper strip, respectively, are different methods to measure the MIC of an organism . The disk diffusion method defines an organism as sensitive or resistant based on the extent of its growth around an antibiotic-containing disk . MIC values are influenced by several laboratory factors . To ensure reproducible results, the laboratory must closely follow methods developed by the National Committee for Clinical Laboratory Standards, which defines standard growth media, incubation temperature and environment, the inoculum and quality control parameters.

J Bacteriol, 2000 Dec, 182(24), 7035 - 43
ModE-dependent molybdate regulation of the molybdenum cofactor operon moa in Escherichia coli; Anderson LA et al.; The expression of the moa locus, which encodes enzymes required for molybdopterin biosynthesis, is enhanced under anaerobiosis but repressed when the bacterium is able to synthesize active molybdenum cofactor . In addition, moa expression exhibits a strong requirement for molybdate . The molybdate enhancement of moa transcription is fully dependent upon the molybdate-binding protein, ModE, which also mediates molybdate repression of the mod operon encoding the high-affinity molybdate uptake system . Due to the repression of moa in molybdenum cofactor-sufficient strains, the positive molybdate regulation of moa is revealed only in strains unable to make the active cofactor . Transcription of moa is controlled at two sigma-70-type promoters immediately upstream of the moaA gene . Deletion mutations covering the region upstream of moaA have allowed each of the promoters to be studied in isolation . The distal promoter is the site of the anaerobic enhancement which is Fnr-dependent . The molybdate induction of moa is exerted at the proximal promoter . Molybdate-ModE binds adjacent to the -35 region of this promoter, acting as a direct positive regulator of moa . The molybdenum cofactor repression also appears to act at the proximal transcriptional start site, but the mechanism remains to be established . Tungstate in the growth medium affects moa expression in two ways . Firstly, it can act as a functional molybdate analogue for the ModE-mediated regulation . Secondly, tungstate brings about the loss of the molybdenum cofactor repression of moa . It is proposed that the tungsten derivative of the molybdenum cofactor, which is known to be formed under such conditions, is ineffective in bringing about repression of moa . The complex control of moa is discussed in relation to the synthesis of molybdoenzymes in the bacterium.

J Biotechnol, 2001 Nov 30, 84(2), 175 - 85
Increased production of human proinsulin in the periplasmic space of Escherichia coli by fusion to DsbA; Winter J et al.; The production of human proinsulin in its disulfide-intact, native form in Escherichia coli requires disulfide bond formation and the periplasmic space is the favourable compartment for oxidative folding . However, the secretory expression of proinsulin is limited by its high susceptibility to proteolysis and by disulfide bond formation, which is rate-limiting for proinsulin folding . In this report we describe a method for the production of high amounts of soluble, native human proinsulin in E . coli . We fused proinsulin to the C-terminus of the periplasmic disulfide oxidoreductase DsbA via a trypsin cleavage site . As DsbA is the main catalyst of disulfide bond formation in E . coli, we expected increased yields of proinsulin by intra- or intermolecular catalysis of disulfide bond formation . In the context of the fusion protein, proinsulin was found to be stabilised, probably due to an increased solubility and faster disulfide bond formation . To increase the yield of DsbA-proinsulin in the periplasm, several parameters were optimised, including host strains and cultivation conditions, and in particular growth medium composition and supplement of low molecular weight additives . We obtained a further, about three-fold increase in the amount of native DsbA-proinsulin by addition of L-arginine or ethanol to the culture medium . The maximum yield of native human proinsulin obtained from the soluble periplasmic fraction after specific cleavage of the fusion protein with trypsin was 9.2 mg g(-1), corresponding to 1.8% of the total cell protein.

J Biol Chem, 2001 Mar 2, 276(9), 6566 - 75 Epub 2000 Nov 17.
The role of phosphomannose isomerase in Leishmania mexicana glycoconjugate synthesis and virulence; Garami A et al.; Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of fructose 6-phosphate and mannose 6-phosphate, which is the first step in the biosynthesis of activated mannose donors required for the biosynthesis of various glycoconjugates . Leishmania species synthesize copious amounts of mannose-containing glycolipids and glycoproteins, which are involved in virulence of these parasitic protozoa . To investigate the role of PMI for parasite glycoconjugate synthesis, we have cloned the PMI gene (lmexpmi) from Leishmania mexicana, generated gene deletion mutants (Delta lmexpmi), and analyzed their phenotype . Delta lmexpmi mutants lack completely the high PMI activity found in wild type parasites, but are, in contrast to fungi, able to grow in media deficient for free mannose . The mutants are unable to synthesize phosphoglycan repeats {-6-Gal beta 1-4Man alpha 1-PO(4)-} and mannose-containing glycoinositolphospholipids, and the surface expression of the glycosylphosphatidylinositol-anchored dominant surface glycoprotein leishmanolysin is strongly decreased, unless the parasite growth medium is supplemented with mannose . The Delta lmexpmi mutant is attenuated in infections of macrophages in vitro and of mice, suggesting that PMI may be a target for anti-Leishmania drug development . L . mexicana Delta lmexpmi provides the first conditional mannose-controlled system for parasite glycoconjugate assembly with potential applications for the investigation of their biosynthesis, intracellular sorting, and function.

Infect Immun, 2000 Dec, 68(12), 7039 - 48
Phagosome acidification has opposite effects on intracellular survival of Bordetella pertussis and B . bronchiseptica; Schneider B et al.; Bordetella pertussis is readily killed after uptake by professional phagocytes, whereas its close relative Bordetella bronchiseptica is not and can persist intracellularly for days . Phagocytosis of members of either species by a mouse macrophage cell line results in transport of the bacteria to a phagosomal compartment positive for the lysosome-associated membrane protein 1, the protease cathepsin D, and the late endosomal vacuolar proton-pumping ATPase but negative for the early endosome antigen 1 and the early endosomal transferrin receptor . In addition, we demonstrate that Bordetella-containing phagosomes rapidly acidify to pH 4.5 to 5.0 . Taken together, these data demonstrate that Bordetella-containing phagosomes rapidly mature to an acidic late endosomal/lysosomal compartment . Following up on this observation, we determined that B . pertussis does not survive in bacterial growth media adjusted to a pH of 4.5, whereas this pH has only minor effects on the growth of B . bronchiseptica . Raising the intracellular pH in infected macrophages by the addition of bafilomycin A(1), ammonium chloride, or monensin increases the survival of acid-sensitive B . pertussis but, surprisingly, decreases that of acid-tolerant B . bronchiseptica . In summary, we hypothesize that the differential survival of B . pertussis and B . bronchiseptica in macrophages is, at least in part, due to the differences in their acid tolerance.

Infect Immun, 2000 Dec, 68(12), 6970 - 8
Essential role for the Legionella pneumophila rep helicase homologue in intracellular infection of mammalian cells; Harb OS et al.; We have previously isolated 32 mutants of Legionella pneumophila that are defective in the infection of mammalian cells but not protozoa . The mutated loci have been designated macrophage-specific infectivity (mil) loci . In this study we characterized the mil mutant GK11 . This mutant was incapable of growth within U937 macrophage-like cells and WI-26 alveolar epithelial cells . This defect in intracellular replication correlated with a defect in cytopathogenicity to these cells . Sequence analysis of the GK11 locus revealed it to be highly similar to rep helicase genes of other bacteria . Since helicase mutants of Escherichia coli are hypersensitive to thymine starvation, we examined the sensitivity of GK11 to thymineless death (TLD) . In the absence of thymine and thymidine, mutant GK11 did not undergo TLD but was defective for in vitro growth, and the defect was partially restored when these compounds were added to the growth medium . In addition, supplementation with thymidine or thymine partially restored the ability of GK11 to grow within and kill U937 macrophage-like cells . The data suggested that the low levels of thymine or thymidine in the L . pneumophila phagosome contributed to the defect of GK11 within macrophages . Using confocal laser scanning microscopy, we determined the effect of the mutation in the Rep helicase homologue on the intracellular trafficking of GK11 within macrophages . In contrast to the wild-type strain, phagosomes harboring GK11 colocalized with several late endosomal/lysosomal markers, including LAMP-1, LAMP-2, and cathepsin D . In addition, only 50% of the GK11 phagosomes colocalized with the endoplasmic reticulum marker BiP 4 h postinfection . Colocalization of BiP with GK11 phagosomes was absent 6 h postinfection, while 90% of the wild-type phagosomes colocalized with this marker at both time points . We propose that the low level of thymine within the L . pneumophila phagosome in combination with simultaneous exposure to multiple stress stimuli results in deleterious mutations that cannot be repaired in the rep helicase homologue mutant, rendering it defective in intracellular replication.

Eur J Biochem, 2000 Dec, 267(23), 6817 - 23
Regulation of pyc1 encoding pyruvate carboxylase isozyme I by nitrogen sources in Saccharomyces cerevisiae; Huet C et al.; In Saccharomyces cerevisiae, the existence of PYC1 and PYC2 encoding cytosolic pyruvate carboxylase isoform I and II is rather puzzling, owing to the lack of potent differential gene regulation by the carbon sources . We report several findings indicating that these two genes are differentially regulated by the nature of the nitrogen source . In wild-type cells, the activity of pyruvate carboxylase, which is the sum of pyruvate carboxylase isoform I and II, was two- to fivefold lower in carbon medium containing aspartate, asparagine, glutamate or glutamine instead of ammonium as the nitrogen source, whereas it was 1.5- to threefold higher when the ammonium source was substituted by arginine, methionine, threonine or leucine . These enzymatic changes were independent of the nature of the carbon source and closely correlated to the changes in beta-galactosidase from PYC1-lacZ gene fusion and in PYC1 transcripts . Transfer of exponentially growing cells of the pyc2 mutant from an aspartate or a glutamate medium to an ammonium medium caused a fivefold increase in PYC1 mRNA in less than 30 min, whereas in the inverse experiment, PYC1 transcripts returned within 30 min to the low levels found in aspartate/glutamate medium . By contrast, these conditions affected neither the pyruvate carboxylase activity encoded by PYC2 nor PYC2 mRNA . Considering that changes in PYC1 expression inversely correlated with changes in alpha-ketoglutarate concentration or in alpha-ketoglutarate/glutamate ratio following the nitrogen shift experiments, and taking into account the pivotal role of this metabolite in ammonium assimilation, it is suggested that changes in alpha-ketoglutarate or in the alpha-ketoglutarate/glutamate ratio might be implicated in triggering the nitrogen effects on PYC1 expression . The physiological significance of the differential sensitivity of PYC1 and PYC2 genes with respect to the nitrogen source in the growth medium is also discussed.

Plant Sci, 2000 Nov 6, 159(2), 213 - 222
In vitro selection and characterisation of a drought tolerant clone of Tagetes minuta; Mohamed MA et al.; Aromatic Tagetes plants produce secondary products which have a biological activity against a wide range of micro-organisms, insects and nematodes . Tagetes oils are also used as pharmaceuticals and as flavour components in the food industry . This study aimed to use somaclonal variation to select drought tolerant plants of Tagetes . Cotyledons cultured on MS medium containing 3 mg l(-1) IAA and 10 mg l(-1) BA (callus growth medium; CGM) with 60 mM mannitol died . Shoot clumps developed on CGM for 6 months and then subcultured onto CGM containing 80 mM mannitol also died . Four shoots were regenerated from 72 shoot clumps on 12 MS medium containing 0.5 mg l(-1) IAA (shoot growth medium; SGM) after culturing on CGM without mannitol for 6 months and then on CGM with 60 mM mannitol for 3 months . Twelve shoots developed from 72 shoot clumps on SGM after culture for 9 months on CGM . Significant variations were observed in biomass amongst regenerated clones when cultured on medium containing mannitol . After growth in greenhouse conditions for 2 months, one clone developed from shoot clumps selected on medium with mannitol exhibited a significant tolerance in vitro in medium containing 90 mM mannitol; this medium completely inhibited growth of control plants . This clone had significantly higher proline content and soluble sugars than the non-stress-selected clone when cultured on medium containing 0 or 30 mM mannitol . When tested for drought tolerance (growth at 40% soil field capacity) in the greenhouse for 2 months, this clone showed a significant tolerance compared with other regenerated and control plants and revealed lower water potential, greater accumulated biomass and a higher relative growth rate.

