What Is Growth Medium?

Microbiological growth medium - also called Culture Medium, or Nutrient Broth, solution freed of all microorganisms by sterilization (usually in an autoclave, where it undergoes heating under pressure for a specific time) and containing the substances required for the growth of microorganisms such as bacteria, protozoans, algae, and fungi. The medium may be solidified by the addition of agar.

Growth medium - a source of nutrients in which a microorganism is placed to permit its growth, cause it to produce substances, or observe its activity under defined conditions; also called culture medium or growth medium . The medium is usually a solution of nutrients in water, or a similar solution solidified with gelatin or agar.

Growth medium (pl. growth media) - a synthetic medium which is filled with nutrients necessary to the growth of microorganisms or cells being cultured in the lab.

The majority of the cell is made of carbon, oxygen, hydrogen, nitrogen and phosphorous and these elements are necessary for forming membranes, proteins, nucleic acids and the other structures of the cell as described in Chapter 2. These nutrients are required in large amounts, consisting of more than 1% of the cells dry weight and are termed macronutrients (Table 6-1). A second tier of elements that are required in lower concentrations are calcium, potassium, magnesium, sulfur, iron and manganese and these are called micronutrients (Table 6-1). They make up between 0.1-1.0% of the cells dry weight micronutrients serve as less common parts of cellular structures or as necessary components for proteins. Although in smaller amounts, they too are absolutely required for the functioning of a living cell. Trace elements (Table 6-2) are compounds necessary in such small amounts that they are difficult to assign a requirement for. These are required in very small amounts (less than 0.1%) and their actual amount is difficult to measure. Growth factors (Table 6-3) are premade organic molecules that are necessary for growth, yet the microbe is unable to synthesize itself. Examples include vitamins, amino acids and nucleotides. These nutrients are the chemical substances used by the organism for biosynthesis and energy generation. Bacteria must acquire all these elements from their environment for growth.

We list here a few examples to give you an idea of the utility of nutritional information.

Bacterial nutrition began with studies directed at testing spontaneous generation. In these instances very simple medium containing chicken or beef broth were used for experimentation. Without these media it would have been impossible to test the validity of the hypothesis. Further work on bacteria led to the isolation of microbes and pure culture techniques. Again, without the ability to grow bacteria under controlled conditions, these experiments would be impossible.

Microorganisms are so small that in most cases large populations of them are needed to perform any experiment. To get these populations the scientist has to be able to grow large quantities of them and to do that, you have to know what they need nutritionally.

Knowing the nutritional requirements of a pathogen not only allows you to culture it in the laboratory for further study, but it can sometimes give you insight into its pathology. Pseudomonas aeruginosa, an important pathogen in burn patients and those with cystic fibrosis, will only express its virulence genes in medium low in iron. The low iron concentration serves as a signal to the microbe that it is inside a host. Understanding this facet of its pathology may help in the treatment of patients.

When trying to grow antibiotic-producing strains in industry, knowledge of the nutritional requirements of the microbe can enhance its growth and creating higher yields of the desired antibiotic. Knowledge of the nutritional needs of a microbe can also save money as just the right amount of each nutrient can be added to the growth medium.

As a final example, the ability of microbes to grow on different media can help to elucidate their metabolic capabilities. This is useful in identifying bacteria and can help us to understand their roles in the environment.

From these examples it should be clear that knowing the nutritional requirements of a microbe are extremely useful, but now the question becomes what are these requirements? One can get a reasonable estimate of this by examining the composition of the average bacterial cell. Bacterial cells contain a cell wall, membranes, ribosomes, proteins, RNA and DNA. If you break these structures down you end up with lipids, amino acids, sugars, nucleic acids and a few other compounds. By examining the chemical formulas of these compounds, it is possible to generate a reasonable estimate of the nutritional requirements of the cell. In this chapter we will look at what makes up the average bacterial cell, and give an insight into those requirements. We will also examine how these things are provided to a bacterium in the laboratory by examining the formulation and synthesis of culture medium.

Mar Biotechnol (NY) . 2005 Jan 17; {Epub ahead of print}
Metabolically Engineered Rhodobacter sphaeroides RV strains for Improved Biohydrogen Photoproduction Combined with Disposal of Food Wastes; Franchi E et al.; Three differently metabolically engineered strains, 2 single PHA(-) and Hup(-) mutants and one double PHA(-)/Hup(-) mutant, of the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides RV, were constructed to improve a light-driven biohydrogen production process combined with the disposal of solid food wastes . These phenotypes were designed to abolish, singly or in combination, the competition of H(2) photoproduction with polyhydroxyalkanoate (PHA) accumulation by inactivating PHA synthase activity, and with H(2) recycling by abolishing the uptake hydrogenase enzyme . The performance of these mutants was compared with that of the wild-type strain in laboratory tests carried out in continuously fed photobioreactors using as substrates both synthetic media containing lactic acid and media from the acidogenic fermentation of actual fruit and vegetable wastes, containing mainly lactic acid, smaller amounts of acetic acia, and traces of higher volatile acids . With the lactic acid-based synthetic medium, the single Hup(-) and the double PHA(-)/Hup(-) mutants, but not the single PHA(-) mutant, exhibited increased rates of H(2) photoproduction, about one third higher than that of the wild-type strain . With the food-waste-derived growth medium, only the single Hup(-) mutant showed higher rates of H(2) production, but all 3 mutants sustained a longer-term H(2) photoproduction phase than the wild-type strain, with the double mutant exhibiting overall the largest amount of H(2) evolved . This work demonstrates the feasibility of single and multiple gene engineering of microorganisms to redirect their metabolism for improving H(2) photoproduction using actual waste-derived substrates.

Can J Microbiol, 2004 Sep, 50(9), 669 - 74
Lipid nutrition of Saccharomyces cerevisiae in winemaking; Belviso S et al.; Biosynthesis of cell membrane lipids is a crucial metabolic pathway for the growth and viability of eucaryotic microorganisms . In Saccharomyces cerevisiae, unsaturated fatty acids and ergosterol synthesis needs molecular oxygen . Stuck and sluggish fermentations are related to this aspect of metabolism and constitute a major problem in the wine industry . Anaerobiosis, when lipids are not available in the growth medium, highly stresses cells . They release lipid biosynthesis metabolites and soon cease to multiply . This paper describes an investigation of the nutritional role of exogenous lipids from inactivated yeast cells (IYCs) . Fermentations were carried out in a nitrogen-rich synthetic medium similar to grape juice with glucose and fructose as carbon sources, without lipid sources, and in anaerobiosis . The effect of the addition of IYC was assessed . Cell growth, cell lipid composition, glucose and fructose consumption, and acetic acid production were measured during fermentation . Addition of IYC boosted cell growth and sugar consumption, whereas acetic acid production decreased . Biomass yield was influenced by ergosterol availability and increased when IYCs were added . Fatty acid composition of yeast cells was changed by IYC addition.

Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao, 2004 Dec, 30(6), 619 - 24
{Effects of 6-BA on Cluster Root Formation and Organic Acid Exudation in White Lupin Grown Under Phosphorus Deficiency.}; Liang RX et al.; Phosphorus deficiency results in cluster root formation and increased organic acid exudation . The regulatory mechanisms for these processes, however, are not yet clear . In the present study, influences of 6-BA (6-benzyl aminopurine) on cluster root formation, exudation of citrate and malate and their concentrations in the root clusters of P-deficient white lupin plants were studied by using non-destructive localized collection method and high-performance liquid chromatography (HPLC) technique . The results showed that application of exogenous 6-BA to P-deficient plants did not influence plant growth and P distribution within the plant, whereas the cluster root formation and organic acid exudation were inhibited . The inhibitory effects could be reversed by omitting 6-BA from the growth medium, and even some stimulatory effects was observed, when lower concentration of 6-BA (10(-8) mol/L) was applied . The inhibitory effects of higher concentration of 6-BA (10(-7) mol/L) were not reversible . Treatment with 6-BA also had some influence on organic acid concentration in the tissue of cluster roots . The possible reasons for the effects on cluster root formation and organic acid exudation by 6-BA are discussed.

Protein Expr Purif, 2005 Feb, 39(2), 296 - 306
Overexpression and purification of Pyrococcus abyssi phosphopantetheine adenylyltransferase from an optimized synthetic gene for NMR studies; Nalezkova M et al.; Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme that catalyses a rate-limiting step in coenzyme A (CoA) biosynthesis in all organisms . This study was conducted to obtain a high amount of pure, soluble, and stable PPAT from the hyperthermophilic archaeon Pyrococcus abyssi with the aim of investigating its structural characterization by NMR . Production of this enzyme from its natural gene in the Escherichia coli classical expression strain (BL21(DE3)) was not possible, most likely due to the presence of a high number of E . coli rare codons . Only a low amount of P . abyssi PPAT was previously obtained in two E . coli strains encoding tRNAs that recognize these rare E . coli codons and only by using a very rich growth medium . It was not possible to use this strategy to prepare labelled samples for the NMR study, thus another solution had to be found . Therefore, a synthetic gene encoding P . abyssi PPAT was constructed for which not only the rare codons were changed but which was also optimized to avoid other expression-limiting factors such as internal ribosome entry sites, RNA secondary structures, and DNA repeats . Gene optimization strongly increased the yield of P . abyssi PPAT in E . coli BL21(DE3) and allowed us to start the structural characterization of the enzyme . Circular dichroism and 2D NMR experiments indicate the presence of a well-ordered structure for P . abyssi PPAT and also confirm the existence of this enzyme as a monomer in solution.

Appl Environ Microbiol, 2005 Jan, 71(1), 58 - 64
Effect of culture conditions on microorganism identification by matrix-assisted laser desorption ionization mass spectrometry; Valentine N et al.; Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been used to identify bacteria based upon protein signatures . This research shows that while some different proteins are produced by vegetative bacteria when they are cultured in different growth media, positive identification with MALDI-TOF MS is still possible with the protocol established at the Pacific Northwest National Laboratory (K . H . Jarman, S . T . Cebula, A . J . Saenz, C . E . Petersen, N . B . Valentine, M . T . Kingsley, and K . L . Wahl, Anal . Chem . 72:1217-1223, 2000) . A core set of small proteins remain constant under at least four different culture media conditions and blood agar plates, including minimal medium M9, rich media, tryptic soy broth (TSB) or Luria-Bertani (LB) broth, and blood agar plates, such that analysis of the intact cells by matrix-assisted laser desorption/ionization mass spectrometry allows for consistent identification.

J Biotechnol, 2005 Feb 9, 115(3), 221 - 37 Epub 2004 Nov 19.
The global gene expression response of Escherichia coli to l-phenylalanine; Polen T et al.; We investigated the global gene expression changes of Escherichia coli due to the presence of different concentrations of phenylalanine or shikimate in the growth medium . The response to 0.5gl(-1) phenylalanine primarily reflected a perturbed aromatic amino acid metabolism, in particular due to TyrR-mediated regulation . The addition of 5gl(-1) phenylalanine reduced the growth rate by half and elicited a great number of likely indirect effects on genes regulated in response to changed pH, nitrogen or carbon availability . Consistent with the observed gene expression changes, supplementation with shikimate, tyrosine and tryptophan relieved growth inhibition by phenylalanine . In contrast to the wild-type, a tyrR disruption strain showed increased expression of pckA and of tktB in the presence of phenylalanine, but its growth was not affected by phenylalanine at the concentrations tested . The absence of growth inhibition by phenylalanine suggested that at high phenylalanine concentrations TyrR-defective strains might perform better in phenylalanine production.

J Biol Chem . 2005 Jan 4; {Epub ahead of print}
Triticum durum metallothionein: Isolation of the gene and structural characterization of the protein using solution scattering and molecular modeling; Bilecen K et al.; A novel gene sequence, with two exons and one intron, encoding a metallothionein (MT) has been identified in durum wheat Triticum durum cv . Balcali85 genomic DNA . Multiple alignment analyses on the cDNA and the translated protein sequences showed that T . durum MT (dMT) can be classified as a Type 1 MT . dMT has three Cys-X-Cys motifs in each of the N- and C-terminal domains and a 42 residues long hinge region devoid of cysteines . dMT was overexpressed in E . coli as a fusion protein (GSTdMT), and bacteria expressing the fusion protein showed increased tolerance to Cd in the growth medium compared to controls . Purified GSTdMT was characterized by SDS- and native- PAGE, size exclusion chromatography and MALDI-TOF-MS . It was shown that the recombinant protein binds 4+/-1 moles of Cd/mole protein and has a high tendency to form stable oligomeric structures . The structure of GSTdMT and dMT was investigated by synchrotron X-ray solution scattering and computational methods . X-ray scattering measurements indicated a strong tendency for GSTdMT to form dimers and trimers in solution and yielded structural models that were compatible with a stable dimeric form in which dMT had an extended conformation . Results of homology modeling and ab initio solution scattering approaches produced an elongated dMT structure with a long central hinge region . The predicted model and those obtained from X-ray scattering are in agreement and suggest that dMT may be involved functions other than metal detoxification.