Genetics, 2000 Nov, 156(3), 1005 - 23
Genetic analysis reveals that FLO11 upregulation and cell polarization independently regulate invasive growth in Saccharomyces cerevisiae; Palecek SP et al.; Under inducing conditions, haploid Saccharomyces cerevisiae perform a dimorphic transition from yeast-form growth on the agar surface to invasive growth, where chains of cells dig into the solid growth medium . Previous work on signaling cascades that promote agar invasion has demonstrated upregulation of FLO11, a cell-surface flocculin involved in cell-cell adhesion . We find that increasing FLO11 transcription is sufficient to induce both invasive and filamentous growth . A genetic screen for repressors of FLO11 isolated mutant strains that dig into agar (dia) and identified mutations in 35 different genes: ELM1, HSL1, HSL7, BUD3, BUD4, BUD10, AXL1, SIR2, SIR4, BEM2, PGI1, GND1, YDJ1, ARO7, GRR1, CDC53, HSC82, ZUO1, ADH1, CSE2, GCR1, IRA1, MSN5, SRB8, SSN3, SSN8, BPL1, GTR1, MED1, SKN7, TAF25, DIA1, DIA2, DIA3, and DIA4 . Indeed, agar invasion in 20 dia mutants requires upregulation of the endogenous FLO11 promoter . However, 13 mutants promote agar invasion even with FLO11 clamped at a constitutive low-expression level . These FLO11 promoter-independent dia mutants establish distinct invasive growth pathways due to polarized bud site selection and/or cell elongation . Epistasis with the STE MAP kinase cascade and cytokinesis/budding checkpoint shows these pathways are targets of DIA genes that repress agar invasion by FLO11 promoter-dependent and -independent mechanisms, respectively.

J Med Chem, 2000 Nov 2, 43(22), 4212 - 8
Inhibition of protein synthesis by didemnins: cell potency and SAR; Ahuja D et al.; Synthetic and naturally occurring didemnins are potent and specific inhibitors of protein synthesis in vitro . Structure-activity analysis indicates a requirement for the intact macrocycle; however, the smaller ring size represented by the didemnin analogue, tamandarin A, is equipotent to didemnin B . Replacement of the N,O-dimethyltyrosine by a N-methylphenylalanine or N-methylleucine residue is also well-tolerated . The rank order for inhibition of protein synthesis in vitro appears to be retained in MCF-7 cells, albeit at much higher potency . This increase in potency is explained for the first time by data indicating that MCF-7 cells can accumulate didemnin B up to 2-3 orders of magnitude compared to the growth medium.

Microbiol Res, 2000 Sep, 155(3), 209 - 14
Effect of water-soluble vitamins on the production of indole-3-acetic acid by Azospirillum brasilense; Zakharova EA et al.; The effects of six water-soluble vitamins on tryptophan-dependent synthesis of indole-3-acetic acid in Azospirillum brasilense were investigated . A multifactorial regression analysis was employed to produce models of indole-3-acetic acid synthesis versus concentrations of tryptophan and the vitamins added to the growth medium . Very low levels of the B-group vitamins added at 10 to 100 microg l(-1) affected production of indole-3-acetic acid in A . brasilense . The largest release of this phytohormone was observed after amendment with pyridoxine and nicotinic acid . Results of the study suggest a role these vitamins may fulfil in the regulation of indole-3-acetic acid synthesis in A . brasilense.

Sheng Wu Gong Cheng Xue Bao, 2000 May, 16(3), 387 - 91
{Production of u-PA with rCHO cell culture on porous microcarriers in serum-free growth medium}; Hu XW et al.; A novel technique was developed to deal with apoptosis in large-scale animal cell culture . By means of replacing part of Cytopore porous microcarriers at regular intervals, a rCHO cell line, which produces urokinase-type plasminogen activitor(u-PA), was cultivated continuously with serum-free medium in a 30 L stirred tank for 91 days . The cell density was maintained at (1.3-2.6) x 10(7)/mL, and > 90% of cells was viable . In order to reduce the effect of cell density on cell growth and expression, a cyclic pressure oscillation was exerted on a 7.5 L reactor headspace to enhance cell expression at high cell density to a certain extent . During the 67 days of medium-replacement culture, the maximal cell density reached 2.64 x 10(7)/mL, and cell viability was always kept above 95% when combined with microcarrier-replacement . Compare to control culture, culture with cyclic pressure oscillation could enhance cell expression level and reduce the ratio of glucose metabolized anaerobically to produce lactate . With four-step purification process, about 80 g u-PA(approximately 90% scu-PA) was recovered from approximately 2100 liters supernatant which contained approximately 135 g u-PA.

Chemosphere, 2000 Nov, 41(10), 1669 - 74
The acute and chronic toxicity of lanthanum to Daphnia carinata; Barry MJ et al.; The rare earth elements (REEs) are increasingly being used as trace supplements in agriculture . This study measured the acute and chronic toxicity of one REE, lanthanum (La), to Daphnia carinata . The 48-h EC50 of La to Daphnia was measured in three media of differing composition and hardness . Lanthanum was most toxic to Daphnia in soft tap water (TW) with an acute 48-h EC50 of 43 microg/l compared with 1180 microg/l in ASTM hard water (ASTM) . In the third daphnid growth medium (DW), based on diluted sea water, the acute 48-h EC50 was 49 microg La/l, however, there was significant precipitation of La in this media . The chronic toxicity of La to Daphnia was measured in the DW and ASTM media . Nominal exposure concentrations were 100, 200, 400, 600, 800, and 1000 microg La/l . Mortality was a more sensitive endpoint than growth or reproduction in both chronic experiments . Very little La was detected in either media after 24 h and the measured concentrations below were estimated by logarithmic mean of nominal and measured values . There was 100% mortality at concentrations > or = 80 microg La/l (400 microg/l nominal) by day six of the experiment using DW media, but no effect on survival growth or reproduction at lower concentrations . In the ASTM media, La caused significant mortality to Daphnia at concentrations > or = 39 microg/l (200 microg/l nominal), however, at least one animal survived to the end of the study at each of the tested concentrations . There was no effect of La on growth of surviving daphnids at concentrations < or = 57 microg/l (400 g/l), however, second brood clutch sizes were significantly increased at 30, 39, and 57 microg/l (100, 200, 400 g/l nominal) compared with controls . Lanthanum also caused a delayed maturation in Daphnia.

Folia Neuropathol, 2000, 38(2), 47 - 53
Aluminum enhances glutamate-mediated neurotoxicity in organotypic cultures of rat hippocampus; Matyja E; Both, glutamate (GLU) and aluminum (Al) have been implicated in neuronal damage and/or death in certain human neurodegenerative disorders . Recent evidence suggests that aluminum (Al) may potentiate the increase in glutamate-induced intracellular calcium overload . The present ultrastructural study was undertaken to determine the effect of Al on the development of GLU-mediated neurotoxicity in tissue culture conditions . The experiments were performed on organotypic cultures of rat hippocampus treated with low, subtoxic concentration of GLU (50 microM) and AlCl3 (400 microM) added to the growth medium separately or in combination . The exposure of cultures to GLU in the presence of Al3+ ions for up to 24 hours resulted in the development of typical excitotoxic neuronal changes, whereas separate GLU treatment at subtoxic doses or single Al application did not produce any apparent tissue damage . The neuronal lesions resulting from the combined application of GLU plus Al consisted predominately of more or less pronounced mitochondrial abnormalities, which are characteristic for early excitotoxic events . Severe swelling of the mitochondria led to the disruption of their internal structure and finally resulted in an apparent microvacuolization of the perikaryal cytoplasm of some pyramidal neurons . The present morphological data evidenced that Al is capable to potentiate the GLU-induced degenerative changes in hippocampal neurons in vitro . This supports the view of a possible role of Al in the process of neurodegeneration and suggests that Al may participate in the development of glutamate-mediated excitotoxic neuronal injury under certain pathological conditions.

Folia Neuropathol, 2000, 38(1), 13 - 21
Extracellular signal-regulated kinase suppression is not involved in apoptosis of neuroblastoma N2a cells induced by protein kinase C inhibition; Bronisz-Kowalczyk A et al.; A model study for the mechanism of cell death in vitro was established on the neuroblastoma N2a cell line . By differential staining of living cells with Hoechst 33258 and propidium iodide, or by Terminal-dUTP-Transferase-Nick-End Labelling (TUNEL), cJun/AP1 immunoreactivity, and DNA laddering, we show that the N2a cell line responded with apoptosis to protein kinase C (PKC) inhibition . Two different classes of PKC inhibitors (staurosporine and Go6976) at concentrations generally regarded as PKC-selective (10 nM and 50 nM, respectively), significantly increased the number of apoptotic cells in N2a cultures . The cells started to die 2-3 hours after the treatment; then, at 6 and/or 24 hours, approximately 30-40% of the cells acquired the apoptotic feature . This response was dependent on neither cell differentiation nor PKC and bcl2 gene and protein expression, but exclusively on the concomitant withdrawal of serum from growth medium . Furthermore, both of the experimental manipulations (serum deprivation and PKC inhibition) synergically, but to different extents, suppressed the extracellular signal regulated kinase (ERK) pathway . Their pro-apoptotic effect, however, was neither mimicked nor modified by an additional inhibition of MEK/ERK kinase by 50 microM PD 98059, resulting in an 80% inhibition of its initial activity . We conclude, therefore, that apoptosis of N2a cells triggered by PKC-inhibition, as well as its abrogation by serum, is totally independent of concomitant modulations of ERK activity also evoked by the above treatments.

Sheng Wu Gong Cheng Xue Bao, 2000 Jul, 16(4), 464 - 8
{The influences of lactose as an inducer on the expression of the recombinant proteins in Escherichia coli BL21 (DE3)}; Zhang Y et al.; The possibility of using lactose as an inducer to substitute the common inducer IPTG in the fermentation process of the recombinant microorganism was deeply investigated . The influences of culture conditions such as lactose concentration, growth medium composition, the point of induction and the duration of the induction phase on the expression of the recombinant protein were analyzed and studied in detail . In the following experiments, lactose was then used in the high cell density culture process of E . coli BL21 (DE3)(pFu) . The final cell density (OD600) was over 40 . The expression level of recombinant protein was about 15% of the total cellular protein . Both the culture density and foreign protein expression level were lower than those induced by IPTG . However, because of the potential toxicity to human beings and the high cost of IPTG, the use of lactose might provide an alternative means of inducing foreign protein expression . This would be more attractive in industrial scale productions of recombinant proteins . The results confirmed that lactose could be used as an inducer in the fermentation process.

Brain Res Dev Brain Res, 2000 Oct 28, 123(2), 151 - 64
Activity-dependent development of spontaneous bioelectric activity in organotypic cultures of rat occipital cortex; Echevarria D et al.; The development of spontaneous bioelectric activity (SBA) in organotypic tissue cultures (OTCs) from rat occipital cortex was studied by means of extracellular recording techniques in OTCs grown normally for 6-51 days in vitro (DIV), and in OTCs in which SBA had been silenced from DIV 4 on for 2 to 3 weeks by elevating the Mg(2+) levels in the growth medium . The proportions of spontaneously active neurones increased from about 25% at 6-14 DIV to more than 80% beyond the third week in vitro . Mature neurones discharged at shorter intervals and more vigorously than immature neurones; the developmental increase in firing rate was not significant, however . In OTCs 6-14 DIV the majority of spontaneously active neurones fired sluggishly in a regular manner . The remaining neurones fired action potentials in the form of discrete bursts resembling interictal activity in vivo . The proportions of these neurones increased from about 40% at 6-14 DIV to more than 80% beyond the third week in vitro . During development in vitro the mean burst duration increased from 3.5 s to about 8 s whereas the mean burst rate (between 0.7-1 bursts/min) remained constant . Activity-deprived neurons had low firing rates and fired action potentials in the form of discrete bursts with a mean burst rate of 0.4/min . The proportions of spontaneously active neurons, the variability of neuronal firing and the viability of the explants either were not altered by the activity blockade or had recovered to control values after 5-6 days in normal growth medium . We conclude that in OTCs of rat neocortex the absence of SBA during development in vitro delays the maturation of excitatory mechanisms responsible for the developmental increase in firing intensity . The development of burst firing modes is less affected by activity blockade.

FEMS Microbiol Lett, 2000 Nov 1, 192(1), 139 - 44
Production of shiga toxin by Escherichia coli measured with reference to the membrane vesicle-associated toxins; Yokoyama K et al.; Production of Shiga toxin (Stx) 1 and 2 from Stx-producing Escherichia coli (STEC) was measured with reference to the membrane vesicle (MV)-associated toxins . An immunoblot analysis method using specific antibodies for Stx1 and Stx2 was developed for the detection of the extracellular toxins . All 46 STEC isolates, studied including 30 O157 and 16 other O-antigenic isolates, released Stx1 and Stx2 as MV-associated and MV-removed fractions under aerobic and anaerobic conditions . Treatment of vesicles with polymyxin B that disrupted MVs increased the release of Stx1 and Stx2 . Therefore, delivery of Stx1 and Stx2 by MVs is a general mechanism in STEC . Stx1 remained within MVs rather than in the MV-removed fraction under an aerobic culture condition . On the other hand, a larger proportion of Stx2 was detected in the MV-removed fraction . The kinetic patterns of the release of the toxins from STEC strains showed that both Stx1 and Stx2 were released into the growth medium during the exponential growth phase . An rpoS-deficient mutation did not have altered levels of extracellular Stx1 and Stx2, supporting the idea that Stx1 and Stx2 are produced during exponential growth phase.

J Dent Res, 1988 Jan, 67(1), 75 - 81
Degradation of starch and its hydrolytic products by oral bacteria; Glor EB et al.; Selected strains of oral bacteria were analyzed for their ability to degrade wheat starch, maltose, maltotriose, and maltoheptaose . S . sanguis IUOM-11M and JC804, S . mutans 6715, S . salivarius IUOM-8, A . viscosus IUOM-62, and A . naeslundii ATCC 12104 degraded all four substrates . S . mutans NCTC 10449 degraded starch, maltose, and maltotriose, while A . viscosus ATCC 15987 degraded starch and maltose, and S . sanguis SS34 degraded only maltose . L . casei IUOM-14 did not degrade any of the substrates . Analysis of starch degradation products from S . sanguis IUOM-11M and A . viscosus IUOM-62 demonstrated oligosaccharides, maltose, and trace amounts of glucose for the former and oligosaccharides, maltotriose, and maltose for the latter . S . sanguis IUOM-11M alpha-glucosidase (EC 3.2.1.20) demonstrated a pH optimum of 6.5 and greatly enhanced activity from maltose-cultured cells as compared with cells cultured in glucose or fructose . The presence of fructose in the growth medium prevented this enhancement of activity by maltose . Maltose inhibited sucrose-dependent synthesis of S . sanguis IUOM-11M insoluble polysaccharide and both primer-dependent and primer-independent synthesis of soluble polysaccharide . Maltoheptaose inhibited primer-dependent but not primer-independent soluble polysaccharide synthesis . Several oral bacteria have the ability to hydrolyze starch and to degrade further the products to acidogenic substrates . These products may also inhibit sucrose-dependent synthesis of polysaccharides, which enhances the production of the acidogenic substrate fructose . The results add further support to the growing body of evidence suggesting that caries-promoting properties of starch may be expressed only when starch is present in diets containing sucrose.

J Gen Virol, 2000 Nov, 81(Pt 11), 2697 - 705
Entry of influenza viruses into cells is inhibited by a highly specific protein kinase C inhibitor; Root CN et al.; Following binding to cell surface sialic acid, entry of influenza viruses into cells is mediated by endocytosis . Productive entry of influenza virus requires the low-pH environment of the late endosome for fusion and release of the virus into the cytoplasm and transport of the virus genome into the nucleus . We investigated novel mechanisms to inhibit influenza virus infection using highly specific inhibitors of protein kinase C . We found that one inhibitor, bisindolylmaleimide I, prevented replication of influenza A virus in a dose-dependent manner when added at the time of infection, but had little specific effect when added 2 h after infection had commenced . Virus yields dropped by more than 3 log units in the presence of micromolar levels of bisindolylmaleimide I . Influenza B virus replication was also inhibited by bisindolylmaleimide at micromolar concentrations . We carried out experiments to determine the point in infection that was blocked by bisindolylmaleimide I, and determined that entry of viral ribonucleoproteins (vRNPs) into the nucleus was prevented . Upon drug washout vRNP nuclear entry resumed, showing that bisindolylmaleimide I is reversible . Bisindolylmaleimide I did not affect virus binding and was apparently not acting as a weak base, because its effects were independent of the pH of the external growth medium . These experiments show that bisindolylmaleimide I blocks replication of different types of influenza virus in a dose-dependent and reversible manner, and that virus entry into the cell is inhibited.

Cell Transplant, 2000 Jul-Aug, 9(4), 519 - 29
A factor implicated in the myogenic conversion of nonmuscle cells derived from the mouse dermis; Goldring K et al.; Using the mdx mouse model for human Duchenne muscular dystrophy we have shown that a cell population residing in the dermis of C57B1/10ScSn mouse skin is capable of converting to a myogenic lineage when implanted into the mdx muscle environment . It was important to determine the characteristics of the converting cell . A previous in vitro study indicated that 10% of cells underwent conversion but only when the cells were grown in medium previously harvested from a myogenic culture . In the present study we cloned cells derived from the dermis to identify the converting cells . Clones grown in normal growth medium showed no conversion, but when grown in medium conditioned by muscle cells around 40% conversion was achieved in several individual clones . We investigated whether the protein beta-galactoside binding protein (betaGBP), which is secreted by myoblasts and acts as a cell growth regulator of fibroblasts . could be a candidate factor responsible for conversion . Medium harvested from COS-1 cells infected with a construct containing betaGBP has been used for this investigation . Growth of dermal fibroblasts in medium enriched with this factor showed a high rate of conversion to cells expressing muscle-specific factors.

Infect Immun, 2000 Nov, 68(11), 6094 - 100
Secretion of RTX leukotoxin by Actinobacillus actinomycetemcomitans; Kachlany SC et al.; Actinobacillus actinomycetemcomitans, the etiologic agent for localized juvenile periodontitis and certain other human infections, such as endocarditis, expresses a leukotoxin that acts on polymorphonuclear leukocytes and macrophages . Leukotoxin is a member of the highly conserved repeat toxin (RTX) family of bacterial toxins expressed by a variety of pathogenic bacteria . While the RTX toxins of other bacterial species are secreted, the leukotoxin of A . actinomycetemcomitans is thought to remain associated with the bacterial cell . We have examined leukotoxin production and localization in rough (adherent) and smooth (nonadherent) strains of A . actinomycetemcomitans . We found that leukotoxin expressed by the rough, adherent, clinical isolate CU1000N is indeed cell associated, as expected . However, we were surprised to find that smooth, nonadherent strains of A . actinomycetemcomitans, including Y4, JP2 (a strain expressing a high level of toxin), and CU1060N (an isogenic smooth variant of CU1000N), secrete an abundance of leukotoxin into the culture supernatants during early stages of growth . After longer times of incubation, leukotoxin disappears from the supernatants, and its loss is accompanied by the appearance of a number of low-molecular-weight polypeptides . The secreted leukotoxin is active, as evidenced by its ability to kill HL-60 cells in vitro . We found that the growth phase and initial pH of the growth medium significantly affect the abundance of secreted leukotoxin, and we have developed a rapid (<2 h) method to partially purify large amounts of leukotoxin . Remarkably, mutations in the tad genes, which are required for tight nonspecific adherence of A . actinomycetemcomitans to surfaces, cause leukotoxin to be released from the bacterial cell . These studies show that A . actinomycetemcomitans has the potential to secrete abundant leukotoxin . It is therefore appropriate to consider a possible role for leukotoxin secretion in the pathogenesis of A . actinomycetemcomitans.

Arch Environ Contam Toxicol, 2000 Nov, 39(4), 440 - 4
Assessment of barium toxicity in bush beans; Llugany M et al.; External and internal lowest observed effect concentrations (LOECs) for Ba in bean plants (Phaseolus vulgaris) were established using a nutrient solution culture system where BaSO(4) precipitation in the growth medium was avoided . This was achieved by alternating every 24 h with a nutrient solution containing Ba (0, 50, 500, or 5000 microM) and all essential elements except S, with another containing S and all other nutrients but no Ba . The external LOEC for acute toxicity symptoms in the form of leaf withering and leaf growth inhibition was 481 microM of free Ba(2+) . This was also the LOEC for the complete inhibition of elongation of secondary roots, while for the elongation of the primary root the external LOEC was 4,821 microM . Barium interfered with both the sulfate transport from roots to shoots and the import of Ca into leaves . However, K was the most Ba-sensitive nutrient . External LOEC for reduced leaf K concentrations was 48 microM free Ba(2+); the corresponding internal LOECs for primary and trifoliolate leaves were 700 and 460 mg kg(-1) DW, respectively.

Biochim Biophys Acta, 2000 Aug 25, 1467(2), 419 - 30
A new cationic liposome for efficient gene delivery with serum into cultured human cells: a quantitative analysis using two independent fluorescent probes; Serikawa T et al.; Cationic liposomes are useful to transfer genes into eukaryotic cells in vitro and in vivo . However, liposomes with good transfection efficiency are often cytotoxic, and also require serum-free conditions for optimal activity . In this report, we describe a new formulation of cationic liposome containing DC-6-14, O,O'-ditetradecanoyl-N-(alpha-trimethylammonioacetyl)diethan olamine chloride, dioleoylphosphatidylethanolamine and cholesterol for gene delivery into cultured human cells . This liposome, dispersed in 5% serum-containing growth medium, efficiently delivered a plasmid DNA for GFP (green fluorescent protein) into more than 80% of the cultured human cell hybrids derived from HeLa cells and normal fibroblasts . Flow cytometric analysis revealed that the efficiency of the GFP gene expression was 40-50% in a tumor-suppressed cell hybrid, while it was greatly reduced in the tumorigenic counterpart . The enhanced GFP expression in tumor-suppressed cell hybrids was quantitatively well correlated with a prolonged presence of the plasmid DNA, which had been labeled with another fluorescent probe, ethidium monoazide, within the cells . These results suggest that a newly developed cationic liposome is useful for gene delivery in serum-containing medium into human cells and the stability of the plasmid DNA inside the cell is a crucial step in this liposome-mediated gene expression . The mechanisms by which cationic liposome mediates gene transfer into eukaryotic cells are also discussed.

Biochem Biophys Res Commun, 2000 Oct 14, 277(1), 255 - 60
Intracellular FGF-2 promotes differentiation in T-47D breast cancer cells; Korah RM et al.; To test the implicated role of basic fibroblast growth factor (bFGF; FGF-2) in promoting differentiation in breast cancer, we enforced the expression of FGF-2 in T-47D breast cancer cells . Expression of FGF-2 conferred an overall less malignant phenotype to T-47D cells as revealed by their reduced proliferative response, impaired capacity for anchorage-independent growth, and invasion through Matrigel . To understand one candidate mechanism for the intracellular FGF-2-mediated anti-invasive effect, we examined the effect of FGF-2 on T-47D cell motility . Addition of recombinant FGF-2 to the growth medium markedly enhanced cell motility while constitutive expression of intracellular FGF-2 significantly inhibited the migratory potential of T-47D cells in a dominant manner . FGF-2-expressing T-47D cells also formed relatively defined branching structures in Matrigel matrices, a characteristic phenotype of differentiation in breast cancer cells . These data suggest a potential role for FGF-2 in promoting functional differentiation of breast epithelial cells .