Mycopathologia, 2004 Nov, 158(4), 465 - 74
Antagonism against Rhizoctonia solani and fungitoxic metabolite production by some Penicillium isolates; Nicoletti R et al.; A number of Penicillium isolates were recovered in association to Rhizoctonia solani strains pathogenic on tobacco and from soil on plates pre-colonized by the pathogen itself . Their antagonism toward R . solaniAG-2-1 was evaluated in dual cultures in vitro . Inhibition of growth was evident to some extent in most pairings, while hyphal interactions referable to mycoparasitic relationships were not observed . However, the occurrence of plasmolysis and/or vacuolisation and the induction of monilioid cells were indicative of the release of bioactive compounds . Therefore, production of fungitoxic metabolites was tested by adding concentrated culture filtrates of each Penicillium isolate to the growth medium of R . solani . Complete and lasting inhibition was incited by culture filtrates of some isolates belonging to P . brevicompactum, P . expansum, and P . pinophilum . Three purified compounds, respectively mycophenolic acid, patulin and 3-O-methylfunicone, which were extracted from culture filtrates, were able to inhibit R . solani in vitro . Their production was also detected in dual cultures of the same Penicilliumstrains with R . solani prepared in sterilized soil and when the Penicilliumstrains were cultured directly on R . solani mycelium harvested from liquid cultures . The possible role of such metabolites in antagonism of the above-mentioned Penicilliumspecies against R . solani is discussed.

Pain, 2005 Jan, 113(1-2), 113 - 22
Tumor necrosis factor alpha and interleukin-1beta stimulate the expression of cyclooxygenase II but do not alter prostaglandin E(2) receptor mRNA levels in cultured dorsal root ganglia cells; Fehrenbacher JC et al.; Tumor necrosis factor alpha (TNFalpha) and interleukin 1beta (IL-1beta) are pro-inflammatory cytokines capable of altering the sensitivity of sensory neurons . Because sensitization elicited by IL-1beta and TNFalpha is blocked by inhibition of the inducible enzyme, cyclooxygenase-II (COX-2), we examined whether these cytokines could increase COX-2 expression in dorsal root ganglion (DRG) cultures . Treatment of cell cultures with either IL-1beta or TNFalpha increases immunoreactive COX-2, as measured by immunoblotting, in a time- and concentration-dependent manner . A 24-h pretreatment with 10ng/ml IL-1beta or 50ng/ml TNFalpha augmented COX-2 expression 50- and 8-fold over basal levels, respectively . Immunohistochemistry established the presence of COX-2-like immunoreactivity in both neuronal and non-neuronal cells in culture . The addition of IL-1 receptor antagonist blocked the induction of COX-2 expression by IL-1beta, but did not alter TNFalpha-stimulated increases in COX-2, indicating that the mechanism of TNFalpha is not limited to increasing the expression of IL-1beta . The basal and TNFalpha-induced expression of COX-2 was not dependent on the presence of NGF in the growth media . IL-1beta and TNFalpha treatment for 24h enhanced prostaglandin E(2) (PGE(2)) production 2-4-fold, which was blocked by pretreatment with the COX-2 inhibitor, NS-398 . Exposing cultures to PGE(2), IL-1beta, or TNFalpha for 24h did not alter PGE(2) receptor (EP) mRNA levels . These results indicate that TNFalpha and IL-1beta induce the functional expression of COX-2 but not EP receptors in DRG cells in culture and suggest that cytokine-induced sensitization of sensory neurons is secondary to prostaglandin production and not alterations in EP receptors.

Plant Physiol, 2005 Jan, 137(1), 274 - 86 Epub 2004 Dec 23.
More than a leak sealant . The mechanical properties of callose in pollen tubes; Parre E et al.; While callose is a well-known permeability barrier and leak sealant in plant cells, it is largely unknown whether this cell wall polymer can also serve as a load-bearing structure . Since callose occurs in exceptionally large amounts in pollen, we assessed its role for resisting tension and compression stress in this cell . The effect of callose digestion in Solanum chacoense and Lilium orientalis pollen grains demonstrated that, depending on the species, this cell wall polymer represents a major stress-bearing structure at the aperture area of germinating grains . In the pollen tube, it is involved in cell wall resistance to circumferential tension stress, and despite its absence at the growing apex, callose is indirectly involved in the establishment of tension stress resistance in this area . To investigate whether or not callose is able to provide mechanical resistance against compression stress, we subjected pollen tubes to local deformation by microindentation . The data revealed that lowering the amount of callose resulted in reduced cellular stiffness and increased viscoelasticity, thus indicating clearly that callose is able to resist compression stress . Whether this function is relevant for pollen tube mechanics, however, is unclear, as stiffened growth medium caused a decrease in callose deposition . Together, our data provide clear evidence for the capacity of cell wall callose to resist tension and compression stress, thus demonstrating that this amorphous cell wall substance can have a mechanical role in growing plant cells.

J Biomol NMR, 2004 Nov, 30(3), 267 - 74
Biosynthetic site-specific (13) C labeling of the light-harvesting 2 protein complex: A model for solid state NMR structure determination of transmembrane proteins; van Gammeren AJ et al.; Partly biosynthetic site-directed isotopically (13)C enriched photosynthetic light-harvesting 2(LH2) complexes have been prepared from Rhodopseudomonas acidophila strain 10050 by using chemically labeled {1,2,3,4-(13)C}, {1,4-(13)C} and {2,3-(13)C} succinic acid as a precursor in the growth medium . Two-dimensional proton driven spin diffusion (PDSD) solid state NMR correlation spectroscopy has been used to trace each individual (13)C isotope from the labeled succinic acid precursor to its destination into the protein and into the embedded major light-absorbing bacteriochlorophyll cofactors . For both the residues of the protein and for the cofactors distinct labeling patterns have been deduced, for protein complexes prepared from {1,4-(13)C}-succinic acid or {2,3-(13)C}-succinic labeled media . All residues, except isoleucine and leucine, have been labeled almost homogeneously by the succinic acid precursor . Carbonyl carbons in the protein backbone were labeled by {1,4-(13)C}-succinic acid, while the Calpha and Cbeta carbons of the residues were labeled by {2,3 (13)C}-succinic acid . Leucine and isoleucine residues were labeled using a uniformly labeled amino acid mixture in the medium . The pattern labeling yields an increase of the resolution and less spectral crowding . The partial labeling technique in combination with conventional solid state NMR methods at ultra high magnetic fields provides an attractive route to resolve chemical shifts for alpha-helical transmembrane protein structures.

Parasitol Res, 2005 Jan, 95(1), 17 - 21 Epub 2004 Nov 18.
The in vitro anti-giardial activity of extracts from plants that are used for self-medication by AIDS patients in southern Thailand; Sawangjaroen N et al.; This study evaluated the anti-giardial activity of chloroform, methanol and water extracts of 12 medicinal plants (39 extracts), commonly used as self medication by AIDS patients in southern Thailand . The plant extracts and a standard drug, metronidazole, were incubated with 2x10(5) trophozoites of Giardia intestinalis per millilitre of growth medium in 96-well tissue culture plates under anaerobic conditions for 24 h . The cultures were examined with an inverted microscope and the minimum inhibitory concentration and the IC(50) value for each extract was determined . The chloroform extracts from Alpinia galanga, Boesenbergia pandurata, Eclipta prostrata, Piper betle, Piper chaba, Zingiber zerumbet, and the methanol extracts from B . pandurata and E . prostrata were classified as "active", i.e . with an IC(50) of <100 mug/ml, whereas the chloroform extract from Murraya paniculata was classified as being "moderately active" . This study shows that extracts from some medicinal plants have potential for use as therapeutic agents against G . intestinalis infections.

Syst Appl Microbiol, 2004 Nov, 27(6), 672 - 80
Emericella venezuelensis, a new species with stellate ascospores producing sterigmatocystin and aflatoxin B1; Frisvad JC et al.; Emericella venezuelensis is a new species, differing from two other species with stellate ascospores, E . variecolor and E . pluriseminata, by triangular flaps on the convex sides of the ascospores, and further from E . variecolor by producing an Aspergillus anamorph only on unconventional growth media . The three species also differ in their profiles of extrolites (secondary metabolites) . Emericella venezuelensis produces aflatoxin B1, sterigmatocystin, and terrein and compounds with chromophores of the shamixanthone, emerin and desertorin type of compounds . E . variecolor produces asteltoxin, shamixanthone, asperthecin, and terrein, in addition to metabolites unequivocally recorded in the literature or tentatively identified here as astellolide A & B, andibenin A, B, C, andilesin A, B, C, anditomin, astellatol, stellatic acid, stellatin, tajixanthone, radixanthone, najamxanthone, ajamxanthone, variecoxanthone A, B, C, isoemericellin, kojic acid, varitriol, varioxiran, dihydroterrein, 7-hydroxyemodin, avariquinone and stromemycin . E . pluriseminata produces several unknown specific extrolites . E . venezuelensis is the first organism of marine origin reported to produce aflatoxin . Aflatoxin production by E . venezuelensis makes this species an attractive model organism for the study of the regulation of this important type of carcinogenic mycotoxins in combination with the knowledge on sterigmatocystin production by E . nidulans, soon to be whole genome sequenced . The isolates were also analyzed cladistically using partial sequences of the beta-tubulin gene . Since three species of Emericella have stellate ascospores, and the type material of E . variecolor is equivocal, this species is epitypified with CBS 598.65 . Emericella species normally do not appear to cause problems for food safety, as they are most often found in litter and soil.

Biochem J . 2004 Dec 20; {Epub ahead of print}
The first cysteine-rich domain of the GFRalpha1 receptor stabilises the binding of GDNF; Virtanen H et al.; The GDNF-binding GFRalpha1 receptor is attached to the membrane by a GPI-anchor and consists of three cysteine-rich domains . The region corresponding to the second and third domain has previously been shown to participate in the ligand binding as well as to interact with RET . No function has so far been found for the N-terminal, first domain (D1) . Here we show that the GPI-anchored full-length receptor binds { 125I}GDNF two times more tightly than the GPI-anchored truncated receptor, lacking D1 . Scintillation proximity assays with purified receptor proteins also show that the GDNF-binding capacity of the soluble full-length GFRalpha1 is two times higher than the GDNF-binding capacity of the soluble D1-truncated GFRalpha1 . As RET stabilises the binding of GDNF equally well to the full-length and truncated receptors, D1 seems not to be involved in the interaction between GFRalpha1 and RET . Moreover, the soluble full-length GFRalpha1 receptor mediates GDNF-promoted neurite outgrowth in PC6-3 cells more efficiently than the soluble truncated GFRalpha1 protein . At low concentrations, the soluble full-length receptor mediates the phosphorylation of RET more efficiently than the soluble truncated receptor . However, when the receptors are over-expressed on the cell surface as GPI-anchored proteins, or added to the growth media at high concentrations as soluble proteins, the full length and truncated GFRalpha1 receptors are indistinguishable in GDNF-dependent RET-phosphorylation assays . High levels of the receptors can thus mask a slightly impaired function in the phosphorylation assay . Based on assays with both GPI-anchored and soluble receptors, we therefore conclude that D1 contributes to the optimal function of the GFRalpha1 receptor by stabilising the interaction between GFRalpha1 and GDNF.

Plant Mol Biol, 2004 Jul, 55(5), 743 - 61
Methyl jasmonate induced expression of the tobacco putrescine N -methyltransferase genes requires both G-box and GCC-motif elements; Xu B et al.; Putrescine N-methyltransferase (PMT) catalyzes the first committed step of nicotine biosynthesis, converting putrescine into N-methylputrescine . A variety of chemical, environmental, and developmental cues have been implicated in its regulation . Here we have examined the differential expression of beta-glucuronidase (GUS) transgenes under the control of the transcriptional regulatory sequences of four distinct members of the NtPMT gene family from tobacco (Nicotiana tabacum L.) . BY-2 cell cultures expressing various NtPMT promoter-GUS constructs were examined for their response to treatment with various combinations of methyl jasmonate (MeJA), auxin (AUX), and ethylene (ETH) . All four NtPMT gene promoters examined were inducible by MeJA, although the extent of the induction varied dramatically, with the NtPMT1a promoter being the most responsive . High AUX levels in the cell growth media repressed NtPMT::GUS transgene expression and inhibited their MeJA-induced transcription . Treatment of BY-2 cells with ETH alone did not result in a significant alteration in NtPMT::GUS expression . However, similar to AUX, ETH treatment led to the suppression of MeJA-induced transcription . Detailed deletion analysis of the NtPMT1a gene promoter showed that as little as 111 bp upstream of the transcriptional start site were sufficient to confer MeJA-responsiveness . Deletion of a conserved G-box element (GCACGTTG) at -103 to -96 bp completely abolished MeJA-responsiveness . Further mutagenesis studies revealed that in addition to a functional G-box, MeJA-responsiveness of the NtPMT1a promoter also required a TA-rich region and a GCC-motif (TGCGCCC) located at -80 to -69 bp and -62 to -56 bp relative to the start site, respectively . A synthetic G-box tetramer (4 X syn G-box) fused to a -83 bp fragment from the NtPMT1a promoter (containing the TA-rich region, GCC-box, and TATA-box) displayed a 30-fold induction by MeJA treatment, whereas when the 4 X syn G-box was fused to a minimal (-46 bp) promoter fragment derived from the CaMV 35S gene, no induction by MeJA treatment was detected . Our results indicate that multiple intersecting signal transduction pathways and different transcriptional regulatory factors are involved in mediating JA-responsiveness of NtPMT expression in tobacco.