Biotechnol Prog, 2000 Sep-Oct, 16(5), 782 - 5
Osmolarity effects on observed insect cell size after baculovirus infection are avoided using growth medium for sample dilution; Rosinski M et al.; Rates of cell size increase are an important measure of success during the baculovirus infection process . Batch and fed batch cultures sustain large fluctuations in osmolarity that can affect the measured cell volume if this parameter is not considered during the sizing protocol . Where osmolarity differences between the sizing diluent and the culture broth exist, biased measurements of size are obtained as a result of the cell osmometer response . Spodoptera frugiperda (Sf9) cells are highly sensitive to volume change when subjected to a change in osmolarity . Use of the modified protocol with culture supernatants for sample dilution prior to sizing removed the observed error during measurement.

Microbiology, 2000 Oct, 146 ( Pt 10), 2715 - 22
Dependence of Trichomonas vaginalis upon polyamine backconversion; Yarlett N et al.; Trichomonas vaginalis grown for 16 h in the presence of {(14)C}spermine formed a high intracellular pool of {(14)C}spermidine and a small but detectable pool of {(14)C}putrescine . When {(3)H}putrescine was added to the growth medium, a large intracellular pool of {(3)H}putrescine was found, but it was not further metabolized, confirming previous studies suggesting the absence of a forward-directed polyamine synthetic pathway in T . vaginalis . Spermidine:spermineN:(1)-acetyltransferase (SSAT) and polyamine oxidase enzyme activities were detected which collectively converted spermine to spermidine . Polyamine oxidase was localized in the hydrogenosome-enriched fraction, whereas SSAT was found predominantly in the cytosolic fraction . In the presence of saturating substrate, the trichomonad SSAT had an activity of 0 . 39+/-0.09 nmol min(-1) (mg protein)(-1) (the mean of five analyses) and an apparent K:(m) for spermine of 1.7 microM . The enzyme was competitively inhibited by di(ethyl)norspermine with a K:(i) of 28 microM . Growth studies indicated that 50 microM di(ethyl)norspermine caused a 68% and 84% reduction in the intracellular concentrations of spermidine and spermine, respectively . The trichomonad polyamine oxidase required FAD as a cofactor and had an apparent K:(m) of 6.0 microM for N(1)-acetylspermine . The potential of bis(alkyl) polyamine analogues as antitrichomonad agents is discussed.

Bone Marrow Transplant, 2000 Sep, 26(5), 505 - 10
Clinical impact of ex vivo differentiated myeloid precursors after high-dose chemotherapy and peripheral blood progenitor cell rescue; Zimmerman TM et al.; The infusion of ex vivo differentiated myeloid precursors may be able to shorten the period of obligatory neutropenia after high-dose chemotherapy and peripheral blood progenitor cell rescue by providing cells capable of differentiating to mature neutrophils within days of infusion . To test this hypothesis, 21 female patients with metastatic breast cancer underwent progenitor cell mobilization with cyclophosphamide, etoposide and G-CSF . CD34+ cells from one to two leukapheresis products were isolated and placed in suspension culture with a serum-free growth medium supplemented with PIXY321 . The cultures were maintained for 12 days with subcultures initiated on day 7 . The remaining leukapheresis products were cryopreserved in an unmanipulated state . Forty-eight hours after completing high-dose cyclophosphamide, thiotepa and carboplatin, the cryopreserved progenitors were infused, followed 1 to 24 h later by infusion of the differentiated myeloid precursors . In one patient, the cultured cells were labeled with Indium-111 with nuclear imaging performed up to 48 h post infusion . The differentiated myeloid precursors were suitable for infusion in 17 of the patients with a median 13-fold expansion of total nucleated cells . A range of 5.6 to 1066 x 10(7) nucleated cells were infused . Morphologically the cells were predominantly of myeloid lineage (63%) with a median 41% of the cells expressing CD15 . No untoward effects were noted with the infusion of the cultured cells . The median days to neutrophil and platelet recovery were 8 and 10 days, respectively . There was a significant relationship (r = 0.67, P = 0.007) between the dose of differentiated myeloid precursors (CD15+ cells) and the depth and duration of neutropenia; a similar relationship, however, was also observed with the dose of cryopreserved CD34+ cells . After infusion of the radiolabeled myeloid precursors, a pattern of distribution similar to radio-labeled granulocytes was noted with uptake detected initially in the lungs and subsequently the reticulo-endothelial system . The impact of differentiated myeloid precursors on neutropenia as an adjunct to high-dose chemotherapy and peripheral blood progenitor cell rescue remains unclear from this study . Further study with controlled doses of cryopreserved progenitors and escalating doses of differentiated myeloid precursors is required.

FEBS Lett, 2000 Sep 29, 482(1-2), 25 - 30
Expression of doubly labeled Saccharomyces cerevisiae iso-1 ferricytochrome c and (1)H, (13)C and (15)N chemical shift assignments by multidimensional NMR; Szabo CM et al.; We have expressed {U-(13)C,(15)N}-labeled Saccharomyces cerevisiae iso-1 cytochrome c C102T;K72A in Escherichia coli with a yield of 11 mg/l of growth medium . Nuclear magnetic resonance (NMR) studies were conducted on the Fe(3+) form of the protein . We report herein chemical shift assignments for amide (1)H and (15)N, (13)C(omicron), (13)C(alpha), (13)C(beta), (1)H(alpha) and (1)H(beta) resonances based upon a series of three-dimensional NMR experiments: HNCA, HN(CO)CA, HNCO, HN(CA)CO, HNCACB, HCA(CO)N, HCCH-TOCSY and HBHA(CBCA)NH . An investigation of the chemical shifts of the threonine residues was also made by using density functional theory in order to help solve discrepancies between (15)N chemical shift assignments reported in this study and those reported previously.

J Biol Chem, 2000 Dec 29, 275(52), 40887 - 96
Regulation of the DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol and growth phase . Inhibition of DGPP phosphatase activity by CDP-diacylglyceron and activation of phosphatidylserine synthase activity by DGPP; Oshiro J et al.; The regulation of the Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol supplementation and growth phase was examined . Addition of inositol to the growth medium resulted in a dose-dependent increase in the level of DGPP phosphatase activity in both exponential and stationary phase cells . Activity was greater in stationary phase cells when compared with exponential phase cells, and the inositol- and growth phase-dependent regulations of DGPP phosphatase were additive . Analyses of DGPP phosphatase mRNA and protein levels, and expression of beta-galactosidase activity driven by a P(DPP1)-lacZ reporter gene, indicated that a transcriptional mechanism was responsible for this regulation . Regulation of DGPP phosphatase by inositol and growth phase occurred in a manner that was opposite that of many phospholipid biosynthetic enzymes . Regulation of DGPP phosphatase expression by inositol supplementation, but not growth phase, was altered in opi1Delta, ino2Delta, and ino4Delta phospholipid synthesis regulatory mutants . CDP-diacylglycerol, a phospholipid pathway intermediate used for the synthesis of phosphatidylserine and phosphatidylinositol, inhibited DGPP phosphatase activity by a mixed mechanism that caused an increase in K(m) and a decrease in V(max) . DGPP stimulated the activity of pure phosphatidylserine synthase by a mechanism that increased the affinity of the enzyme for its substrate CDP-diacylglycerol . Phospholipid composition analysis of a dpp1Delta mutant showed that DGPP phosphatase played a role in the regulation of phospholipid metabolism by inositol, as well as regulating the cellular levels of phosphatidylinositol.

Mikrobiologiia, 2000 Jul-Aug, 69(4), 518 - 26
{Effect of nitrogen sources on biosynthesis, chemical composition, and structure of exopolysaccharides from Aureobasidium pullulans (de Bary) Arnaud}; Kudriashova OA et al.; The effect of the nitrogen source and the C/N ratio of the growth medium on the biosynthesis, composition, and structure of the exopolysaccharides (EPSs) of Aurebasidium pullulans (de Bary) Arnaud var . aubasidani Yurlova var . nov . and A . pullulans var . pullulans was studied . A . pullulans var . pullulans and A . pullulans var . aubasidani strains synthesized the maximum amounts of EPSs in the presence of, respectively, a reduced nitrogen source ((NH4)2SO4) and an oxidized nitrogen source (NaNO3) in the medium . The data presented confirm the validity of using the chemical composition and structure of the major cetavlon-precipitated fraction of A . pullulans EPSs for the characterization of intraspecies taxa.

Arch Tierernahr, 2000, 53(3), 241 - 52
Fermentation of carbohydrates and yield of microbial protein in mixed cultures of rabbit caecal microorganisms; Marounek M et al.; Fermentation pattern and yields of microbial protein were investigated in cultures of the rabbit caecal contents supplied with glucose, xylose, starch, pectin and xylan . Rabbits at the age of 4 weeks (before weaning) and 3 months were slaughtered, their caecal contents added at 1.1% to growth media and incubated anaerobically at 39 degrees C for 18 h . Caecal microorganisms of 4-week-old rabbits produced no methane and caproate, less butyrate, but more propionate than microorganisms of 3-month-old rabbits . In both groups of rabbits, fermentation of xylose produced significantly more propionate and less butyrate than fermentation of glucose . More propionate and less acetate was formed from starch than from pectin . In caecal cultures from 4-week-old rabbits with pectin, the molar percentages of acetate was significantly higher and percentages of other short-chain fatty acids (SCFA) lower than in cultures with starch or xylan . In cultures from 3-month-old rabbits, fermentation of pectin and xylan produced similar SCFA profiles, different from SCFA molar composition in cultures with starch . Average production of microbial protein was 129 mg per 1 g of carbohydrate digested (range 110 to 141 mg/g) . Protein yields were the same on glucose and xylose, but nonsignificantly higher on starch than on pectin and xylan . It can be concluded that the characteristics of substrate affected fermentation pattern in mixed cultures of rabbit caecal microorganisms . Substrate effects on protein yields were not statistically significant, due to high variation.

NMR Biomed, 2000 Oct, 13(6), 349 - 60
Metabolism of alternative substrates and the bioenergetic status of EMT6 tumor cell spheroids; Wehrle JP et al.; In order to evaluate the ability of EMT6/Ro multicellular spheroids to utilize various pathways of energy production, (13)C and (31)P MRS have been employed to monitor the metabolism of glucose, glutamine, acetate and propionate . EMT6/Ro spheroids perfused with culture medium containing 5.5 mM glucose maintain stable levels of nucleotide triphosphates (NTP) and phosphocreatine (PCr) for up to 48 h, even in the absence of glutamine . The metabolism of 1-(13)C-glucose was almost entirely to 3-(13)C-lactate (88 +/- 12%, n = 7), even though the perfusion medium was equilibrated with 95% O(2) . Labeling was also observed in other glycolytic metabolites, primarily alanine and alpha-glycerolphosphate . A low level of (13)C labeling in glutamate, indicative of mitochondrial oxidative metabolism (TCA cycle), was consistently detected when spheroids were perfused with 1-(13)C-glucose, almost exclusively in the C4 position of glutamate . Labeling of glutamate C2 and C3 was always less than 20% of the labeling in C4 and was usually undetectable . No evidence of adjacent carbon labeling in individual glutamate molecules (indicative of multiple cycles of label incorporation) was found, even in high-resolution (13)C NMR spectra of extracts from cells or spheroids . Despite the predominantly glycolytic metabolism of glucose, the mitochondrial substrate glutamine (2 mM, in the presence of < or =0.5 mM glucose from fetal bovine serum), supported stable levels of NTP and PCr in the tumor cells for up to 12 h . In the presence of 2.5 mM acetate, the bioenergetic status of cells in EMT6 spheroids declined slowly but measurably, and no incorporation of label from 2-(13)C-acetate into other metabolites was detected either in intact perfused spheroids or in high-resolution spectra of extracts . In contrast, when the anaplerotic TCA cycle substrate 3-(13)C-propionate replaced acetate, the high-energy phosphate levels in EMT6/Ro spheroids were somewhat reduced, but stabilized at a new lower level . Incubation of spheroids with 3-(13)C-propionate (with natural abundance glucose and glutamine) resulted in label detectable in the C2 and C3 of glutamate, but the primary labeled compound was methylmalonate, an intermediate in propionate metabolism . Addition of vitamin B(12), a cofactor for methylmalonyl CoA reductase, to the growth medium 24 h prior to perfusion with propionate resulted in the elimination of the methylmalonate resonance . A variety of 2- and 3-labeled metabolites were detected, including succinate, malate and glutamate . Labeling of C2 and C3 of lactate implicated cytoplasmic malic enzyme activity .