Plant Mol Biol, 2004 Jul, 55(5), 727 - 41
Transcriptional regulation and functional properties of Arabidopsis Pht1;4, a high affinity transporter contributing greatly to phosphate uptake in phosphate deprived plants; Misson J et al.; Phosphate mobilization into the plant is a complex process requiring numerous transporters for absorption and translocation of this major nutrient . In the genome of Arabidopsis thaliana, nine closely related high affinity phosphate transporters have been identified but their specific roles remain unclear . Here we report the molecular, histological and physiological characterization of Arabidopsis pht1;4 high affinity phosphate transporter mutants . Using GUS-gene trap and in situ hybridization, Pht1;4 was found mainly expressed in inorganic phosphate (Pi) limiting medium in roots, primarily in the epidermis, the cortex and the root cap . In addition to this, expression was also observed at the lateral root branch points on the primary root and in the stele of lateral roots, suggesting a role of Pht1;4 in phosphate absorption and translocation from the growth medium to the different parts of the plant . Pi-starved pht1;4 plantlets exhibited a strong reduction of phosphate uptake capacity (40) . This phenotype appears only related to the pht1;4 mutation as there were no obvious changes in the expression of other Pht1 family members in the mutants background . However, after 10 days of growth on phosphate deficient or sufficient medium, the Pi content in the mutants was not significantly different from that of the corresponding wild type controls . Furthermore, the mutants did not display any obvious growth defects or visible phenotypes when grown on a low phosphate containing medium . The work described here offers a first step in the complex genetic dissection of the phosphate transport system in planta.

Dermatology, 2005, 210(1), 45 - 8
An improved organ culture for regeneration of pure autologous keratinocytes from small split-thickness skin specimens; Liu JY et al.; BACKGROUND: Failure of autologous keratinocyte culture from small split-thickness skin specimens or contamination of the keratinocyte culture by melanocytes represents practical problems in basic medical research and clinical studies . PURPOSE: To establish a simple and reliable method of harvesting pure autologous keratinocytes from a small split-thickness skin specimen . METHODS: Split-thickness (0.3 mm) skin explants (1 x 2 mm) were firstly cultured in DMEM containing 10% FCS till formation of keratinocyte strips, then cultured in serum-free keratinocyte growth medium or cocultured with lethally irradiated 3T3 fibroblasts (J2) in a mixture of DMEM and Ham's F12 (DF) medium . RESULTS: Pure autologous keratinocyte culture is easily and reliably established by this organ culture technique . CONCLUSION: Culturing of skin explants in serum-free keratinocyte growth medium or coculturing of the skin explants with lethally irradiated 3T3 cells in DF medium is proved to be a useful, simple and reliable method of harvesting pure autologous keratinocytes from a small split-thickness skin biopsy.

Environ Sci Technol, 2004 Dec 1, 38(23), 6307 - 13
Photosensitizers neutral red (type I) and rose bengal (type II) cause light-dependent toxicity in Chlamydomonas reinhardtii and induce the Gpxh gene via increased singlet oxygen formation; Fischer BB et al.; The connection between the mode of toxic action and the genetic response caused by the type I photosensitizer and photosynthesis inhibitor neutral red (NR) and the type II photosensitizer rose bengal (RB) was investigated in the green alga Chlamydomonas reinhardtii . For both photosensitizers, a light intensity-dependent increase in toxicity and expression of the glutathione peroxidase homologous gene (Gpxh) was found . The toxicity of RB was reduced by the singlet oxygen (1O2) quenchers 1,4-diazabicyclo{2.2.2}octane and L-histidine, and the RB-induced Gpxh expression was stimulated in deuterium oxide-supplemented growth medium . These observations clearly indicate the involvement of 1O2 in both toxicity and the genetic response caused by RB . NR up-regulated the expression of typical oxidative and general stress response genes, probably by a type I mechanism, and also strongly induced the Gpxh expression . The stimulating effect of deuterium oxide in the growth medium suggested the involvement of 1O2 also in the NR-induced response . Indeed, an increased 1O2 formation was detected with EPR-spin trapping in NR-treated spinach thylakoids . However, none of the 102 quenchers could reduce the light-dependent toxicity of NR in C . reinhardtii, indicating that NR has a different mode of toxic action than RB.

Langmuir, 2004 Dec 21, 20(26), 11433 - 42
Elucidation of functional groups on gram-positive and gram-negative bacterial surfaces using infrared spectroscopy; Jiang W et al.; Surface functional group chemistry of intact Gram-positive and Gram-negative bacterial cells and their isolated cell walls was examined as a function of pH, growth phase, and growth media (for intact cells only) using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy . Infrared spectra of aqueous model organic molecules, representatives of the common functional groups found in bacterial cell walls (i.e., hydroxyl, carboxyl, phosphoryl, and amide groups), were also examined in order to assist the interpretation of the infrared spectra of bacterial samples . The surface sensitivity of the ATR-FTIR spectroscopic technique was evaluated using diatom cells, which possess a several-nanometers-thick layer of glycoprotein on their silica shells . The ATR-FTIR spectra of bacterial surfaces exhibit carboxyl, amide, phosphate, and carbohydrate related features, and these are identical for both Gram-positive and Gram-negative cells . These results provide direct evidence to the previously held conviction that the negative charge of bacterial surfaces is derived from the deprotonation of both carboxylates and phosphates . Variation in solution pH has only a minor effect on the secondary structure of the cell wall proteins . The cell surface functional group chemistry is altered neither by the growth phase nor by the growth medium of bacteria . This study reveals the universality of the functional group chemistry of bacterial cell surfaces.

Yeast, 2005 Jan 15, 22(1), 13 - 9
Cloning and characterization of the Hansenula polymorpha PEP4 gene encoding proteinase A; Bae JH et al.; The Hansenula polymorpha PEP4 gene encoding proteinase A was cloned by Southern blot hybridization using the Saccharomyces cerevisiae PEP4 gene as probe and characterized by gene disruption and overexpression . Nucleotide sequence analysis revealed an open reading frame (ORF) of 1239 nucleotides corresponding to a polypeptide of 413 amino acids, sharing about 67.2% sequence similarity with that of S . cerevisiae proteinase A . That the cloned H . polymorpha PEP4 gene encodes proteinase A was supported by a gene disruption experiment, which showed that the H . polymorpha pep4 mutant strain showed significantly reduced level of carboxypeptidase Y activity when assayed with an artificial substrate . When the PEP4 gene is overproduced in pep4 mutant strain, mature proteinase A could be found in the growth medium . N-terminal amino acid sequencing of extracellular proteinase A revealed the presence of a putative propeptide of 55 amino acids ending with a dibasic peptide (Lys-Arg), probably processed by Kex2p-like endopeptidase of H . polymorpha . The nucleotide sequence of the H . polymorpha PEP4 gene has been submitted to GenBank under Accession No . U67173 . Copyright (c) 2004 John Wiley & Sons, Ltd.

Exp Neurol, 2005 Jan, 191(1), 163 - 73
Regulated sarcolemmal localization of the muscle-specific ClC-1 chloride channel; Papponen H et al.; The skeletal muscle-specific ClC-1 is a voltage-gated chloride channel protein . Specific antibodies against ClC-1 revealed in muscle sections a sarcolemmal staining that was absent in the myotonic arrested development of righting response (ADR) mouse muscle . The intensity of the sarcolemmal staining varied from one type of muscle to another and in lateral sections showed a typical mosaic pattern that colocalized with beta-dystroglycan and left the transverse tubule openings clear . Surprisingly, in isolated myofibers, the ClC-1 protein was absent from the sarcolemma . Instead, it localized to intracellular I band areas as soon as the myofibers were isolated . When the isolated myofibers were incubated with the kinase inhibitor staurosporine, the ClC-1 protein shifted back to the sarcolemma . Electric stimulation of the cultivated fibers had a similar effect . Also, myofibers infected with a recombinant Semliki Forest virus (SFV) expressing myc-tagged ClC-1 showed intracellular localization of the protein . The virally expressed mycClC-1 reached the Golgi apparatus but sarcolemmal staining remained nondetectable, and addition of staurosporine into the growth medium recruited the mycClC-1 to the sarcolemma . These data indicate that sarcolemmal targeting of the ClC-1 requires specific signals that are provided by the physiological environment.

Biochem J . 2004 Dec 9; {Epub ahead of print}
Regulation of activity in vitro and in vivo of three phospholipases B from Saccharomyces cerevisiae; Merkel O et al.; The genome of the yeast, Saccharomyces cerevisiae, contains three highly homologous genes coding for phospholipases B/lysophospholipases . These enzymes behave differently with respect to substrate preference in vitro and relative contribution to phospholipid catabolism in vivo (Merkel, Fido, Mayr, Pruger, Raab, Zandonella, Kohlwein, Paltauf (1999) J Biol Chem, 274, 28121-28127) . It is shown in this study that in vitro pH strongly affects substrate preference of phospholipase B1 (Plb1p) and phospholipase B2 (Plb2p), but not of phospholipase B3 (Plb3p) . At the pH optimum of 2.5-3.5 the substrate preference of Plb1p and Plb2p is PtdSer>PtdIns>PtdCho>PtdEtn . At pH 5 and higher the substrate preference changes to PtdCho=PtdEtn for Plb1p and PtdSer=PtdEtn for Plb2p . Accordingly, with cultured cells the ratio of PtdIns vs . PtdCho breakdown, as reflected in the ratio GroPIns/GroPCho released into the culture medium, is inversely related to the pH of the growth medium . This effect is ascribed to the pH response of Plb1p because Plb2p does not contribute to PtdIns and PtdCho degradation in vivo (Merkel, Fido, Mayr, Pruger, Raab, Zandonella, Kohlwein, Paltauf (1999) J Biol Chem, 274, 28121-28127) . Di- and trivalent cations activate phospholipases B at pH 5.5, but inhibit at pH 2.5 . Al 3+ at 20 mM concentration increases Plb1p activity in vitro 8-fold and leads to a 9-fold increase in GroPCho release by whole cells . In vivo cycoheximide (CHI) strongly inhibits breakdown of PtdIns and to a lesser extent of PtdCho . However, Al 3+-stimulated GroPCho release is almost completely inhibited by CHI . Deletion of PLB3 leads to increased sensitivity towards toxic Al 3+ . Addition of SDS or melittin to cultured cells leads to a significant increase in phospholipid degradation, which is insensitive to inhibition by CHI . Deletion mutants defective in the PLB1 gene are significantly more resistant towards SDS than wild type cells.

Biomaterials, 2005 May, 26(15), 2441 - 2453
Quantitative analysis of macrophage apoptosis vs . necrosis induced by cobalt and chromium ions in vitro; Catelas I et al.; The potential toxicity of metal ions in tissues surrounding metal-metal hip replacements is a cause for concern . Previous studies conducted in our laboratory demonstrated that Co(2+) and Cr(3+) induce TNF-alpha secretion in macrophages, as well as cell mortality . However, the degree of apoptosis and necrosis remained to be investigated . The aim of the present study was to quantify the rate of macrophage mortality by apoptosis vs . necrosis induced by Co(2+) and Cr(3+) . J774 mouse macrophages were incubated in growth medium containing 0-10ppm Co(2+) and 0-500ppm Cr(3+) for 24 and 48h under conventional cell culture conditions . Transmission electron microscopy, flow cytometry (Annexin-V fluorescein isothiocyanate/propidium iodide assay) and a specific cell death detection ELISA were used to illustrate cell death and differentiate between apoptotic and necrotic cells . Cell culture exposed to low concentrations of Co(2+) (0-6ppm) revealed a low degree of mortality . In contrast, at the highest concentrations (8-10ppm), late apoptosis occurred within 24h . After 48h, however, there was a clear evidence for an increase in the rate of necrosis while apoptosis occurred at much lower rate . Macrophages exposed to Cr(3+) demonstrated a predominance of apoptosis after 24h . At concentrations lower than 250ppm, early and late apoptosis occurred at the same rate . At higher concentrations (250-500ppm), the number of early apoptotic cells decreased in favor of late apoptosis . After 48h, lower concentrations of Cr(3+) (150ppm) induced a higher degree of early apoptosis than after 24h, and some necrosis . At higher concentrations, the percentage of early apoptotic cells decreased, while necrosis became predominant over late apoptosis . In conclusion, this study demonstrates that macrophage mortality induced by metal ions depends on the type and concentration of metal ions as well as the duration of their exposure . Overall, apoptosis was predominant after 24h with both Co(2+) and Cr(3+) ions, but high concentrations induced mainly necrosis at 48h . These results point to the potential for these ions of inducing tissue damage by necrosis if present in large concentrations in vivo.