Am J Physiol Endocrinol Metab, 2000 Oct, 279(4), E782 - 90
Activation of members of the mitogen-activated protein kinase family by glucose in endothelial cells; Liu W et al.; To better understand the molecular mechanisms for hyperglycemia-induced proatherogenic changes in endothelial cells, the effect of high glucose on activation of members of the mitogen-activated protein kinase (MAPK) family, including c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase (ERK)-1, -2, and -5, and p38 kinase, was examined in bovine pulmonary artery endothelial cells (PAEC) . Glucose, fructose, and raffinose induced a concentration-dependent decrease in PAEC growth . Addition of 25 mM glucose, fructose, or raffinose to normal growth medium stimulated an approximately twofold increase in JNK1 activity that was maximal after 24 h, whereas only glucose markedly increased ERK5 activity . Neither ERK1/2 nor p38 kinase activity was increased by glucose, fructose, or raffinose . The antioxidant N-acetylcysteine partially abrogated the glucose-induced increase in ERK5 activity but had no effect on the increase in JNK1 activity . In contrast, azaserine, which prevents increased flux through the hexosamine pathway, decreased glucose-induced JNK1 activity but had no effect on fructose- or raffinose-induced JNK1 activity . Consistent with this finding, glucosamine stimulated a 2.4-fold increase in JNK1 activity and reproduced the inhibitory effect of glucose on PAEC growth . In summary, glucose activates different members of the MAPK family in PAEC via distinct mechanisms . Moreover, the correlation between the ability of different sugars to activate JNK1 and inhibit cell growth suggests that activation of this signaling pathway may contribute to the growth inhibitory effect of glucose in endothelial cells.

J Microbiol Methods, 2000 Sep, 42(1), 49 - 55
Relating growth dynamics and glucoamylase excretion of individual Saccharomyces cerevisiae cells; Porro D et al.; We have developed a novel flow cytometric procedure that allows determinations of properties of protein excretion in the growth medium on a cell-by-cell basis in Saccharomyces cerevisiae . The procedure is based on labelling of a periplasmically secreted protein with antibodies conjugated to a fluorescent marker such as fluorescein isothiocyanate (FITC) . The staining conditions did not perturb cell growth after resuspension of stained cells in growth medium . Decrease in fluorescence was found to correlate with excretion of glucoamylase into the growth medium . The analysis of the staining pattern over time provides information on the behaviour of individual cells belonging to different cell-cycle phases and can be used to calculate the specific excretion rate of the overall population.

Mol Pharmacol, 2000 Oct, 58(4), 709 - 18
Cellular resistance to the antitumor DNA topoisomerase II inhibitor S16020-2: importance of the N-{2(Dimethylamino)ethyl}carbamoyl side chain; Le Mee S et al.; The new olivacine derivative S16020-2 (NSC-659687) is a DNA topoisomerase II inhibitor endowed with a remarkable antitumor activity against various experimental tumors . In vitro physicochemical properties of this compound, in particular its interaction with DNA and DNA topoisomerase II, were very similar to those of ellipticine derivatives, except for a strictly ATP-dependent mechanism of cleavable complex induction . From the Chinese hamster lung fibroblast cell line DC-3F, a subline resistant to S16020-2, named DC-3F/S16, was selected by adding stepwise increasing concentrations of the drug to the cell growth medium . Whereas DC-3F/9-OH-E cells, a DC-3F subline resistant to 9-hydroxy-ellipticine, are cross-resistant to S16020-2, DC-3F/S16 cells are only very weakly cross-resistant to ellipticine derivatives, indicating that, despite their structural similarity, these compounds may differ in their mechanisms of action . Uptake and efflux rates of S16020-2 were identical in the resistant and the sensitive cells . Topoisomerase IIalpha was expressed at the same level in both sensitive and resistant cells, whereas expression of the beta-enzyme was approximately 50% lower in the resistant cells . Sequencing of both alpha- and beta-isoform cDNAs revealed a point mutation that converts Arg(486) to a Gly in the alpha cDNA, whereas the beta cDNA was not modified . This amino acid substitution in a highly conserved sequence of the enzyme appears to be responsible for the resistance to S16020-2 . Comparative analysis of the properties of the ellipticine and S16020-2-resistant cells suggests that S16020-2, which is a DNA intercalator, might also interact with this enzyme amino acid sequence through its side chain.

Toxicol Lett, 2000 Aug 16, 116(3), 199 - 207
Increased susceptibility of cells to inducible apoptosis during growth from early to late log phase: an important caveat for in vitro apoptosis research; Washo-Stultz D et al.; The physiologic mode of cell death known as apoptosis has become a major focus of scientific research due to its biologic importance . Much of this research involves cells grown in culture, where induction of apoptosis is achieved through a variety of agents . We report that cell cultures in late log growth phase exhibit an increased susceptibility to apoptosis compared with cultures in early log growth phase when apoptosis is induced by sodium deoxycholate (NaDOC), anti-Fas antibody and cytosine-b-D-arabino-furanoside (Ara-C), three agents which induce apoptosis through different upstream mechanisms . We show that this phenomenon occurs in Jurkat lymphocytes, HT-29 and HCT-116 colon epithelial cells . We also present evidence that cell density alone does not affect NaDOC-induced apoptosis, but rather that the growth media plays a key role in increased susceptibility of cells in late log growth phase to NaDOC-induced apoptosis . These results indicate that growth phase is a variable that must be controlled in order to obtain reliable apoptosis data.

Environ Exp Bot, 2000 Oct 1, 44(2), 115 - 124
Root system responses of Japanese red cedar saplings to acidic conditions; Hirano Y et al.; Stemflow from Japanese red cedar (Cryptomeria japonica) enters forest soil at a low pH . We evaluated the responses of the root system of Japanese red cedar saplings to acidic conditions, used to simulate this situation, in two different growth media, a brown forest soil (BS) and a Yahagi sand (YS) . Soils were acidified by the addition of solutions at pH 2.0, 3.0 and 5.5 (control) . Root morphology, root surface area index, root respiration activity and root biomass were measured . In the pH 3.0 treatment, no significant effects were found on the root systems compared with the controls in either soil, except for a slight difference in root-tip diameter in the Yahagi sand . In the pH 2.0 treatment, the surface area index and dry weight ratios of the whole root in the Yahagi sand were significantly lower than those in the other treatments . No significant effects on the whole root were observed in the brown forest soil . These results suggest that detrimental effects of acidic solutions on the root systems would be less significant in brown forest soil, which contains humus, than in the Yahagi sand, which lacks humus . They also suggest that the threshold pH value causing visible morphological changes on the roots of Japanese red cedar saplings falls in the pH range between 2 and 3 . White roots in the pH 2.0 treatment had low respiration activity and showed visible morphological changes in both soils . These responses were presumably related to the effects of excess Al in the soil solution . White roots in the pH 2.0 treatment typically produced exodermis . The results suggest that stemflow with a pH of 3.0 has no effects on the root systems of Japanese red cedar, and that the morphology of white roots was adversely affected not by treatment at pH 2.0 but by excess water-soluble Al in the soil.

Plant Sci, 2000 Sep 8, 158(1-2), 33 - 39
Molecular cloning and characterization of a plant homologue of the origin recognition complex 1 (ORC1); Kimura S et al.; By using the rice EST database, we have isolated a 2.8 kb cDNA, termed Oryza sativa ORC1 (OsORC1), from rice (O . sativa) encoding a protein that shows homology with the eukaryotic ORC1 proteins . Alignment of the OsORC1 protein sequence with the sequence of ORC1 from human and yeasts S . cerevisiae and S . pombe showed a high degree of sequence homology (38.7, 32.9 and 35.0% identity, respectively), particularly around the C-terminal region containing the CDC-NTP domain . Interestingly, the OsORC1 protein had an A+T hook-like motif, which was not present in the human or yeast genes . Genomic analysis indicated that OsORC1 existed as a single copy per genome . OsORC1 transcripts were expressed strongly in root tips and weakly in young leaves containing root apical meristem and marginal meristem, respectively . No expression was detected in the mature leaves . The level of OsORC1 expression was significantly reduced when cell proliferation was temporarily halted by the removal of sucrose from the growth medium . When the growth-halted cells began to re-grow following addition of sucrose to the medium, OsORC1 was again expressed at high levels . These results suggested that OsORC1 is required for cell proliferation . The role of OsORC1 in plant DNA replication will be discussed.

J Agric Food Chem, 2000 Sep, 48(9), 4432 - 8
Growth and anthraquinone production of Morinda elliptica cell suspension cultures in a stirred-tank bioreactor; Abdullah MA et al.; The effects of medium strategy, number of impellers, aeration mode, and mode of operation on Morinda elliptica cell suspension cultures in a stirred-tank bioreactor are described . A lower number of impellers and continuous aeration contributed toward high cell growth rate, whereas a higher number of impellers reduced cell growth rate, although not anthraquinone yield . The semicontinuous mode could indirectly imitate the larger scale version of production medium strategy and improved anthraquinone production even with 0 . 012% (v/v) antifoam addition . Production medium promoted both growth (maximum dry cell weight of 24.6 g/L) and anthraquinone formation (maximum content of 19.5 mg/g of dry cell weight), without any necessity for antifoam addition . Cultures in production medium or with higher growth rate and anthraquinone production were less acidic than cultures in growth medium or with lower growth rate and anthraquinone production . Using the best operating variables, growth of M . elliptica cells (24.6 g/L) and anthraquinone yield (0.25 g/L) were 45% and 140%, respectively, lower than those using a shake flask culture after 12 days of cultivation.

Planta, 2000 Aug, 211(3), 335 - 44
Photosynthetic apparatus organization and function in the wild type and a chlorophyll b-less mutant of Chlamydomonas reinhardtii . Dependence on carbon source; Polle JE et al.; The assembly, organization and function of the photosynthetic apparatus was investigated in the wild type and a chlorophyll (Chl) b-less mutant of the unicellular green alga Chlamydomonas reinhardtii, generated via DNA insertional mutagenesis . Comparative analyses were undertaken with cells grown photoheterotrophically (acetate), photomixotrophically (acetate and HCO3-) or photoautotrophically (HCO3-) . It is shown that lack of Chl b diminished the photosystem-II (PSII) functional Chl antenna size from 320 Chl (a and b) to about 95 Chl a molecules . However, the functional Chl antenna size of PSI remained fairly constant at about 290 Chl molecules, independent of the presence of Chl b . Western blot and kinetic analyses suggested the presence of inner subunits of the Chl a-b light-harvesting complex of PSII (LHCII) and the entire complement of the Chl a-b light-harvesting complex of PSI (LHCI) in the mutant . It is concluded that Chl a can replace Chl b in the inner subunits of the LHCII and in the entire complement of the LHCI . Growth of cells on acetate as the sole carbon source imposes limitations in the photon-use efficiency and capacity of photosynthesis . These are manifested as a lower quantum yield and lower light-saturated rate of photosynthesis, and as lower variable to maximal (Fv/Fmax) chlorophyll fluorescence yield ratios . This adverse effect probably originates because acetate shifts the oxidation-reduction state of the plastoquinone pool, and also because it causes a decrease in the amount and/or activity of Rubisco in the chloroplast . Such limitations are fully alleviated upon inclusion of an inorganic carbon source (e.g . bicarbonate) in the cell growth medium . Further, the work provides evidence to show that transformation of green algae can be used as a tool by which to generate mutants exhibiting a permanently truncated Chl antenna size and a higher (per Chl) photosynthetic productivity of the cells.

J Bacteriol, 2000 Oct, 182(19), 5521 - 9
Characterization of ssfR and ssgA, two genes involved in sporulation of Streptomyces griseus; Jiang H et al.; In the presence of cefoxitin, which inhibits septum formation during sporulation, Streptomyces griseus is unable to sporulate, retaining the sonication sensitivity of nonsporulating hyphae . Cefoxitin- and sonication-resistant mutant SKK2600 was isolated and showed many morphological differences from its parental strain . A 3.6-kb DNA fragment that complemented the mutations of SKK2600 contained two open reading frames (ORFs), either of which could complement SKK2600 . One ORF, designated ssfR, encoded a protein containing a potential DNA-binding helix-turn-helix motif close to its N terminus . SsfR is similar to members of a large family of transcriptional regulators, particularly IclR of Escherichia coli . The second ORF was identified as ssgA, a previously described sporulation gene from S . griseus (S . Kawamoto and J . C . Ensign, Actinomycetology 9:136-151, 1995) . A point mutation of C to T seven nucleotides upstream of the UGA stop codon of ssfR was responsible for the phenotype of isolated mutant strain SKK2600 . Surprisingly, this mutation should not change the primary structure of SsfR . The ssfR and ssgA disruption mutants were constructed and showed the "white" mutant phenotype, with some growth medium dependence . In addition, the ssfR null mutant sporulated ectopically in phosphate starvation medium.

J Biomed Mater Res, 2000 Sep, 53(5), 467 - 74
Fatigue properties of hydroxyapatite-coated dental implants after exposure to a periodontal pathogen; Mukherjee DP et al.; We studied the fatigue properties of rods (4 mm diameter) of hydroxyapatite-coated, titanium alloy implant material after it was exposed to a periodontal pathogen, Actinobacillus actinomycetemcomitans (Aa) . We varied the crystallinity of the hydroxyapatite (HA) coating in these rods to the levels of, 60.5%, 52.8%, and 47.8% . Each rod was first inoculated with Aa in the log phase of its growth cycle . After 48 h, we counted the adhered cells . We measured the dissolution of HA coating due to bacterial exposure alone by determining the calcium and phosphate concentrations in the bacterial growth media . Once the adherent bacteria were removed from these rods, we subjected them to 5 million cycles of fatigue testing after immersion in Lactated Ringer's solution . We then determined the calcium and phosphate concentrations in the fatigue media . We found additional coating loss after fatiguing of the samples . This coating loss was a cumulative effect of bacterial exposure and fatigue loading of the hydroxyapatite-coated dental implant alloy . The lower crystallinity sample showed a higher loss of coating within the range of crystallinity studied here . The HA coating in implants during clinical use may undergo such changes, because they are exposed to the same bacteria .

Curr Microbiol, 2000 Oct, 41(4), 227 - 31
Degradation of glycinebetaine by betaine-homocysteine methyltransferase in Aphanothece halophytica: effect of salt downshock and starvation; Incharoensakdi A et al.; We have investigated conditions leading to the degradation of glycinebetaine in Aphanothece halophytica and have shown the activity of betaine-homocysteine methyltransferase (BHMT) . The intracellular glycinebetaine level was decreased approximately 50% after 36 h salt downshock from 2.0 m NaCl medium to 0.5 m NaCl medium . A slight additional decrease of glycinebetaine occurred when salt downshock was combined with dark treatment . The omission of carbon and nitrogen sources in the growth medium further decreased intracellular glycinebetaine . The activity of BHMT increased from 0 to 460 nmol h(-1)mg(-1) after 3 h salt downshock . Higher strength of salt downshock resulted in higher activity of the enzyme . Small increase of the enzyme activity was also observed when A . halophytica was deprived of carbon and nitrogen sources in the growth medium.

Mol Plant Microbe Interact, 2000 Sep, 13(9), 951 - 61
Interactions of Pseudomonas syringae pv . glycinea with host and nonhost plants in relation to temperature and phytotoxin synthesis; Budde IP et al.; Pseudomonas syringae pv . glycinea PG4180 causes bacterial blight of soybean and produces the phytotoxin coronatine (COR) in a temperature-dependent manner . COR consists of a polyketide, coronafacic acid (CFA), and an amino acid derivative, coronamic acid, and is produced optimally at 18 degrees C whereas no detectable synthesis occurs at 28 degrees C . We investigated the impact of temperature on PG4180 during compatible and incompatible interactions with soybean and tobacco plants, respectively . After spray inoculation, PG4180 caused typical bacterial blight symptoms on soybean plants when the bacteria were grown at 18 degrees C prior to inoculation but not when derived from cultures grown at 28 degrees C . The disease outcome was quantified by determination of bacterial populations in planta . The temperature effect was not observed when PG4180 was artificially infiltrated into soybean leaves, indicating that the pre-inoculation temperature and phytotoxin synthesis were important for bacterial invasion via natural plant openings . In the incompatible interaction, PG4180 elicited the hypersensitive response (HR) on tobacco plants regardless of the bacterial pre-inoculation temperature . However, the HR was significantly delayed when tobacco plants were treated with cells of the CFA-overproducing derivative, PG4180.N9, which were derived from cultures grown at 18 degrees C, compared with parallels incubated at 28 degrees C . CFA biosynthesis by PG4180.N9 was optimal at 18 degrees C and negligible at 28 degrees C . The impact of CFA synthesis on the HR was studied with different growth media, mutants, and transconjugants of PG4180, indicating that the amount of synthesized CFA but not that of COR influenced the outcome of the HR . Feeding experiments with purified coronafacoyl compounds suggested that the observed delay of the HR was mediated by CFA, shedding further light on CFA's putative role as a molecular mimic of the plant signaling molecule, jasmonic acid.

Cardiovasc Res, 2000 Sep, 47(4), 749 - 58
Low density lipoproteins in human plasma make vascular smooth muscle cells resistant to growth inhibition by heparin; Underwood PA et al.; OBJECTIVE: Vascular smooth muscle cell hyperplasia plays a role in atherosclerosis and restenosis . While heparin has shown promise as an inhibitor of smooth muscle cell proliferation in vitro and in some animal models, it has failed to reduce restenosis in clinical trials . We have previously shown that human smooth muscle cells grown in the presence of human serum are heparin resistant, whereas in the presence of bovine serum they are heparin sensitive . In this report, we demonstrate that the heparin resistance factor is present in human plasma as well as serum, and characterise it further . METHODS: Human vascular smooth muscle cells were cultured as explants from the media of redundant adult internal mammary or umbilical cord arteries . They were tested for sensitivity to heparin at 100 microg/ml in the growth medium, in the presence of foetal bovine serum, human-plasma-derived serum, human whole blood serum, or fractions derived from these . RESULTS: In the presence of foetal bovine serum, heparin inhibited cell proliferation, while human-plasma-derived or whole blood sera conveyed heparin resistance . This activity was contained within the fraction of plasma/serum which bound to heparin Sepharose, and the sub-fraction which was retained by a membrane filter of molecular weight cut off of 100000 . All the heparin resistance in this latter fraction was supplied by lipoproteins . LDL prepared directly from human plasma conveyed similar heparin resistance to the lipoproteins from the above sub-fraction . CONCLUSION: LDL in human plasma/serum conveys resistance to the anti-proliferative effects of heparin upon vascular smooth muscle cells . This activity may interfere with potential therapeutic effects of heparin as an anti-restenosis agent.

Mol Microbiol, 2000 Sep, 37(5), 1248 - 57
A defined sequence within the 3' UTR of the areA transcript is sufficient to mediate nitrogen metabolite signalling via accelerated deadenylation; Morozov IY et al.; Nitrogen metabolism in Aspergillus nidulans is regulated by AREA, a member of the GATA family of transcription factors . One mechanism that modulates AREA activity involves the rapid degradation of the areA transcript when sufficient NH4+ or Gln are available . This signalling mechanism has been shown to require a region of 218 nucleotides within the 3' untranslated region of areA mRNA . We demonstrate that this region functions independently in a heterologous transcript and acts to accelerate degradation of the poly(A) tail, which in turn leads to rapid transcript degradation in response to the addition of NH4+ or Gln to the growth medium . areA transcript degradation is inhibited by cycloheximide, but this is not a general consequence of translational inhibition . We believe that this is the first reported example in which specific physiological signals, acting through a defined sequence within a transcript, have been shown to promote accelerated poly(A) degradation, which in turn triggers transcript degradation.

Virus Res, 2000 Jul, 68(2), 175 - 81
Bovine herpesvirus 1 glycoprotein G is required for viral growth by cell-to-cell infection; Nakamichi K et al.; The bovine herpesvirus 1 (BHV-1) US4 gene encodes glycoprotein G (gG), which is conserved in the majority of alphaherpesviruses . In order to identify the role of BHV-1 gG in the viral infection cycle, a gG minus BHV-1 mutant and its gG-positive revertant were constructed and their growth characteristics in Madin-Darby bovine kidney (MDBK) cells were compared . The gG minus mutant formed smaller plaques than the gG-positive BHV-1 in MDBK cells . When a monolayer culture of MDBK cells was infected with BHV-1 at a low multiplicity of infection and overlaid with semi-solid growth medium, under which adsorption of the mature virion released in the medium was inhibited, gG-positive BHV-1 multiplied, while the growth of the gG negative BHV-1 was severely inhibited . These data suggest that BHV-1 gG functions in direct cell-to-cell transmission mechanism of BHV-1 in tissue culture.

Aquat Toxicol, 2000 Sep 1, 50(3), 275 - 284
The effect of creosote on membrane ion leakage in Myriophyllum spicatum L; McCann JH et al.; Creosote is a complex chemical mixture used as a wood preservative that has the potential to contaminate aquatic systems via spills or leaching from treated wood structures . Aquatic macrophytes are important components of aquatic systems, which may be adversely affected by creosote contamination . Several chemicals that are constituents of creosote are known to affect cell membranes in various organisms . Therefore, the effect of creosote on the membrane permeability of the aquatic macrophyte Myriophyllum spicatum was investigated . Apical meristems from axenic Myriophyllum plants were exposed for 4 days to 8 creosote treatments (ranging from 0.1 to 92 mg creosote/l) plus controls . Following the exposure, the ion leakage from the cellular membranes was determined via conductivity measurements . The concentration of 15 polycyclic aromatic hydrocarbons (PAHs) in the growth medium and in the plant tissue was also determined . A significant increase in ion leakage was observed at all creosote concentrations, even those in which no biological effects were observed on plant growth . However, saturation of the growth medium with PAHs was observed, thus indicating that nominal creosote concentrations may over-estimate the actual exposure.

Mol Cell Biol, 2000 Sep, 20(18), 7024 - 36
MyoD-dependent induction during myoblast differentiation of p204, a protein also inducible by interferon; Liu C et al.; p204, an interferon-inducible p200 family protein, inhibits rRNA synthesis in fibroblasts by blocking the binding of the upstream binding factor transcription factor to DNA . Here we report that among 10 adult mouse tissues tested, the level of p204 was highest in heart and skeletal muscles . In cultured C2C12 skeletal muscle myoblasts, p204 was nucleoplasmic and its level was low . During myoblast fusion this level strongly increased, p204 became phosphorylated, and the bulk of p204 appeared in the cytoplasm of the myotubes . Leptomycin B, an inhibitor of nuclear export that blocked myoblast fusion, inhibited the nuclear export signal-dependent translocation of p204 to the cytoplasm . The increase in the p204 level during myoblast fusion was a consequence of MyoD transcription factor binding to several MyoD-specific sequences in the gene encoding p204, followed by transcription . Overexpression of p204 (in C2C12 myoblasts carrying an inducible p204 expression plasmid) accelerated the fusion of myoblasts to myotubes in differentiation medium and induced the fusion even in growth medium . The level of p204 in mouse heart muscle strongly increased during differentiation; it was barely detectable in 10 . 5-day-old embryos, reached the peak level in 16.5-day-old embryos, and remained high thereafter . p204 is the second p200 family protein (after p202a) found to be involved in muscle differentiation . (p202a was formerly designated p202 . The new designation is due to the identification of a highly similar protein-p202b {H . Wang, G . Chatterjee, J . J . Meyer, C . J . Liu, N . A . Manjunath, P . Bray-Ward, and P . Lengyel, Genomics 60:281-294, 1999}.) These results reveal that p204 and p202a function in both muscle differentiation and interferon action.