Scand J Immunol, 2004 Dec, 60(6), 584 - 91
Serum concentration of the growth medium markedly affects monocyte-derived dendritic cells' phenotype, cytokine production profile and capacities to stimulate in MLR; Jakobsen MA et al.; We have investigated how the maturation of monocyte-derived dendritic cells (Mo-DC) is affected by the serum concentration of the culture medium . Day 6 DC cultured in 1% human serum were a heterogeneous population of CD1a(-) and CD1a(+) DC that were separated by flow sorting . In contrast, Mo-DC generated in 10% human serum formed a homogenous population of CD1a(-) cells . Other phenotypically immature characteristics also varied, and three subsets were still distinguishable upon maturation in LPS . Furthermore, CD1a(-) DC and CD1a(+) DC from 1% culture conditions were excellent stimulators in MLR, while DC cultured in 10% serum were poor stimulators . Similarly, different cytokine profiles of the three subsets were identified . DC cultured in 1% serum had low expression of interleukin-12 (IL-12) p40 and IL-10 mRNA at day 6 . Upon maturation, expression of IL-12 p40 mRNA was upregulated in CD1a(+) DC, whereas the level remained relatively low in CD1a(-) DC . In contrast, DC cultured in 10% had high levels of IL-10 mRNA at day 6 that was downregulated upon maturation . We conclude that the differentiation of monocytes into DC is significantly influenced by the serum concentration of the growth medium with effects on phenotype, cytokine profile and stimulatory activity.

Cell Biochem Funct . 2004 Dec 6; {Epub ahead of print}
Transient transfection of epidermal growth factor receptor gene into MCF7 breast ductal carcinoma cell line; Alokail MS; Epidermal growth factor receptor (EGFR) is activated by autocrine growth factors in many types of tumours, including breast tumours . This receptor has been linked to a poor prognosis in breast cancer and may promote proliferation, migration, invasion, and cell survival as well as inhibition of apoptosis . Human breast ductal carcinoma MCF7 cells were transfected using FuGENEtrade mark 6 with 1 mug of pcDNA3-EGFR containing the full-length human EGFR promoter or 1 mug of the vectors alone (pcDNA3) . The transfected cells were transferred into a 25-cm(2) flask containing growth medium and G418 . Confluent cultures were lysed, total protein levels measured and electrophoresed . The electrophoresed samples were transferred to nitrocellulose and incubated overnight at 4 degrees C with either anti-EGFR or anti-phospho-ERK and immunoreactive bands were visualized using HRP-linked secondary antibody . We created a model system of EGFR overexpression in MCF7 clones with stably transfected pcDNA3/EGFR plasmid . These cells have been shown to promote substantial phosphorylation of both ERK1 and ERK2 . The high level of EGFR and ERK1/2 phosphorylation was not seen in the pcDNA3 vector control cells or in non-transfected cells . In this article we describe successful transient transfection experiments on MCF7 cells using the FuGENEtrade mark 6 Transfection Reagent . The overexpression of EGFR could be a mediated stress response and a survival signal that involves ERK1 and ERK2 phosphorylation . Copyright (c) 2004 John Wiley & Sons, Ltd.

J Clin Microbiol, 2004 Dec, 42(12), 5875 - 6
Evaluation of decontamination methods and growth media for primary isolation of Mycobacterium ulcerans from surgical specimens; Yeboah-Manu D et al.; We evaluated four decontamination methods and one nondecontamination procedure in combination with four egg-based media for the primary isolation of Mycobacterium ulcerans from tissue specimens . With mycobacterial recovery and contamination rates of 75.6 and 2.4%, respectively, the combination of the oxalic acid decontamination method with Lowenstein-Jensen medium supplemented with glycerol yielded the best results.

Microbiology, 2004 Dec, 150(Pt 12), 3979 - 87
Siderophore and haem iron use by Tritrichomonas foetus; Sutak R et al.; The ability of the parasitic flagellate Tritrichomonas foetus to use various iron sources for its physiological requirements was studied . The siderophores ferrioxamine B, ferrichrome, triacetylfusarinine, coprogen, enterobactin and pyoverdine sustained growth of the cells under iron-limited conditions, and siderophore iron was incorporated into the major iron protein of T . foetus, ferredoxin . The kinetics of siderophore uptake by the cells indicated that a non-saturable transport is involved, unlike the uptake of a ferrous salt . Siderophore uptake by the cells did not involve extracellular reductive dissociation of the ferric chelates, although T . foetus cells had some ferrireductase activity on ferric citrate . Fluorescent analogues of siderophores were used to show that the siderophores taken up by the cells were in small intracellular vesicles . The fluorescence emission maximum of pyoverdine in these intracellular vesicles shifted from 460 nm to 530 nm, indicating a very acidic environment . The results suggest that a wide range of chemically unrelated siderophores can be taken up non-specifically and efficiently used by T . foetus; the mechanism involved may be pinocytosis and removal of the iron from the siderophores in acidic intracellular vesicles . Haemin also sustained the growth of T . foetus cells under iron-limited conditions . The use of haemin iron by the cells probably involves haem oxygenase, since traces of biliverdin were found in the medium when haemin was the iron source . The iron uptake and ferrireductase activities of the cells do not seem to be regulated by the amounts of iron and copper in the growth medium.

Int J Food Microbiol, 2005 Jan 1, 97(3), 247 - 57
Use of an electronic tongue and HPLC with electrochemical detection to differentiate molds in culture media; Soderstrom C et al.; A study was conducted to further evaluate an electronic tongue, using high-performance liquid chromatography (HPLC) with electrochemical (EC) and UV detection as a reference method . The electronic tongue consisted of four working electrodes made of different metals and arranged in a standard three-electrode configuration . Pulses of voltage were applied to the metals, and the current responses were sampled and collected in a data matrix . The objectives of the present investigation were to examine the ability of the electronic tongue to distinguish between two mold species growing in three different media, and to obtain support for the hypothesis that the device actually discriminates between different redox-active metabolites produced by the molds . Peak areas in EC and UV HPLC chromatograms were collected in a data matrix . The electronic tongue data and the EC and UV data were then subjected to principal component analysis (PCA) . A number of peaks in the HPLC-EC chromatograms indicated that the growth media contained redox-active metabolites . Moreover, PCA of peak areas in EC chromatograms revealed differences between the distribution of redox-active metabolites produced by the two species and between the three culture media . The same pattern was apparent in a PCA score plot of electronic tongue data . The peaks in the UV and EC chromatograms differed, and these were also shown by the PCA score plots.

Curr Issues Mol Biol, 2005 Jan, 7(1), 109 - 17
Control of ribosome synthesis during the cell division cycles of E . coli and Synechococcus; Asato Y; The regulation of ribosome synthesis has been investigated for nearly five decades . In earlier studies, the control of rRNA synthesis in bacteria was found to be dependent on nutrient composition of the growth media or cell growth rates, and these observations led to the growth rate-dependent regulation model . Also developed were stringent control, feedback ribosome synthesis, passive regulation, and antitermination models . Current evidence indicates that upstream (UP) element, molecular effectors, ppGpp and iNTP (initiating nucleoside triphosphate), and trans-acting proteins, Fis and H-NS, play important roles in the control of rRNA synthesis in response to changing nutritional environments . The mechanisms for the ribosome feedback regulation, and growth rate-dependent controls of rRNA synthesis remain to be determined despite numerous investigations . r-protein synthesis can be controlled by translational coupling, translation repression, or premature transcription termination . In Synechococcus, a photoautotroph, ribosome synthesis occurs early in the cell cycle as programmed events under conditions that support balanced growth . Periods of r-protein synthesis occur before rRNA synthesis periods, and rRNA synthesis is stimulated by a light-activated gene regulatory protein . These observations suggest that gene regulatory proteins are involved in the coordinate regulation of ribosome assembly in Synechococcus.

Gene Ther . 2004 Nov 25; {Epub ahead of print}
Novel dextran-spermine conjugates as transfecting agents: comparing water-soluble and micellar polymers; Eliyahu H et al.; Recently, a novel cationic polymer, dextran-spermine (D-SPM) was developed for gene delivery . An efficient transfection was obtained using this polycation for a variety of genes and cell lines in serum-free or serum-poor medium . However, transfection using the water-soluble D-SPM-based polyplexes decreased with increasing serum concentration in cell culture in a concentration-dependent manner, reaching 95% inhibition at 50% serum in the cell growth medium . In order to overcome this obstacle, oleyl derivatives of D-SPM (which form micelles in aqueous phase) were synthesized at 1, 10, and 20 mol% of oleyl moiety to polymer varepsilon-NH(2) to form N-oleyl-D-SPM (ODS) . Polyplexes based on ODS transfected well in medium containing 50% serum . Comparison with polyplexes based on well-established polymers (branched and linear polyethyleneimine) and with DOTAP/Cholesterol lipoplexes showed that regarding beta-galactosidase transgene expression level and cytotoxicity in tissue culture, the D-SPM and ODS compare well with the above polyplexes and lipoplexes . Intracellular trafficking using FITC-labeled ODS and Rhodamine-labeled pGeneGrip plasmid cloned with hBMP2 monitored by confocal microscopy revealed that during the transfection process the fluorescent-labeled polymer concentrates in the Golgi apparatus and around the nucleus, while the cell cytoplasm was free of fluorescent particles, suggesting that the polyplexes move in the cell toward the nucleus by vesicular transport through the cytoplasm and not by a random diffusion . We found that the plasmids penetrate the cell nucleus without the polymer . Preliminary results in zebra fish and mice demonstrate the potential of ODS to serve as an efficient nonviral vector for in vivo transfection.Gene Therapy advance online publication, 25 November 2004; doi:10.1038/sj.gt.3302395.

Am J Physiol Renal Physiol . 2004 Nov 23; {Epub ahead of print}
Biotin uptake by human proximal tubular epithelial cells: Cellular and molecular aspects; Balamurugan K et al.; Cellular and molecular regulation of the renal biotin uptake in human is not well defined . In addition, the contribution of the human sodium-dependent multivitamin transporter, hSMVT, toward carrier-mediated biotin uptake by human proximal tubular epithelial cells is not clear . The aims of this study were, therefore, to address these issues using the human-derived proximal tubular epithelial HK-2 cells as a model . First, we characterized the mechanism of biotin uptake by these cells and obtained evidence for the involvement of a Na(+), temperature- and energy-dependent carrier-mediated uptake system . This system was inhibited by the biotin structural analogue desthiobiotin, pantothenic acid and lipoate . These findings suggest the involvement of the hSMVT system in the uptake process . This was confirmed by demonstrating that the hSMVT system is indeed expressed in HK-2 cells at the protein and mRNA levels, and by selectively silencing the hSMVT gene with the use of gene-specific small interfering RNAs (siRNAs) which lead to specific and significant inhibition in carrier-mediated biotin uptake . Of the two recently cloned promoters of the hSMVT gene, promoter 1 was found to be more active than promoter 2 in these cells . Pre-treatment of the HK-2 cells with modulators of protein kinase C (PKC) and Ca(2+)/calmodulin (Ca(2+)/CaM)- mediated pathways {but not those that modulate protein kinase A (PKA), protein tyrosine kinase (PTK), or nitric oxide(NO)- mediated pathways} lead to significant alterations in biotin uptake . Maintaining the HK-2 cells in a biotin-deficient growth medium lead to a marked up-regulation in biotin transport, which was associated with an increase in hSMVT protein and RNA levels as well as by an increase in the activity of the hSMVT promoters . These results demonstrate that biotin uptake by human renal epithelial cells occurs via the hSMVT system and that the process is under the regulation of an intracellular PKC and Ca(2+)/CaM-mediated pathways . In addition, the uptake process appears to be adaptively regulated by extracellular biotin level and that this regulation involves transcriptional regulatory mechanism(s).

Nucleosides Nucleotides Nucleic Acids, 2004 Oct, 23(6-7), 927 - 30
Release of nucleic acids by eukaryotic cells in tissue culture; Morozkin ES et al.; Extracellular nucleic acids in cultures of A431 and HeLa cells were investigated . The data obtained demonstrate the presence of high weight DNA and RNA in the extracellular medium . Temporal changes of extracellular nucleic acids levels in growth medium were investigated.

Mycorrhiza . 2004 Nov 19; {Epub ahead of print}
Gene expression of the ericoid mycorrhizal fungus Oidiodendron maius in the presence of high zinc concentrations; Vallino M et al.; A heavy metal tolerant strain of the ericoid mycorrhizal species Oidiodendron maius, isolated from roots of Vaccinium myrtillus growing in soil heavily contaminated with zinc, was previously shown to tolerate high concentrations of zinc and cadmium ions in the growth medium . We have investigated the genetic basis of this fungal strain tolerance to high zinc concentrations by using an untargeted approach . From a cDNA library constructed by using mRNA from Zn-treated O . maius mycelia, 444 clones were randomly selected and 318 were sequenced . Sequence analysis identified 219 unique clones: 117 showed homology to previously identified genes, 26 matched unknown protein coding regions found in other organisms, and 76 were novel . Variation in the gene expression level after a 20-day treatment with high concentrations of Zn was monitored on 130 unigenes by reverse northern blot hybridisation . Sixteen unigenes were shown to be either up- (9) or down- (7) regulated . The putative function of these genes and their involvement in stress tolerance is discussed.

Gene, 2004 Sep 29, 340(1), 11 - 8
Bacterial expression system with tightly regulated gene expression and plasmid copy number; Bowers LM et al.; A new Escherichia coli host/vector system has been engineered to allow tight and uniform modulation of gene expression and gamma origin (ori) plasmid copy number . Regulation of gamma ori plasmid copy number is achieved through arabinose-inducible expression of the necessary Rep protein, pi, whose gene was integrated into the chromosome of the host strain under control of the P(BAD) promoter . gamma ori replication can be uniformly modulated over 100-fold by changing the concentration of l-arabinose in the growth medium . This strain avoids the problem of all-or-nothing induction of P(BAD) because it is deficient in both arabinose uptake and degradation genes . Arabinose enters the cell by a mutant LacY transporter, LacYA177C, which is expressed from the host chromosome . Although this strain could be compatible with any gamma ori plasmid, we describe the utility of a gamma ori expression vector that allows especially tight regulation of gene expression . With this host/vector system, it is possible to independently modulate gene expression and gene dosage, facilitating the cloning and overproduction of toxic gene products . We describe the successful use of this system for cloning a highly potent toxin, Colicin E3, in the absence of its cognate immunity protein . This system could be useful for cloning genes encoding other potent toxins, screening libraries for potential toxins, and maintaining any gamma ori vector at precise copy levels in a cell.

Arch Microbiol, 2005 Jan, 183(1), 9 - 18 Epub 2004 Nov 11.
FieF (YiiP) from Escherichia coli mediates decreased cellular accumulation of iron and relieves iron stress; Grass G et al.; The Escherichia coli yiiP gene encodes an iron transporter, ferrous iron efflux (FieF), which belongs to the cation diffusion facilitator family (CDF) . Transcription of fieF correlated with iron concentration; however, expression appeared to be independent of the ferrous iron uptake regulator Fur . Absence of FieF led to decreased growth of E . coli cells in complex growth medium but only if fur was additionally deleted . The presence of EDTA was partially able to relieve this growth deficiency . Expression of fieF in trans rendered the double deletion strain more tolerant to iron . Furthermore, E . coli cells exhibited reduced accumulation of (55)Fe when FieF was expressed in trans . FieF catalyzed active efflux of Zn(II) in antiport with protons energized by NADH via the transmembrane pH gradient in everted membrane vesicles . Using the iron-sensitive fluorescent indicator PhenGreen-SK encapsulated in proteoliposomes, transmembrane fluxes of iron cations were measured with purified and reconstituted FieF by fluorescence quenching . This suggests that FieF is an iron and zinc efflux system, which would be the first example of iron detoxification by efflux in any organism.

Radiat Res, 2004 Dec, 162(6), 677 - 86
Induction of replication protein a in bystander cells; Balajee AS et al.; The bystander effect is a biological phenomenon whereby cells not directly targeted by DNA-damaging agents elicit a response similar to that of targeted cells . Understanding the mechanisms underlying the bystander effect is important not only for radiation risk assessment but also for evaluation of protocols for radiotherapy of tumors . Identification of DNA repair and signal transduction proteins that are induced specifically in bystander cells may help in deducing the molecular mechanism(s) responsible for this complex phenomenon . With this objective, we have studied the expression of replication protein A (RPA), which is involved in various DNA metabolic activities such as replication, repair and recombination . We analyzed RPA expression by immunofluorescence and Western blot techniques in both gamma-irradiated primary human fibroblast cells and bystander cells that were recipients of conditioned growth medium harvested from gamma-irradiated cell cultures . A two- to threefold induction of RPA was observed in bystander MRC5 cells treated with conditioned medium collected from gamma-irradiated WI38 or MRC5 cells . Lack of induction of RPA in sham-manipulated MRC5 cells treated with irradiated medium alone (without cells) indicates that the signal elicited from the irradiated cells is responsible for induction of RPA in bystander cells . RPA was induced more effectively in bystander cells than in irradiated cells at the earliest time analyzed (30 min), and the RPA level declined to that of sham-treated control cells by 24 h after treatment . In addition to RPA, apurinic/apyrimidinic endonuclease (APE, a key enzyme of the base excision repair pathway) also showed enhanced expression in bystander cells . Our findings suggest that the induction of RPA and APE is due to a combination of DNA strand breaks and oxidized base lesions in the genomic DNA of bystander cells.

J Bacteriol, 2004 Dec, 186(23), 7888 - 95
Rubrerythrin from the hyperthermophilic archaeon Pyrococcus furiosus is a rubredoxin-dependent, iron-containing peroxidase; Weinberg MV et al.; Rubrerythrin was purified by multistep chromatography under anaerobic, reducing conditions from the hyperthermophilic archaeon Pyrococcus furiosus . It is a homodimer with a molecular mass of 39.2 kDa and contains 2.9 +/- 0.2 iron atoms per subunit . The purified protein had peroxidase activity at 85 degrees C using hydrogen peroxide with reduced P . furiosus rubredoxin as the electron donor . The specific activity was 36 micromol of rubredoxin oxidized/min/mg with apparent K(m) values of 35 and 70 microM for hydrogen peroxide and rubredoxin, respectively . When rubrerythrin was combined with rubredoxin and P . furiosus NADH:rubredoxin oxidoreductase, the complete system used NADH as the electron donor to reduce hydrogen peroxide with a specific activity of 7.0 micromol of H(2)O(2) reduced/min/mg of rubrerythrin at 85 degrees C . Strangely, as-purified (reduced) rubrerythrin precipitated when oxidized by either hydrogen peroxide, air, or ferricyanide . The gene (PF1283) encoding rubrerythrin was expressed in Escherichia coli grown in medium with various metal contents . The purified recombinant proteins each contained approximately three metal atoms/subunit, ranging from 0.4 Fe plus 2.2 Zn to 1.9 Fe plus 1.2 Zn, where the metal content of the protein depended on the metal content of the E . coli growth medium . The peroxidase activities of the recombinant forms were proportional to the iron content . P . furiosus rubrerythrin is the first to be characterized from a hyperthermophile or from an archaeon, and the results are the first demonstration that this protein functions in an NADH-dependent, hydrogen peroxide:rubredoxin oxidoreductase system . Rubrerythrin is proposed to play a role in the recently defined anaerobic detoxification pathway for reactive oxygen species.

Plant J, 2004 Dec, 40(5), 799 - 812
Chloride fluxes in lily pollen tubes: a critical reevaluation; Messerli MA et al.; Microelectrodes, made from a Cl(-)-selective liquid ion exchanger previously used to measure putative Cl- fluxes in Lilium longiflorum pollen tubes, were characterized . The electrodes were poorly selective, possessing only about 10-fold selectivity for Cl- over other anions tested . They had only 2.4-fold selectivity for Cl- over the anionic form of the H+ buffer, MES, indicating that the electrode can indirectly detect H+ gradients . Apparent anion influx was detected along the pollen tube shafts and at the grains while apparent anion efflux was detected near the tip of the tube . During oscillating growth, the peak of the oscillating apparent anion efflux at the tip occurred, on average, 7.9 sec after the peak of the growth oscillations . Consideration of the previously characterized H+ fluxes in lily pollen grains and tubes, as well as the poor anion selectivity of the Cl- electrodes, indicates that the putative Cl- fluxes are in fact changes in the anionic concentration of the buffer resulting from H+ gradients and not changes in Cl- concentration . The claim of a central role for Cl- in lily pollen tube growth is further undermined by the fact that these tubes grow at the same rate if the Cl- content of the growth medium is reduced to trace levels (< or =31 microM), and that the grains have only small reserves of Cl- . These results lead to the conclusion that Cl- fluxes are not a significant component of pollen tube growth and Cl- itself is not required for growth.

J Mol Biol, 2004 Dec 3, 344(4), 1147 - 57
Neurospora crassa CyPBP37: a cytosolic stress protein that is able to replace yeast Thi4p function in the synthesis of vitamin B1; Faou P et al.; Recently, we identified CyPBP37 of Neurospora crassa as a binding partner of cyclophilin41 . CyPBP37 function had not yet been described, although orthologs in other organisms have been implicated in the biosynthesis of the thiazole moiety of thiamine (vitamin B1) and/or stress-related pathways . Here, CyPBP37 is characterized as an abundant cytosolic protein with a functional NAD-binding site . Saccharomyces cerevisiae mutants lacking Thi4p (the CyPBP37 ortholog) are auxotrophic for vitamin B1 (thiamine) but can grow in the presence of the thiazole moiety of thiamine, suggesting a role for Thi4p in the biosynthesis of thiazole . N.crassa CyPBP37 is able to functionally replace Thi4p in yeast thiazole synthesis . Cellular fractionation studies revealed that Thi4p is a cytosolic protein in S.cerevisiae, like its ortholog CyPBP37 in N.crassa . This implies that thiamine synthesis takes place in the cytosol of both organisms and not in the mitochondria, as suggested . The expression of CyPBP37 and Thi4p is repressed by thiamine but not by thiazole in the growth medium . In addition to its function in thiazole synthesis, CyPBP37 is a stress-inducible protein . N.crassa cyclophilin41 can chaperone the folding of CyPBP37, its own binding partner.

J Photochem Photobiol B, 2004 Dec 2, 77(1-3), 63 - 9
Light-induced modulation of Porphyromonas gingivalis growth; Izzo AD et al.; The bacterium Porphyromonas gingivalis is a clinically significant agent in periodontitis, a disease for which there is no definitive cure . Several groups have attempted to kill this bacterium using low levels of light in the absence of a photosensitizer, with conflicting results . We hypothesize that it is not possible to kill P . gingivalis by targeting endogenous porphyrins for a photochemical reaction . We demonstrated that irradiation of P . gingivalis with 455 or 625 nm light emitting diodes did not induce a photochemical killing of the cultures . Controlled temperature experiments indicate that irradiation at either wavelength did not significantly impact the growth of P . gingivalis cultures, as compared to non-irradiated controls . Rather, the irradiation caused a temperature increase in the growth medium, which altered the growth of the cultures . These results indicate that heat-induced killing of P . gingivalis could be the mechanism behind successful irradiation experiments with this bacterium.

Chem Res Toxicol, 2004 Nov, 17(11), 1434 - 44
Experimental and theoretical studies on the pharmacodynamics of cisplatin in jurkat cells; Tacka KA et al.; For Jurkat cells in culture exposed to cisplatin (1), we measured the number of platinum adducts on DNA and showed that it is proportional to the AUC, the area under the concentration vs time curve, for cisplatin . The number of platinum-DNA adducts is measured immediately following exposure to drug . The AUC is calculated either as the product of the initial cisplatin concentration and the exposure time or as the integral under the concentration vs time curve for the unreacted dichloro species, which decreases exponentially . We also show that the number of adducts correlates with decreases in respiration, with the amount of DNA fragmentation, and with cell viability, all measured 24 h after exposure to the drug . To study the reactions of cisplatin at concentrations approaching clinical relevance (65 microM), we use two-dimensional {1H15N}HSQC NMR and the 15N-labeled form of the drug, cis-Pt(15NH3)2Cl2, 1 . In the absence of cells, 1 reacts with components of the growth medium and also transforms slowly (k(h) = 0.205 h-1 at 37 degrees C) into the chloro-aquo species, cis-{Pt(15NH3)2Cl(H2O)}+ (2), which at the pH of the medium (pH 7.15), is mainly in the deprotonated chloro-hydroxy form, cis-Pt(15NH3)2Cl(OH) (4) . The concentration of 2 (4), as measured by HSQC NMR, decreases due to reaction with components of the medium . In the presence of 5 million or more cells, the concentration of 1 decreases with time, but the NMR signal for 2 (4) is not seen because it is rapidly removed from solution by the cells, keeping its concentration very low . These experiments confirm that the species preferentially removed from the medium by cells is 2 (4) and not 1 . Our findings are discussed in the context of a kinetic model for platination of nuclear DNA by cisplatin, which includes aquation of cisplatin outside the cell, passage of 2 (4) through the cell membrane, reaction of reactive platinum species (RPS) in the cytosol with thiols, formation of adducts between RPS and accessible sites on genomic DNA, and removal of platinum from DNA by repair . Some of the rate constants involved are measured, but others can only be estimated . Calculations with this model show that little of the platinum reacts with intracellular thiols before reaching the nuclear DNA, indicating that binding to thiols is not important in cisplatin resistance . The model also predicts the circumstances under which the amount of platination of nuclear DNA is proportional to AUC.