Prenat Diagn, 2000 Aug, 20(8), 640 - 7
Differential effects of interleukin-3 on fetal and adult erythroid cells in culture: implications for the isolation of fetal cells from maternal blood; Bohmer RM et al.; Fetal clonogenic erythroid cells may be present in maternal blood and serve as a source of fetal DNA for prenatal genetic diagnosis . Proliferating nucleated red cells in cultures from first and second trimester fetal blood contain only fetal haemoglobin (HbF; F+A- cells), whereas nucleated red cells from adult blood contain also adult haemoglobin (HbA; F+A+ or F-A+ cells) . Thus, fetal red cells can be identified and flow sorted . However, a few adult cells are also F+A-, which reduces the purity of fetal cell isolation . Culture media optimized for erythropoiesis contain interleukin-3 (IL3) . We show here that IL3 strongly stimulates the growth of F+ cells (both F+A- and F+A+) in cultures from adult blood but has only a comparatively small effect on F+A- cells in cultures from fetal blood . This difference is maintained in the unified conditions of co-cultures of adult and fetal cells, so that the purity of fetal cell sorts can be increased by omitting IL3 from the culture medium . We further show that IL3 accelerates the exhaustion of the long-term division potential of adult cells, allowing fetal secondary colonies to be identified by their size following a two-stage culture scheme . Thus, the choice to omit or include IL3 in the growth medium of maternal blood cultures should depend on whether fetal nucleated red cells are to be isolated by flow sorting after one week, or by picking secondary colonies after a later secondary culture stage .

J Biol Chem, 2000 Nov 3, 275(44), 34041 - 5
Expression and characterization of soluble and membrane-bound human nucleoside triphosphate diphosphohydrolase 6 (CD39L2); Hicks-Berger CA et al.; Ecto-nucleoside-triphosphate diphosphohydrolase-6 (eNTPDase6(1), also known as CD39L2) cDNA was expressed in mammalian COS-1 cells and characterized using nucleotidase assays as well as size exclusion, anion exchange, and cation exchange chromatography . The deduced amino acid sequence of eNTPDase6 is more homologous with the soluble E-type ATPase, eNTPDase5, than other E-type ATPases, suggesting it may also be soluble . To test this possibility, both the cell membranes and the growth media from eNTPDase6-transfected COS-1 cells were assayed for nucleotidase activities . Activity was found in both the membranes and the media . Soluble eNTPDase6 preferentially exhibits nucleoside diphosphatase activity, which is dependent on the presence of divalent cations . Western blot analysis of eNTPDase6 treated with PNGase-F indicated both soluble and membrane-bound forms are glycosylated . However, unlike some membrane-bound ecto-nucleotidases, the eNTPDase6 activity was not specifically inhibited by deglycosylation with peptide N-glycosidase F . Soluble eNTPDase6 hydrolyzed nucleoside triphosphates poorly and nucleoside monophosphates not at all . Analysis of the relative rates of hydrolysis of nucleoside diphosphates (GDP = IDP > UDP > CDP >> ADP) suggests that soluble eNTPDase6 is a diphosphatase most likely not involved in regulation of ADP levels important for circulatory hemostasis.

Infect Immun, 2000 Sep, 68(9), 5408 - 11
Uptake and killing of Leptospira interrogans and Borrelia burgdorferi, spirochetes pathogenic to humans, by reticuloendothelial cells in perfused rat liver; Marangoni A et al.; In situ-perfused rat livers were infused with a single dose of 1.5 x 10(7) radiolabeled cells of Leptospira interrogans serovar icterohaemorrhagiae, the agent of leptospirosis, or with Borrelia burgdorferi IRS, the agent of Lyme disease . Significant (P<0.0001) differences in the liver uptake of L . interrogans and of B . burgdorferi were observed, the uptakes being 37.4%+/-2.3% for L . interrogans and 60.5%+/-3.1% for B . burgdorferi . Leptospires, in contrast to borreliae, were recovered from the livers when liver samples were cultured in growth medium . Leptospires but not borreliae were recovered in bile within 30 min of infusion . The association of leptospires and borreliae with reticuloendothelial cells of the liver was demonstrated by immunohistochemistry . Leptospires and borreliae were found to be associated with vimentin-positive cells and not with desmin-positive cells . Few leptospires but no borreliae were also seen associated with vimentin- and desmin-negative cells, suggesting the presence of leptospires outside the sinusoidal spaces, in the liver parenchyma.

J Lab Clin Med, 2000 Aug, 136(2), 100 - 9
Endothelial cell-mediated type I collagen gel contraction is regulated by hemin; Liu XD et al.; The contraction of three-dimensional type I collagen gels is regarded as a model of contraction during wound healing and tissue remodeling . Because such a process could contribute to vessel narrowing, we hypothesized that endothelial cells may be able to mediate gel contraction . To demonstrate this, type I collagen was extracted from rat tail tendon and used to prepare collagen gels . Bovine arterial endothelial cells (BAECs) or human pulmonary artery endothelial cells (HPAECs) were then plated on the top of the gels in serum-free Ham's F-12 medium or 2% fetal calf serum-endothelium growth medium-2 (FCS-EGM2), respectively . After 48 hours of attachment, gels were released and floated in 0.2% FCS-Ham's F-12 medium (BAECs) or 2% FCS-EGM2 (HPAECs) . Gel size was measured with an image analyzer daily for 5 consecutive days . Gels were then digested with collagenase to quantify DNA and hydroxyproline . BAECs contracted the gels in a time-dependent manner over the 5 days . Contraction was dependent on cell density (gel size was 100% of initial size after 5 days with no cells vs . 66.4%+/-0.5% with 0.9x10(4) cells/cm2 and 22.1%+/-0.3% with 7.5x10(4) cells/cm2) and was inversely related to collagen concentration (gel size was 22.3%+/-0.05%, 46.4%+/-0.9%, 72.3%+/-0.4%, and 87.4% +/-0.3% of initial size for gels prepared with 0.5 mg/mL, 0.75 mg/mL, 1 mg/mL, and 2 mg/mL of collagen, respectively) . Hemin (a precursor for CO) and cytochalasin D inhibited collagen gel contraction mediated by both bovine and human endothelial cells without changing cell number or hydroxyproline content . In contrast, prostaglandin E2, an inhibitor, and transforming growth factor-beta1, a stimulator of fibroblast-mediated gel contraction, had no effect on endothelial cell-mediated contraction . These findings demonstrate that endothelial cells are able to contract native type I collagen gels and that this process can be modulated by exogenous mediators . Such a capability may cause remodeling of subjacent matrix of endothelial cells and may contribute to vessel narrowing.

Cytometry, 2000 Sep 1, 41(1), 31 - 5
Efficient generation of stably electrotransfected human hematopoietic cell lines without drug selection by consecutive FACsorting; Van Tendeloo VF et al.; BACKGROUND: Current methods to establish stably transfected cell lines by nonviral techniques involve coselection for a drug selection marker . However, this approach suffers from several drawbacks . We developed a fluorescence-activated cell sorting (FACS)-based protocol for the selection and isolation of stable hematopoietic electrotransfectants without the need for selective growth conditions . METHODS: Leukemic K562 cells were electroporated with the enhanced green fluorescent protein (EGFP) reporter gene and FACsorted to obtain stably EGFP-expressing cells . Stable EGFP(+) clones were established by single-cell sorting . RESULTS: Efficiency of stable EGFP gene expression increased steadily in function of number of consecutive FACsorts . Stable transfectants (>99% EGFP(+)) were obtained after four FACsorts . Furthermore, several single-cell derived clones with variable levels of stable EGFP expression were isolated and cultured without the use of selective growth media . CONCLUSIONS: EGFP is an effective selection marker for the generation and isolation of stably transfected hematopoietic cell clones without the need for selection in toxic media that could create a potentially undesirable stress environment for stably transfected cells .

Exp Cell Res, 2000 Aug 25, 259(1), 266 - 73
Induction of ribosomal subunits misassembly by antisense RNAs to control cell growth; Mangiarotti G; The assembly of ribosomal subunits starting from free ribosomal RNA and protein of Dictyostelium discoideum was induced in vitro in the presence of several oligoribonucleotides complementary to defined sequences of ribosomal RNA . The reconstituted particles had a full complement of ribosomal proteins, but did not function in an in vitro protein synthesis system and were disassembled following interaction with mRNA . The same result was obtained in vivo by fusing the oligodeossiribonucleotides coding for the selected oligoribonucleotides to the promoter of the gene coding for contact site A protein . This gene is expressed only in the first part of development . Transfected growing cells, transferred in developing buffer in the presence of pulses of cAMP, accumulated significant amounts of the oligoribonucleotides . When retransferred to the growth medium, they grew progressively more slowly, until their doubling time doubled, apparently due to the availability of a limiting amount of functional ribosomes . To avoid disassembly of misassembled subunits (G . Mangiarotti et al., 1997, J . Biol . Chem . 272, 27818-27822), two oligoribonucleotides complementary to sequences present at the 5' ends of pre-17S and pre-26S RNAs were also induced to accumulate during early development with the same technique . When transfected cells were retransferred to the growth medium, their rate of growth declined rapidly to zero and cells died, apparently because they were unable to disassemble misassembled ribosomal subunits and avoid their entry into polyribosomes . This technique to perturb protein synthesis, arrest cell growth, and cause cell suicide will be tested in abnormally growing animal cells .

J Bacteriol, 2000 Sep, 182(17), 4970 - 8
Understanding the growth phenotype of the yeast gcr1 mutant in terms of global genomic expression patterns; Lopez MC et al.; The phenotype of an organism is the manifestation of its expressed genome . The gcr1 mutant of yeast grows at near wild-type rates on nonfermentable carbon sources but exhibits a severe growth defect when grown in the presence of glucose, even when nonfermentable carbon sources are available . Using DNA microarrays, the genomic expression patterns of wild-type and gcr1 mutant yeast growing on various media, with and without glucose, were compared . A total of 53 open reading frames (ORFs) were identified as GCR1 dependent based on the criterion that their expression was reduced twofold or greater in mutant versus wild-type cultures grown in permissive medium consisting of YP supplemented with glycerol and lactate . The GCR1-dependent genes, so defined, fell into three classes: (i) glycolytic enzyme genes, (ii) ORFs carried by Ty elements, and (iii) genes not previously known to be GCR1 dependent . In wild-type cultures, GCR1-dependent genes accounted for 27% of the total hybridization signal, whereas in mutant cultures, they accounted for 6% of the total . Glucose addition to the growth medium resulted in a reprogramming of gene expression in both wild-type and mutant yeasts . In both strains, glycolytic enzyme gene expression was induced by the addition of glucose, although the expression of these genes was still impaired in the mutant compared to the wild type . By contrast, glucose resulted in a strong induction of Ty-borne genes in the mutant background but did not greatly affect their already high expression in the wild-type background . Both strains responded to glucose by repressing the expression of genes involved in respiration and the metabolism of alternative carbon sources . Thus, the severe growth inhibition observed in gcr1 mutants in the presence of glucose is the result of normal signal transduction pathways and glucose repression mechanisms operating without sufficient glycolytic enzyme gene expression to support growth via glycolysis alone.