J Biol Chem, 2005 Jan 21, 280(3), 2165 - 75 Epub 2004 Nov 10.
Nuclear Calpain Regulates Ca2+-dependent Signaling via Proteolysis of Nuclear Ca2+/Calmodulin-dependent Protein Kinase Type IV in Cultured Neurons; Tremper-Wells B et al.; Accumulating evidence indicates that calpains can reside in or translocate to the cell nucleus, but their functions in this compartment remain poorly understood . Dissociated cultures of cerebellar granule cells (GCs) demonstrate improved long-term survival when their growth medium is supplemented with depolarizing agents that stimulate Ca(2+) influx and activate calmodulin-dependent signaling cascades, notably 20 mm KCl . We previously observed Ca(2+)-dependent down-regulation of Ca(2+)/calmodulin-dependent protein kinase (CaMK) type IV, which was attenuated by calpain inhibitors, in GCs supplemented with 20 mm KCl (Tremper-Wells, B., Mathur, A., Beaman-Hall, C . M., and Vallano, M . L . (2002) J . Neurochem . 81, 314-324) . CaMKIV is highly enriched in the nucleus and thought to be critical for improved survival . Here, we demonstrate by immunolocalization/confocal microscopy and subcellular fractionation that the regulatory and catalytic subunits of m-calpain are enriched in GC nuclei, including GCs grown in medium containing 5 mm KCl . Calpain-mediated proteolysis of CaMKIV is selective, as several other nuclear and non-nuclear calpain substrates were not degraded under chronic depolarizing culture conditions . Depolarization and Ca(2+)-dependent down-regulation of CaMKIV were associated with significant alterations in other components of the Ca(2+)-CaMKIV signaling cascade: the ratio of phosphorylated to total cAMP response element-binding protein (a downstream CaMKIV substrate) was reduced by approximately 10-fold, and the amount of CaMK kinase (an upstream activator of CaMKIV) protein and mRNA was significantly reduced . We hypothesize that calpain-mediated CaMKIV proteolysis is an autoregulatory feedback response to sustained activation of a Ca(2+)-CaMKIV signaling pathway, resulting from growth of cultures in medium containing 25 mm KCl . This study establishes nuclear m-calpain as a regulator of CaMKIV and associated signaling molecules under conditions of sustained Ca(2+) influx.

Folia Microbiol (Praha), 2004, 49(4), 491 - 6
Secreted aspartate proteinases, a virulence factor of Candida spp.: occurrence among clinical isolates; Hamal P et al.; Production of secreted aspartate proteinases was determined in a set of 646 isolates of Candida and non-Candida yeast species collected from 465 patients of the University Hospital in Olomouc (Czechia) in the period 1995-2002, and Candida samples obtained from 64 healthy volunteers using solid media developed for this purpose . Using random amplified polymorphic DNA analysis (RAPD) 79 Candida isolates from blood were analyzed to show potential relationships between clustering of the fingerprints and extracellular proteolytic activity of these strains . C . albicans, C . tropicalis and C . parapsilosis possess always proteolytic activity while non-Candida species did not display any proteolysis . A tight relationship between fingerprints and extracellular proteolysis in the Candida isolates was not shown . A remarkable consistency between fingerprint clusters and proteolysis occurred in a subset of C . parapsilosis samples . Suboptimal pH of the growth medium was shown to facilitate the investigation of potential co-incidence of genotypic and phenotypic traits.

Biofizika, 2004 Sep-Oct, 49(5), 866 - 71
{The causes of the biological action of electrochemically activated solutions by changes in the growth of Escherichia coli cells}; Regulation of aflatoxin synthesis by FadA/cAMP/protein kinase A signaling in Aspergillus parasiticus; Department of Food Science and Human Nutrition, Michigan State University (MSU), USAAnalysis of fadA and pkaA mutants in the filamentous fungus Aspergillus nidulans demonstrated that FadA (Galpha) stimulates cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity resulting, at least in part, in inhibition of conidiation and sterigmatocystin (ST) biosynthesis . In contrast, cAMP added to the growth medium stimulates aflatoxin (AF) synthesis in Aspergillus parasiticus . Our goal was to explain these conflicting reports and to provide mechanistic detail on the role of FadA, cAMP, and PKA in regulation of AF synthesis and conidiation in A . parasiticus . cAMP or dibutyryl-cAMP (DcAMP) were added to a solid growth medium and intracellular cyclic nucleotide levels, PKA activity, and nor-1 promoter activity were measured in A . parasiticus D8D3 (nor1::GUS reporter) and TJYP1-22 (fadAGA2R, activated allele) . Similar to Tice and Buchanan {34}, cAMP or DcAMP stimulated AF synthesis (and conidiation) associated with an AflR-dependent increase in nor-1 promoter activity . However, treatment resulted in a 100-fold increase in intracellular cAMP/DcAMP accompanied by a 40 to 80 fold decrease in total PKA activity . ThefadAG42R allele in TJYP1-22 decreased AF synthesis and conidiation, increased basal PKA activity 10 fold, and decreased total PKA activity 2 fold . In TJYP1-22, intracellular cAMP increased 2 fold without cAMP or DcAMP treatment; treatment did not stimulate conidiation or AF synthesis . Based on these data, we conclude that: (1) FadA/PKA regulate toxin synthesis and conidiation via similar mechanisms in Aspergillus spp.; and (2) intracellular cAMP levels, at least in part, mediate a PKA-dependent regulatory influence on conidiation and AF synthesis.

Biochim Biophys Acta, 2004 Nov 4, 1659(1), 32 - 45
Respiratory terminal oxidases in the facultative chemoheterotrophic and dinitrogen fixing cyanobacterium Anabaena variabilis strain ATCC 29413: characterization of the cox2 locus; Pils D et al.; Upon nitrogen step-down, some filamentous cyanobacteria differentiate heterocysts, cells specialized for dinitrogen fixation, a highly oxygen sensitive process . Aerobic respiration is one of the mechanisms responsible for a microaerobic environment in heterocysts and respiratory terminal oxidases are the key enzymes of the respiratory chains . We used Anabaena variabilis strain ATCC 29413, because it is one of the few heterocyst-forming facultatively chemoheterotrophic cyanobacteria amenable to genetic manipulation . Using PCR with degenerate primers, we found four gene loci for respiratory terminal oxidases, three of which code for putative cytochrome c oxidases and one whose genes are homologous to cytochrome bd-type quinol oxidases . One cytochrome c oxidase, Cox2, was the only enzyme whose expression, tested by RT-PCR, was evidently up-regulated in diazotrophy, and therefore cloned, sequenced, and characterized . Up-regulation of Cox2 was corroborated by Northern and primer extension analyses . Strains were constructed lacking Cox1 (a previously characterized cytochrome c oxidase), Cox2, or both, which all grew diazotrophically . In vitro cytochrome c oxidase and respiratory activities were determined in all strains, allowing for the first time to estimate the relative contributions to total respiration of the different respiratory electron transport branches under different external conditions . Especially adding fructose to the growth medium led to a dramatic enhancement of in vitro cytochrome c oxidation and in vivo respiratory activity without significantly influencing gene expression.

J Cell Sci, 2004 Nov 15, 117(Pt 24), 5759 - 70 Epub 2004 Oct 26.
Translocation of the Dictyostelium TRAP1 homologue to mitochondria induces a novel prestarvation response; Morita T et al.; Dd-TRAP1 is a Dictyostelium homologue of tumor necrosis factor receptor-associated protein 1 (TRAP-1) . Dd-TRAP1 is located in the cortex of cells growing at a low density, but was found to be translocated to mitochondria with the help of a novel prestarvation factor that was accumulated in growth medium along with increased cell densities . The knockdown mutant of Dd-TRAP1 (TRAP1-RNAi cells) exhibited a significant defect in prestarvation response . Although TRAP1-RNAi cells showed normal expressions of classical prestarvation genes {dscA (discoidin I) and car1 (carA; cAMP receptor)}, the expression of differentiation-associated genes (dia1 and dia3) induced by the prestarvation response were markedly repressed . By contrast, transformants overexpressing Dd-TRAP1 showed an early prestarvation response and also increased expression of dia1 and dia3 in a cell-density-dependent manner . Importantly, introduction of Dd-TRAP1 antibody into D . discoideum Ax-2 cells by electroporation inhibited the translocation of Dd-TRAP1 from the cortex to mitochondria and greatly inhibited the initiation of differentiation . Taken together, these results indicate that Dd-TRAP1 is translocated to mitochondria by sensing the cell density in growth medium and enhances the early developmental program through a novel prestarvation response.

J Cancer Res Clin Oncol . 2004 Oct 16; {Epub ahead of print}
Heterocyclic complexes of ruthenium(III) induce apoptosis in colorectal carcinoma cells; Kapitza S et al.; PURPOSE . The ruthenium complex salt indazolium trans-{tetrachlorobisindazole-ruthenate(III)} (KP1019) and the analogous sodium salt KP1339 are effective tumor-inhibiting drugs in experimental therapy of autochthonous colorectal carcinomas in rats . This paper examines the cell biological mechanisms underlying their antineoplastic effects . METHODS . Colorectal tumor cell lines were used to analyze uptake of the ruthenium(III) complexes into the cells and the mechanism as well as the efficacy of their cytotoxic effects . RESULTS . KP1019 and KP1339 are efficiently taken up into the cells: 100 microM ruthenium(III) complex in the growth medium led to the uptake of 120-160 ng ruthenium per 10(6) cells within 30 min . Uptake of KP418 was tenfold lower correlating with its lower cytotoxic efficiency . KP1019 and KP1339 induced apoptosis in SW480 and HT29 cells predominantly by the intrinsic mitochondrial pathway as indicated by loss of mitochondrial membrane potential . Correspondingly sensitivity of the cells paralleled expression of bcl(2) while it was only slightly affected by mutations in Ki-ras . CONCLUSIONS . Our data demonstrate that trans-{tetrachlorobisindazole-ruthenate(III)} complex salts are promising candidate drugs in the second-line treatment of colorectal cancers resistant to other cytostatic drugs and has been introduced into phase I clinical trials.

Biophys J, 2005 Jan, 88(1), 132 - 46 Epub 2004 Oct 22.
Near-Critical Behavior of Aminoacyl-tRNA Pools in E . coli at Rate-Limiting Supply of Amino Acids; Elf J et al.; The rates of consumption of different amino acids in protein synthesis are in general stoichiometrically coupled with coefficients determined by codon usage frequencies on translating ribosomes . We show that when the rates of synthesis of two or more amino acids are limiting for protein synthesis and exactly matching their coupled rates of consumption on translating ribosomes, the pools of aminoacyl-tRNAs in ternary complex with elongation factor Tu and GTP are hypersensitive to a variation in the rate of amino acid supply . This high sensitivity makes a macroscopic analysis inconclusive, because it is accompanied by almost free and anticorrelated diffusion in copy numbers of ternary complexes . This near-critical behavior is relevant for balanced growth of Escherichia coli cells in media that lack amino acids and for adaptation of E . coli cells after downshifts from amino-acid-containing to amino-acid-lacking growth media . The theoretical results are used to discuss transcriptional control of amino acid synthesis during multiple amino acid limitation, the recovery of E . coli cells after nutritional downshifts and to propose a robust mechanism for the regulation of RelA-dependent synthesis of the global effector molecule ppGpp.

Microvasc Res, 2004 Nov, 68(3), 179 - 87
A murine model of ex vivo angiogenesis using aortic disks grown in fibrin clot; Berger AC et al.; The rat aortic ring model is well utilized for evaluation of angiogenesis . We report here an alternative assay employing an ex vivo mouse aorta angiogenesis model that can be extensively manipulated and serially evaluated using digital-assisted image analysis . Mouse aortas were harvested, cut into 2-mm disks, and cultured in fibrin matrix with growth media . Radial vascular outgrowths arose from the cut edge of the aortic disk and were digitally photographed and serially quantified . A variety of culture conditions were evaluated to determine their ability to alter angiogenesis in this model . Vessel outgrowth became apparent on day 3 and continued through day 10 with linear growth occurring between days 3 and 6 . Increasing concentrations of serum from 0% to 40% resulted in stimulation of angiogenesis after day 3 . Suramin and endostatin dramatically inhibited angiogenesis, which was more profound when applied at day 0 than when linear growth could be identified (day 3) . Cells isolated from vessel outgrowths were predominantly endothelial in origin by immunocytochemistry and FACS analysis . We demonstrate that angiogenesis in an ex vivo murine model can be easily quantified using digital image analysis, responds appropriately to stimulation and inhibition, and exhibits differential results based on time of inhibitor administration . Antiangiogenic agents may be most effective if administered before development of accelerated vessel growth.

Plant J, 2004 Nov, 40(4), 575 - 85
WRINKLED1 encodes an AP2/EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis; Cernac A et al.; The accumulation of storage compounds during seed development ensures the survival of the young seedling, and also provides nutrition to humans and animals in the form of foods and feeds . The putative AP2/EREBP transcription factor WRINKLED1 (WRI1) is involved in the regulation of seed storage metabolism in Arabidopsis . A splicing mutant allele, wri1-1, caused the reduction of seed oil accumulation . Glycolysis was compromised in this mutant, rendering developing embryos unable to efficiently convert sucrose into precursors of triacylglycerol biosynthesis . Expression of the WRINKLED1 cDNA under the control of the cauliflower mosaic virus 35S-promoter led to increased seed oil content . Moreover, the ectopic expression of the WRINKLED1 cDNA caused the accumulation of triacylglycerols in developing seedlings . This effect depended upon the presence of glucose in the growth medium or other sugars readily metabolized to glucose . Oil-accumulating seedlings showed aberrant development consistent with a prolonged embryonic state.

J Control Release, 2004 Nov 5, 100(1), 121 - 33
A new approach to the in vivo and in vitro investigation of drug release from locoregionally delivered microspheres; Cheung RY et al.; The purpose of this work was to determine the in vivo release profile of doxorubicin (Dox) delivered locoregionally by dextran-based microspheres (MS) and to develop an in vitro method for predicting in vivo drug release from MS-- in vitro-in vivo correlation (IVIVC) . For the determination of in vivo Dox release, drug-loaded MS were placed into hollow fibers (HF) and implanted subcutaneously into C3H mice . Samples were retrieved at various times following implantation, MS removed from HF, and the amount of Dox remaining determined via ultraviolet/visible (UV/Vis) spectrophotometry . Various in vitro systems were designed and investigated for their ability to link in vivo and in vitro release profiles, including an open system (e.g . a column) with continuous flow of release medium at different flow rates and closed systems (e.g . a cuvette) using different release media and conditions . About 34% of loaded Dox was released from MS in vivo at 48 h . Only an incremental release was observed over the ensuing 72 h . The release kinetics of Dox from MS using three of the investigated in vitro systems, column system and HF immersed in a buffer solution or growth medium gave release profiles that were highly correlated with the in vivo release profile (r(2)>0.9) . The relationships, both linear and non-linear, suggest that Level A IVIVC models can be developed for Dox release from locoregionally delivered MS using specially designed release systems.

J Virol Methods, 2004 Dec 1, 122(1), 9 - 15
Neuraminidase activity assays for monitoring MDCK cell culture derived influenza virus; Nayak DP et al.; Three assay methods were investigated for monitoring the time-course of neuraminidase (NA) activity of tissue culture derived equine influenza A virus from large-scale microcarrier cultivation and several steps of downstream processing required for the production of inactivated vaccines . Measurements of neuraminidase activity by a thiobarbituric acid (TBA) and a fluorometric method using Amplex Red as a fluorogen (FL-AR) did not correlate with the increase of hemagglutinin (HA) during virus replication . Samples analysed by the TBA method showed unspecific interference from low molecular weight compounds (< 3 kDa) of cell growth medium and virus maintenance medium . Further investigations showed that this was probably caused by interfering reactions between reducing sugars and amino acids that can be overcome by dialysis of samples . On the other hand, the sensitivity of the FL-AR method was not sufficient for the required measuring range . However, a reliable and sensitive fluorometric assay method (FL-MU-NANA) was obtained using 4-methylumbelliferyl-alpha-d-N-acetylneuraminic acid (4-MU-NANA) as a substrate, which allowed the detection of neuraminidase activities as low as 0.09 mU/mL . In this assay, time-course of neuraminidase activities correlated well with increasing hemagglutinin activities during virus replication in a bioreactor . Analysis of samples from various downstream processing steps comprising of clarification, inactivation, ultrafiltration (UF) and size-exclusion chromatography for the purification of influenza virus showed that neuraminidase activity was preserved at comparatively high levels . Based on the hemagglutinin and neuraminidase activity of the clarified and inactivated virus harvest, the overall recovery after gel filtration was about 34.4% and 119.5%, respectively.

J Gen Virol, 2004 Nov, 85(Pt 11), 3473 - 82
Cell-surface retention of PrPC by anti-PrP antibody prevents protease-resistant PrP formation; Kim CL et al.; The C-terminal portion of the prion protein (PrP), corresponding to a protease-resistant core fragment of the abnormal isoform of the prion protein (PrP(Sc)), is essential for prion propagation . Antibodies to the C-terminal portion of PrP are known to inhibit PrP(Sc) accumulation in cells persistently infected with prions . Here it was shown that, in addition to monoclonal antibodies (mAbs) to the C-terminal portion of PrP, a mAb recognizing the octapeptide repeat region in the N-terminal part of PrP that is dispensable for PrP(Sc) formation reduced PrP(Sc) accumulation in cells persistently infected with prions . The 50% effective dose was as low as approximately 1 nM, and, regardless of their epitope specificity, the inhibitory mAbs shared the ability to bind cellular prion protein (PrP(C)) expressed on the cell surface . Flow cytometric analysis revealed that mAbs that bound to the cell surface during cell culture were not internalized even after their withdrawal from the growth medium . Retention of the mAb-PrP(C) complex on the cell surface was also confirmed by the fact that internalization was enhanced by treatment of cells with dextran sulfate . These results suggested that anti-PrP mAb antagonizes PrP(Sc) formation by interfering with the regular PrP(C) degradation pathway.

Biomaterials, 2005 Apr, 26(12), 1423 - 30
Micropatterned polymer substrates control alignment of proliferating Schwann cells to direct neuronal regeneration; Schmalenberg KE et al.; Microcontact printed polymeric substrates were evaluated for their ability to control Schwann cell attachment and direct proliferation, as Schwann cell guidance is a crucial factor in directing peripheral nerve regeneration . Elastomeric stamps of poly(dimethylsiloxane) were "inked" with laminin, a permissive protein for Schwann cell adhesion, and stamped onto poly(methyl methacrylate) substrates to create patterns of lines and intervals varying from 10 to 50 microm wide . Schwann cells were seeded onto the substrates in serum-free media . After 4h, media was replaced with serum-containing growth media and changed daily thereafter . The addition of growth media to stimulate proliferation initially caused some loss in cell orientation relative to the laminin pattern, but when monolayer formation was complete, a high degree of cell orientation was observed . As both cell-cell contacts and surface coverage were maximized, the Schwann cells achieved an even higher order of orientation than observed during the early stages of proliferation . Significantly, smaller pattern widths increased the degree of orientation, regardless of interval width . Our results indicate that patterned polymeric substrates may enhance peripheral nerve regeneration by creating a highly ordered Schwann cell matrix for guidance of neurons.

Pol J Microbiol, 2004, 53(2), 117 - 20
Optimization of carbon-nitrogen ratio for production of gibberellic acid by Pseudomonas sp; Basiacik Karakoc S et al.; In this study, favorable carbon-nitrogen ratio for high yields of gibberellic acid (GA3) production from Pseudomonas sp . was investigated . First of all, optimum carbon (glucose, maltose, sucrose, fructose, lactose) and nitrogen (KNO3, NH4Cl, NaNO3, urea, glycine) sources among the others were chosen . The highest yield of GA3 productivity was found in growth medium supplemented with fructose (168.5 mg/L) . NaNO3 was found as a suitable nitrogen source (141 mg/L) . Then, in order to determine the optimum carbon-nitrogen ratio, different concentrations of carbon (from 50 mM to 150 mM) and nitrogen (from 17 mM to 47 mM) sources were added in culture media . As a result, optimum carbon-nitrogen ratio for GA3 production from Pseudomonas sp . was found to be 100:17 mM.

FEMS Immunol Med Microbiol, 2004 Nov 1, 42(3), 291 - 7
A growth study of Coxiella burnetii Nine Mile Phase I and Phase II in fibroblasts; Miller JD et al.; Coxiella burnetii, a slow-growing, gram-negative, obligate intracellular bacterium, is the causative agent of Q fever in humans . The avirulent Phase II C . burnetii Nine Mile strain can invade and establish persistent infections in a wide variety of laboratory cell lines, and is generally considered to be easier to grow in culture than the wild-type Phase I organism . Efforts to improve Phase I organism yield in the BHK-21 cell line demonstrated that high CO2 conditions and the use of Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/l glucose supplementation resulted in higher organism yields . Phase II organisms grown in the same cell line and conditions showed lower growth rates . Analysis revealed that increased average numbers of C . burnetii Phase I organisms within fibroblasts was due to higher growth rates within the hosts rather than to increased uptake or to increased cell-to-cell spreading . Addition of the nucleoside cytidine to the growth medium stimulated growth of Phase II but not Phase I organisms.

Exp Cell Res, 2004 Nov 1, 300(2), 365 - 78
p18INK4c and p27KIP1 are required for cell cycle arrest of differentiated myotubes; Myers TK et al.; Myogenic differentiation is characterized by permanent and irreversible cell cycle withdrawal and increased resistance to apoptosis . These functions correlate with changes in expression and activity of several cyclin-dependent kinase inhibitors, including p18, p21, and p27 . In this study, we examined the requirements for p18, p21, and p27 in initiating growth arrest in multinucleated myotubes under differentiation conditions and in maintaining terminal arrest upon restimulation of differentiated myotubes with mitogenic signals . Under differentiation conditions, only p27(-/-) or p18(-/-)p27(-/-) myotubes are capable of reentering the cell cycle and synthesizing DNA at a very low frequency . Escape from cell cycle arrest was significantly greater in p18(-/-)p27(-/-) myotubes than in p27(-/-) myotubes . Stimulation of differentiated cultures with a mitogen-rich growth medium enhances p18(-/-)p27(-/-) myotube proliferation to encompass approximately half of the nuclei . p18(-/-)p21(-/-) and p21(-/-)p27(-/-) myotubes remain terminally arrested . Nuclei within individual restimulated p18(-/-)p27(-/-) myotubes can be found in all phases of the cell cycle, and a myotube can be multiphasic without any obvious deleterious effects . Increasing the time of differentiation or serum stimulation of p18(-/-)p27(-/-) myotubes neither increases the proliferation index of the myotube nuclei, nor does it alter the percentage of nuclei in each of the cell cycle phases . During the first 24 h of serum stimulation, the p18(-/-)p27(-/-) myotube nuclei that escape G0 arrest will rearrest in either S or G2 phase, without either mitosis or endoreplication . Apoptosis is increased in restimulated p18(-/-)p27(-/-) myotube nuclei, but is not specific for any cell cycle phase . These results suggest a collaborative role for p18 and p27 in initiating and maintaining G0 arrest during myogenic differentiation . While p18 and p27 appear to be essential in initiating G0 arrest in a proportion of postmitotic myotube nuclei, there must be another cell cycle inhibitor protein that functions with p18 and p27 in maintaining terminal arrest . We propose that the combined rate-limiting expressions of p18, p27, and this other inhibitor determine whether the myotube nuclei will remain postmitotic, or reenter the cell cycle, and if the nuclei escape G0 arrest, in which phase of the cell cycle the nuclei will ultimately rearrest.

J Biol Chem, 2004 Dec 24, 279(52), 54510 - 7 Epub 2004 Oct 07.
Overexpression of enzymatically active human cytosolic and mitochondrial thioredoxin reductase in HEK-293 cells . Effect on cell growth and differentiation; Nalvarte I et al.; The mammalian thioredoxin reductases (TrxR) are selenoproteins containing a catalytically active selenocysteine residue (Sec) and are important enzymes in cellular redox control . The cotranslational incorporation of Sec, necessary for activity, is governed by a stem-loop structure in the 3'-untranslated region of the mRNA and demands adequate selenium availability . The complicated translation machinery required for Sec incorporation is a major obstacle in isolating mammalian cell lines stably overexpressing selenoproteins . In this work we report on the development and characterization of stably transfected human embryonic kidney 293 cells that overexpress enzymatically active selenocysteine-containing cytosolic TrxR1 or mitochondrial TrxR2 . We demonstrate that the overexpression of selenium-containing TrxR1 results in lower expression and activity of the endogenous selenoprotein glutathione peroxidase and that the activity of overexpressed TrxRs, rather than the protein amount, can be increased by selenium supplementation in the cell growth media . We also found that the TrxR-overexpressing cells grew slower over a wide range of selenium concentrations, which was an effect apparently not related to increased apoptosis nor to fatally altered intracellular levels of reactive oxygen species . Most surprisingly, the TrxR1- or TrxR2-overexpressing cells also induced novel expression of the epithelial markers CK18, CK-Cam5.2, and BerEP4, suggestive of a stimulation of cellular differentiation.

Microbiology, 2004 Oct, 150(Pt 10), 3315 - 26
The cell wall stress response in Aspergillus niger involves increased expression of the glutamine : fructose-6-phosphate amidotransferase-encoding gene (gfaA) and increased deposition of chitin in the cell wall; Ram AF et al.; Perturbation of cell wall synthesis in Saccharomyces cerevisiae, either by mutations in cell wall synthesis-related genes or by adding compounds that interfere with normal cell wall assembly, triggers a compensatory response to ensure cell wall integrity . This response includes an increase in chitin levels in the cell wall . Here it is shown that Aspergillus niger also responds to cell wall stress by increasing chitin levels . The increased chitin level in the cell wall was accompanied by increased transcription of gfaA, encoding the glutamine : fructose-6-phosphate amidotransferase enzyme, which is responsible for the first and a rate-limiting step in chitin synthesis . Cloning and disruption of the gfaA gene in A . niger showed that it was an essential gene, but that addition of glucosamine to the growth medium could rescue the deletion strain . When the plant-pathogenic fungus Fusarium oxysporum and food spoilage fungus Penicillium chrysogenum were subjected to cell wall stress, the transcript level of their gfa gene increased as well . These observations suggest that cell wall stress in fungi may generally lead to activation of the chitin biosynthetic pathway.