J Biol Chem, 2000 Nov 10, 275(45), 35256 - 63
Rapid induction of histone hyperacetylation and cellular differentiation in human breast tumor cell lines following degradation of histone deacetylase-1; Zhou Q et al.; Quinidine inhibits proliferation and promotes cellular differentiation in human breast tumor epithelial cells . Previously we showed quinidine arrested MCF-7 cells in G(1) phase of the cell cycle and led to a G(1) to G(0) transition followed by apoptotic cell death . The present experiments demonstrated that MCF-7, MCF-7ras, T47D, MDA-MB-231, and MDA-MB-435 cells transiently differentiate before undergoing apoptosis in response to quinidine . The cells accumulated lipid droplets, and the cytokeratin 18 cytoskeleton was reorganized . Hyperacetylated histone H4 appeared within 2 h of the addition of quinidine to the medium, and levels were maximal by 24 h . Quinidine-treated MCF-7 cells showed elevated p21(WAF1), hypophosphorylation and suppression of retinoblastoma protein, and down-regulation of cyclin D1, similar to the cell cycle response observed with cells induced to differentiate by histone deacetylase inhibitors, trichostatin A, and trapoxin . Quinidine did not show evidence for direct inhibition of histone deacetylase enzymatic activity in vitro . HDAC1 was undetectable in MCF-7 cells 30 min after addition of quinidine to the growth medium . The proteasome inhibitors MG-132 and lactacystin completely protected HDAC1 from the action of quinidine . We conclude that quinidine is a breast tumor cell differentiating agent that causes the loss of HDAC1 via a proteasomal sensitive mechanism.

J Mol Microbiol Biotechnol, 2000 Jan, 2(1), 95 - 100
The cause of "acid-crash" and "acidogenic fermentations" during the batch acetone-butanol-ethanol (ABE-) fermentation process; Maddox IS et al.; Experiments were performed to determine the cause of "acid crash", a phenomenon which occasionally occurs in pH-uncontrolled batch fermentations resulting in premature cessation of ABE (acetone butanol) production . The results indicate that "acid crash" occurs when the concentration of undissociated acids in the broth exceeds 57 - 60 mmol/l . Prevention can be achieved by introducing some limited pH control to minimize the concentration of undissociated acids or by slowing the metabolic rate, and thus the rate of acid production, by, for example, lowering the fermentation temperature . "Acidogenic fermentations", which occur when batch fermentations are performed at pH values close to neutrality, are due to rapid production of acids followed by inhibition of solventogenesis when the total acid concentration reaches 240 - 250 mmol/l . Solventogenesis can be achieved at these pH values by lowering the glucose uptake rate / acid production rate by use of e.g . elevated glucose or lowered yeast extract concentrations in the growth medium.

Biotechnol Prog, 2000 Jul-Aug, 16(4), 650 - 6
Influence of baculovirus-host cell interactions on complex N-linked glycosylation of a recombinant human protein; Joshi L et al.; The conditions required for mammalian-type complex N-linked glycosylation of human proteins produced in insect cells with the baculovirus expression vector system were investigated . Marked alterations to N-linked glycosylation of human placental secreted alkaline phosphatase (SEAP) were observed with different baculovirus species, insect cell lines, and cell culture media . When a recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) was used to produce SEAP in Trichoplusia ni (Tn-4h) cells cultured in serum-free medium, structural analyses indicated <1% hybrid and no complex oligosaccharides attached to SEAP, a typical result with the baculovirus expression vector system . However, when fetal bovine serum was added to the culture medium, 48 +/- 4% of the oligosaccharides were hybrid or complex (but asialylated) glycans . When a recombinant T . ni nucleopolyhedrovirus (TnSNPV) was similarly used to express SEAP in Tn-4h cells cultured in serum-containing medium, only 24 +/- 3% of the glycans contained terminal N-acetylglucosamine and/or galactose residues . In contrast, SEAP produced in Sf9 cells grown in serum-containing medium with AcMNPV contained <1% hybrid oligosaccharides and no complex oligosaccharides . The results illustrate that baculovirus type, host cell type, and the growth medium all have a strong influence on the glycosylation pathway in insect cells, resulting in significant alterations in structures and relative abundance of N-linked glycoforms . Although the addition of sialic acid residues to the SEAP glycans was not detected, possible approaches to obtain sialylated glycans are discussed.

Microbiology, 2000 Aug, 146 ( Pt 8), 1949 - 58
Threshold level of protein kinase A activity and polarized growth in Mucor rouxii; Pereyra E et al.; A model system to study the involvement of cAMP-mediated regulation of a cellular process such as hyphal morphogenesis was investigated . Impairment of polarized growth was observed when Mucor rouxii sporangiospores were grown in the presence of N(6)-cAMP analogues and of SQ 65,442, a cAMP phosphodiesterase inhibitor . Together with an effect on isodiametric growth, there was increased pigmentation, increased cell fragility and loss of cell adhesiveness . The total effect on morphology was attained even after adding the compounds shortly before germ-tube emergence; when added after this time growth continued in a non-polarized form and rounding of the germ tip was observed . The morphological effect was observed under all the nutritional and environmental conditions studied (aerobic conditions and defined medium with maltose or glucose, Casamino acids medium with glucose, or rich medium; anaerobic conditions with rich medium; and following a shift from anaerobiosis to aerobiosis) . The time of germ-tube emergence, and the size of the cell at this time, was dependent on the growth medium . Protein kinase A (PKA) specific activity was followed during the germination process under three growth conditions . It was found that the total activity of PKA correlated with differentiation and not with growth, and that the total specific activity at the time of germination was the same, independent of the culture medium . The time of germ-tube emergence correlated with the time of attainment of a threshold level of PKA total specific activity . The concentration of dibutyryl-cAMP needed to promote isodiametric growth correlated with the total units of activity of PKA to be activated per cell . It was concluded that PKA is involved in the morphogenetic process of the fungus grown under all the nutritional and ambient conditions tested.

Microbiology, 2000 Aug, 146 ( Pt 8), 1933 - 40
Generation of lys-gingipain protease activity in Porphyromonas gingivalis W50 is independent of Arg-gingipain protease activities; Aduse-Opoku J et al.; Porphyromonas gingivalis, a black-pigmenting anaerobe implicated in the aetiology of periodontal disease, contains two loci, rgpA and rgpB, encoding the extracellular Arg-X specific proteases (RGPs, Arg-gingipains), and kgp, which encodes a Lys-X specific protease (KGP, Lys-gingipain) . The rgpA and kgp genes encode polyproteins comprising pro-peptide and catalytic domain with large N- and C-terminal extensions which require proteolytic processing at several Arg and Lys residues to generate mature enzymes . The product of rgpB contains only a pro-peptide and the catalytic domain which requires processing at an Arg residue to generate active enzyme . An rgpA rgpB double mutant (E8) of P . gingivalis was constructed to study the role of RGPs in the processing of KGP . A kgp mutant (K1A) was also studied to investigate the role of KGP in the generation of RGPs . E8 was stable in the absence of the antibiotics tetracycline and clindamycin (selection markers for rgpA and rgpB, respectively) and exhibited the same pigmentation, colony morphology and identical growth rates to the parent W50 strain in the absence of antibiotics, in both complex and chemically defined media . The KGP activity of E8, grown in the absence of tetracycline, in whole cultures and in culture supernatants (up to 6 d) was identical to levels in W50 . However, in the presence of tetracycline in the growth medium, the level of KGP was reduced to 50% of levels present in whole cultures of W50 . Since tetracycline had no effect on RGP or KGP activity when incorporated into assay buffer, this effect is most likely to be on the synthesis of Kgp polypeptide . K1A was also stable in the absence of antibiotics but was unable to pigment, and remained straw-coloured throughout growth . RGP activity in whole cultures of K1A was identical to levels in W50, but RGP activity in 6 d culture supernatants was reduced to 50% of levels present in W50 . Thus, although KGP is not required for generation of RGP activity from RgpA and RgpB polypeptides, its absence affects the release/transport of RGP into culture supernatant.

J Steroid Biochem Mol Biol, 2000 Jun, 73(3-4), 147 - 52
Steroid-inducible transcription of the 3beta/17beta-hydroxysteroid dehydrogenase gene (3beta/17beta-hsd) in Comamonas testosteroni; Cabrera JE et al.; The expression of the Comamonas testosteroni gene, encoding 3beta/17beta-hydroxysteroid dehydrogenase enzyme (3beta/17beta-HSD), was analyzed at the transcriptional level . Northern blot analysis detected a 1 kb transcript in bacterial cells grown in minimum media supplemented with Casamino acids and testosterone . Also this transcript was observed when cells were grown in presence of 1-dehydrotestosterone, androstenedione and 1,4-androstadien-3, 17dione, but not in presence of acetate, citrate, cholic acid, cholesterol, and cortisol . In addition, this effect was dependent on the presence of another carbon source in the growth medium used, revealing catabolite repression.

Planta, 2000 Jun, 211(1), 91 - 7
Characterization of prosystemin expressed in the baculovirus/insect cell system reveals biological activity of the systemin precursor; Vetsch M et al.; Tomato (Lycopersicon esculentum Mill.) prosystemin in fusion with a viral signal peptide was expressed in Sf21 insect cell cultures after infection with recombinant baculoviruses . Prosystemin was purified from culture supernatants and its identity was confirmed by N-terminal sequence and mass-spectral analyses . Recombinant prosystemin was found to be equally active as compared to systemin in inducing the expression of wound-response genes in tomato plants . In cultured cells of L . peruvianum, prosystemin elicited a rapid alkalinization of the growth medium . The timing and dose-dependence of the alkalinization response were found to be identical for prosystemin and systemin, respectively . Prosystemin-triggered defense responses were inhibited by a competitive antagonist of systemin activity, indicating that the systemin sequence within the primary structure of prosystemin determines its activity.

FEMS Microbiol Ecol, 2000 Jul 1, 33(1), 1 - 9
Starvation-induced changes in the cell surface of Azospirillum lipoferum; Castellanos T et al.; Three starvation regimes (a deficient culture medium, a saline buffer solution and distilled water) were evaluated for their possible effect on cell surface characteristics of Azospirillum lipoferum 1842 related to the initial adsorption of the bacterium to surfaces . The bacteria survived for 7 days in all media although they did not multiply . Upon transfer from a rich growth medium (nutrient agar) to starvation conditions, cell surface hydrophobicity dropped sharply but recovered its initial value within 24 to 48 h, except in phosphate-buffered saline, the length of the recovery period depending on the starvation medium . Starvation affected the sugar affinity of the A . lipoferum cell surface mainly towards p-aminophenyl-alpha-D-mannopyranoside, to a lesser extent to glucose, but not to other monosaccharides tested . Starvation changed the concentration of several cell surface proteins but did not induce the synthesis of new ones . The cell surface hydrophobic protein (43 kDa) of A . lipoferum 1842 was unaffected by any starvation treatment for a period of up to 48 h, but later disappeared . These data showed that starvation is not a major factor in inducing changes in the cell surface which lead to the primary phase of attachment of Azospirillum to surfaces.

Protein Expr Purif, 2000 Aug, 19(3), 335 - 42
Dictyostelium discoideum as expression host: isotopic labeling of a recombinant glycoprotein for NMR studies; Cubeddu L et al.; The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies . Growth medium containing {(15)N}NH(4)Cl and {(13)C}glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D . discoideum cells in simple Mes buffer . A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol . Following single-step purification by anion-exchange chromatography, 8 mg of uniformly (13)C,(15)N-labeled protein secreted by approximately 10(10) D . discoideum cells was isolated from 3.3 liters of supernatant . Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label . The two-dimensional (1)H-(13)C HSQC spectrum confirms (13)C labeling of both glycan and amino acid residues of the glycoprotein . All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination . Copyright 2000 Academic Press.

What Is Molecular Microbiology?, What Is Rhizobia?, What Is Bioreactor?, What Is Genetic Engineering?, What Is Biofilm?, e, Microorganism, c, Microorganisms, a, Bacteriology, o, Microbe, r, Bacteria, i, Candida albicans, n, Bacteriological, a, Escherichia coli, o, Multidrug resistant, e, Activated sludge, o, Bactericidal, o, Staphylococcus, r, Candida albicans, r, Phage, r, Escherichia coli, a, Streptococci, s, Escherichia coli, r, Streptococci, o, Candida albicans, r, Culture medium, i, Vibriosis, s, Microbiological, c, Gram negative, s, Escherichia coli, c, Antimicrobial, i, Staphylococcus

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

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

Last modified: May 25, 2005