Microbiology, 2004 Oct, 150(Pt 10), 3145 - 50
Increased mortality of Saccharomyces cerevisiae cell wall protein mutants; Teparic R et al.; The yeast cell wall contains an unusually high number of different mannoproteins . The physiological role of most of them is unknown and gene disruptions leading to depletion of different proteins do not affect major functions of the wall . In this work the phenotype of different single and multiple cell wall protein mutants was observed at the level of individual cells . It was found that the lack of the non-covalently bound wall proteins Scw4p, Scw10p and Bgl2p increases the mortality of Saccharomyces cerevisiae cells grown exponentially under standard laboratory conditions, as assayed by methylene blue staining . Mutation of SCW11, however, suppressed the phenotype of scw4scw10, or scw4scw10bgl2, indicating that Scw4p, Scw10p and Bgl2p act synergistically while Scw11p has an activity antagonistic to that of the other three proteins . Mutants lacking major covalently bound proteins, either all four described Pir-proteins or the five most abundant glycosylphosphatidylinositol (GPI)-anchored proteins (Ccw12p, Ccw13p/Dan1p, Ccw14p/Icwp1p, Tip1p and Cwp1p), also had increased mortalities, the first somewhat more and the latter less than that of scw4scw10bgl2 . In all cases the observed phenotype was suppressed by the addition of an osmotic stabilizer to the growth medium, indicating that cells died due to decreased osmotic stability . If cells were grown to stationary phase, Scw-mutants showed only slightly increased mortality, but mutants lacking Pir- or GPI-anchored proteins had significantly increased sensitivity, suggesting that their physiological function is primarily expressed in stationary-phase cells . In many cases structures consisting of a living ccw5ccw6ccw7ccw8 (multiple Pir-protein mutant) mother with two methylene blue-stained daughters could be seen.

Med J Malaysia, 2004 May, 59 Suppl B, 194 - 5
Human serum provided additional values in growth factors supplemented medium for human chondrocytes monolayer expansion and engineered cartilage construction; Chua KH et al.; We have previously formulated an optimized human chondrocytes growth medium based on 2% fetal bovine serum supplementation . For clinical usage, the animal serum must be replaced by patient own serum . We investigated the effects of human serum concentration for human nasal septum chondrocytes monolayer culture and cartilage reconstruction . Human serum demonstrated a dose dependent manner in promoting chondrocytes growth and cartilage engineering.

Cell Transplant, 2004, 13(4), 385 - 91
Propagation of adult rat bone marrow-derived hepatocyte-like cells by serial passages in vitro; Miyazaki M et al.; Previously, we found that hepatocyte growth factor receptor (c-Met)-and alpha-fetoprotein (AFP)-expressing cells were present in adult rat bone marrow, and that these cells also expressed hematopoietic stem cell markers, such as CD34, Thy-1, and c-Kit . When bone marrow cells were cultured in a hepatocyte growth medium (HGM) with HGF and EGF, colonies composed of polygonal cells resembling mature hepatocytes appeared by 2 weeks and grew very slowly because of overgrowth of stromal cells . At days 34-41, 2-mm2 sheets of hepatocyte-like cells were cut out of their colonies by scratching with an injection needle under observation with a phase contrast microscope, transferred into wells of 24-well plates, and cultured in the HGM medium in the presence or absence of HGF and EGF . When cells reached confluence, cells were detached with trypsin and EDTA and transferred step by step into bigger culture vessels . Thus, hepatocyte-like cells were expanded 1000-fold during less than 4 months . These cells were immunocytochemically stained for albumin and also for AFP and the hematopoietic stem cell markers described above, showing characteristics of oval cells . By RT-PCR, we detected mRNAs of tryptophan-2,3-dioxygenase and tyrosine aminotransferase, markers of hepatocytes at a terminal differentiation stage . The present culture system may be useful for supply of hepatocyte resources for cell transplantation therapy.

Macromol Biosci, 2004 Mar 15, 4(3), 243 - 54
Mass spectrometry feedback control for synthesis of polyhydroxyalkanoate granule microstructures in Ralstonia eutropha; Pederson EN et al.; Polyhydroxyalkanoate (PHA) granules with core-shell layered microstructure were synthesized in Ralstonia eutropha using periodic feeding of valeric acid into a growth medium containing excess fructose . The O2 consumption and CO2 evolution rates, determined by off-gas mass spectrometry, have been used as sensitive measures to indicate the type of nutrients utilized by R . eutropha during PHA synthesis . Domains of poly-3-hydroxybutyrate (PHB) were formed during polymer storage conditions when only fructose was present . Feeding of valeric acid (pentanoic acid) resulted in the synthesis of hydroxyvalerate (HV) monomers, forming a poly-3-hydroxybutyrate-co-valerate (PHBV) copolymer . The synthesis of desired polymer microstructures was monitored and controlled using online mass spectrometry (MS) . The respiratory quotient (RQ) was unique to the type of polymer being synthesized due to increased O2 consumption during PHBV synthesis . MS data was used as the control signal for nutrient feeding strategies in the bioreactor . The core-shell structures synthesized were verified in cells using transmission electron microscopy after thin sectioning and staining with RuO4 . It was demonstrated that the synthesis of core-shell microstructures can be precisely controlled utilizing a MS feedback control system.

Can J Microbiol, 2004 Aug, 50(8), 623 - 31
Determining the environmental fate of a filamentous fungus, Trichoderma reesei, in laboratory-contained intact soil-core microcosms using competitive PCR and viability plating; Providenti MA et al.; Trichoderma spp . are used extensively in industry and are routinely disposed of in landfill sites as spent biomass from fermentation plants . However, little is known regarding the environmental fate of this biomass . We tracked the survival of T . reesei strain QM6A#4 (a derivative of strain QM6A marked with a recombinant construct) over a 6-month period in laboratory-contained, intact soil-core microcosms incubated in a growth chamber . Survival was tested in 3 different soils and the effect of a plant rhizosphere (bush lima beans, Phaseolus limensis) was investigated . Levels and viability of the fungus were determined, respectively, by quantitative competitive polymerase chain reaction analysis of total soil DNA extracts and dilution-plating of soil on a semiselective growth medium . Whereas chemically killed QM6A#4 became undetectable within 3 d, QM6A#4 added as a live inoculum decreased approximately 4- to approximately 160-fold over the first 1-3 months and then reached a steady state . After 4 months, soil cores were subjected to a 1.5-month simulated winter period, which did not significantly affect QM6A#4 levels . Throughout the experiment, QM6A#4 remained viable . These results indicate that, following release into the environment, live T . reesei will persist in soil for at least 2 seasons.

J Cell Sci, 2004 Oct 15, 117(Pt 22), 5393 - 404 Epub 2004 Oct 05.
Skeletal muscle satellite cells can spontaneously enter an alternative mesenchymal pathway; Shefer G et al.; We show that muscle satellite cells, traditionally considered as committed myogenic precursors, are comprised of Pax7-expressing progenitors that preserve a mesenchymal repertoire extending beyond a mere myogenic potential . Mouse satellite cells from freshly isolated single myofibers, cultured individually in serum-rich growth medium, produced myogenic and non-myogenic clones . Only the myogenic clones expressed muscle-specific transcription factors and formed myotubes . Pax7 was initially expressed in all clones, but subsequently was associated only with the myogenic clones . Some cells in the non-myogenic clones expressed alpha-smooth muscle actin and nestin whereas others differentiated into mature adipocytes . This type of cell composition mirrors characteristics of mesenchymal stem cell progeny . Overall, individual myofibers persistently gave rise to both clonal phenotypes, but the ratio of myogenic to non-myogenic clones randomly varied among fibers . This randomness indicates that clonal dichotomy reflects satellite cell suppleness rather than pre-fated cell heterogeneity . We conclude that satellite cells possess mesenchymal plasticity, being able to commit either to myogenesis or to a mesenchymal alternative differentiation (MAD) program.

Plant Physiol, 2004 Oct, 136(2), 3134 - 47 Epub 2004 Oct 01.
Uncoupling the effects of abscisic acid on plant growth and water relations . Analysis of sto1/nced3, an abscisic acid-deficient but salt stress-tolerant mutant in Arabidopsis; Ruggiero B et al.; We have identified a T-DNA insertion mutation of Arabidopsis (ecotype C24), named sto1 (salt tolerant), that results in enhanced germination on both ionic (NaCl) and nonionic (sorbitol) hyperosmotic media . sto1 plants were more tolerant in vitro than wild type to Na(+) and K(+) both for germination and subsequent growth but were hypersensitive to Li(+) . Postgermination growth of the sto1 plants on sorbitol was not improved . Analysis of the amino acid sequence revealed that STO1 encodes a 9-cis-epoxicarotenoid dioxygenase (similar to 9-cis-epoxicarotenoid dioxygenase GB:AAF26356 {Phaseolus vulgaris} and to NCED3 GB:AB020817 {Arabidopsis}), a key enzyme in the abscisic acid (ABA) biosynthetic pathway . STO1 transcript abundance was substantially reduced in mutant plants . Mutant sto1 plants were unable to accumulate ABA following a hyperosmotic stress, although their basal ABA level was only moderately altered . Either complementation of the sto1 with the native gene from the wild-type genome or supplementation of ABA to the growth medium restored the wild-type phenotype . Improved growth of sto1 mutant plants on NaCl, but not sorbitol, medium was associated with a reduction in both NaCl-induced expression of the ICK1 gene and ethylene accumulation . Osmotic adjustment of sto1 plants was substantially reduced compared to wild-type plants under conditions where sto1 plants grew faster . The sto1 mutation has revealed that reduced ABA can lead to more rapid growth during hyperionic stress by a signal pathway that apparently is at least partially independent of signals that mediate nonionic osmotic responses.

Biotechnol Prog, 2004 Sep-Oct, 20(5), 1345 - 51
Screening of cyanobacterial species for calcification; Lee BD et al.; Species of cyanobacteria in the genera Synechococcus and Synechocystis are known to be the catalysts of a phenomenon called "whitings", which is the formation and precipitation of fine-grained CaCO3 particles . Whitings occur when the cyanobacteria fix atmospheric CO2 through the formation of CaCO3 on their cell surfaces, which leads to precipitation to the ocean floor and subsequent entombment in mud . Whitings represent one potential mechanism for CO2 sequestration . Research was performed to determine the ability of various strains of Synechocystis and Synechococcus to calcify when grown in microcosms amended with 2.5 mM HCO(3-) and 3.4 mM Ca2+ . Results indicated that although all strains tested have the ability to calcify, only two Synechococcus species, strains PCC 8806 and PCC 8807, were able to calcify to the extent that a CaCO3 precipitate was formed . Enumeration of the cyanobacterial cultures during testing indicated that cell density did not appear to have a direct effect on calcification . Factors that had the greatest effect on calcification were CO2 removal and subsequent generation of alkaline pH . Whereas cell density was similar for all strains tested, differences in maximum pH were demonstrated . As CO2 was removed, growth medium pH increased and soluble Ca2+ was removed from solution . The largest increases in growth medium pH occurred when CO2 levels dropped below 400 ppmv . Research presented demonstrates that, under the conditions tested, many species of cyanobacteria in the genera Synechocystis and Synechococcus are able to calcify but only two species of Synechococcus were able to calcify to an extent that led to the precipitation of calcium carbonate.

Ann Biomed Eng, 2004 Aug, 32(8), 1120 - 30
Oriented Schwann cell monolayers for directed neurite outgrowth; Thompson DM et al.; Schwann cells are an important component of the peripheral nervous system and participate in peripheral nerve regeneration . They create a supportive environment for neurite outgrowth by releasing trophic factors and up-regulating permissive molecules on their surface . In addition, Schwann cells are able to self-organize into linear arrays in vitro and in vivo, suggesting a possible role in neurite guidance . Previously, we showed that Schwann cell placement and orientation in subconfluent cultures can be controlled using microlithographically patterned laminin substrates (Thompson, D . M., and H . M . Buettner . Tissue Eng . 7(3):247-266, 2001) . In the current study, these substrates were used to create oriented Schwann cell monolayers . Both Schwann cell orientation and coverage were quantified in response to seeding density, culture medium, and micropattern dimensions . In serum-free medium, increasing the seeding density yielded a linear increase in coverage of the substrate area but decreased cell alignment . In an alternate approach, Schwann cells were first seeded in serum-free medium at moderate seeding density, allowed to align, then expanded in serum-containing growth medium . This produced complete coverage without large seeding densities while preserving alignment to the micropattern . Alignment and coverage were unaffected by micropattern dimensions . This work provides a useful methodology for investigating Schwann cell guidance effects on growing neurites.

J Cell Physiol, 2004 Dec, 201(3), 429 - 38
Adenosine stimulation of proliferation of breast carcinoma cell lines: evaluation of the {3H}thymidine assay system and modulatory effects of the cellular microenvironment in vitro; Mujoomdar M et al.; The purine nucleoside adenosine is produced at increased levels in the tissues of solid cancers as a result of local hypoxia . Adenosine inhibits the cell-mediated anti-tumor immune response, promotes tumor cell migration and angiogenesis, and stimulates the proliferation of tumor cells . We examined the stimulatory effect of adenosine on DNA synthesis, cell cycle progression, and cell proliferation in MCF7 and T-47D breast carcinoma cell lines in culture, and identified factors that modulate the growth response . The ability of adenosine to stimulate DNA synthesis, as measured by the incorporation of {(3)H}thymidine, was independent of the total radioactivity of the {(3)H}thymidine up to 10 microCi/ml, t