J Bacteriol, 1996 May, 178(9), 2593 - 7 Glucose sensing and signalling properties in Saccharomyces cerevisiae require the presence of at least two members of the glucose transporter family; Walsh MC et al.; The kinetics of glucose transport in a number of different mutants of Saccharomyces cerevisiae with multiple deletions in the glucose transporter gene family were determined . The deletions led to differences in maximal rate and affinity for glucose uptake by the cells, dependent on the growth conditions . At the same time, there were changes in glucose repression, as determined by expression of invertase activity . Only in the strain with genes HXT1-4 and SNF3 deleted but carrying HXT6/7 were glucose uptake kinetics and invertase activity independent of the presence or concentration of glucose in the growth medium . Some degree of glucose sensitivity was recovered if the SNF3 or HXT2 gene was present in the multiple-deletion background . It is hypothesized that during growth on glucose, both modulation of the kinetics of glucose uptake and derepression of invertase activity require the presence of more than one active gene of the glucose transporter family. J Mol Biol, 1996 Apr 26, 258(1), 25 - 36 Compensating effects of opposing changes in putrescine (2+) and K+ concentrations on lac repressor-lac operator binding: in vitro thermodynamic analysis and in vivo relevance; Capp MW et al.; Ion concentrations (K+, Glu-) in the cytoplasm of growing Escherichia coli cells increase strongly with increases in the osmolarity of a defined growth medium . While in vitro experiments demonstrate that the extent of protein-nucleic acid interactions (PNAI) depends critically on salt concentration, in vivo measurements indicate that cells maintain a relatively constant extent of PNAI independent of the osmolarity of growth . How do cells buffer PNAI against changes in the cytoplasmic environment? At high osmolarity, the increase in macromolecular crowding which accompanies the reduction in amount of cytoplasmic water in growing cells appears quantitatively sufficient to compensate for the increase in inverted question markK+ inverted question mark . At low osmolarity, however, changes in crowding appear to be insufficient to compensate for changes in inverted question markK+ inverted question mark, and additional mechanisms must be involved . Here we report quantitative determinations of in vivo total concentrations of polyamines (putrescine(2+), spermidine(3+)) as a function of osmolarity (OsM) of growth, and in vitro binding data on the effects of putrescine concentration on a specific PNAI (lac repressor-lac operator) as a function of inverted question markK+ inverted question mark . The total concentration of putrescine in cytoplasmic water decreases at least eightfold from low osmolarity (approximately 64 mmol (l H2O)-1 at 0.03 OsM) to high osmolarity (approximately 8 mmol (l H2O)-1 at 1.02 OsM) . Over this osmotic range the total inverted question markK+ inverted question mark increases from approximately 0.2 mol (l H2O)-1 to approximately 0.8 mol (lH2O)-1 . We find that the effect of putrescine concentration on the repressor-operator interaction in vitro is purely competitive and is quantitatively described by a simple competition formalism in which lac repressor behaves a a specific-binding oligocation (ZR = 8+/-3) . We demonstrate that this thermodynamic result is consistent with a structural analysis of the number of positively charged side-chains on two DNA binding domains of repressor which interact with the phosphodiester backbone of the operator site . Since this oligocation character of the binding surface of DNA-binding proteins appears to be general, we propose the competitive effects of putrescine and K+ concentrations on the strength of specific binding are general . At low osmolarity, compensating changes in putrescine and K+ concentration in response to changes in external osmolarity provide a general mechanism for E . coli to vary cytoplasmic osmolarity while maintaining a constant extent of PNAI. Brain Res, 1996 Apr 22, 717(1-2), 44 - 54 Clenbuterol protects mouse cerebral cortex and rat hippocampus from ischemic damage and attenuates glutamate neurotoxicity in cultured hippocampal neurons by induction of NGF; Semkova I et al.; It has been shown previously that clenbuterol, a beta 2-adrenergic receptor agonist, enhances NGF synthesis in adult rat brain . Since NGF is able to protect neurons against damage, we tried to find out whether clenbuterol can rescue cultured hippocampal neurons from excitotoxic damage by induction of NGF . The neuroprotective activity of clenbuterol on neurons in the vulnerable CA1 subfield of the hippocampus was tested in a rat model of transient forebrain ischemia . Additionally, in the mouse model of focal cerebral ischemia the ability of clenbuterol to reduce the infarct size was examined . Exposure of mixed neuronal/glial hippocampal cultures to clenbuterol (1 to 100 microM) enhanced significantly the content of NGF measured in the culture medium by two-site ELISA . The excitotoxic injury was induced in the same type of cells after 14 days in vitro by exposure to 1 mM L-glutamate for 1 h in serum-free medium . NGF itself (0.15 to 100 ng/ml) added to the growth medium 4 h before until 18 h after induction of injury (the point of glutamate-toxicity measurement), protected hippocampal neurons from excitotoxic damage . Clenbuterol (1 to 100 microM) provided similar neuroprotection as NGF under the same experimental conditions . The neuroprotective activity of clenbuterol (100 microM) against glutamate-induced damage in hippocampal cultures was blocked by anti-NGF monoclonal antibodies (0.5 microgram/ml) added to the medium during the clenbuterol exposure, demonstrating that the neuronal rescue is mediated by NGF . Propranolol, a beta-adrenergic receptor antagonist (10 microM) added 20 min before and kept in the medium during exposure of the cultures to clenbuterol (1 microM) reversed the neuroprotective activity, suggesting that the induction of NGF and neuroprotection caused by clenbuterol are mediated via beta-adrenergic receptor activation . The capacity of clenbuterol to protect hippocampal neurons was also demonstrated in vivo in a rat model of transient forebrain ischemia . Clenbuterol (4 x 1 mg/kg) administered intraperitoneally increased the number of viable neurons in CA1 subfield of the rat hippocampus . Furthermore, clenbuterol (0.3 and 1 mg/kg, i.p . and 1 mg/kg, s.c.) reduced significantly the infarct area on the mouse brain surface after occlusion of the middle cerebral artery . The present data demonstrate that clenbuterol induces NGF synthesis in cultured hippocampal cells and protects hippocampal neurons from excitotoxic damage . The neuroprotective activity of clenbuterol is also demonstrated in vivo in two rodent models of cerebral ischemia . The results offer strong evidence that the neuroprotective activity of clenbuterol is caused by activation of beta-adrenergic receptors and the subsequent increased expression of NGF. Proc Natl Acad Sci U S A, 1996 Apr 16, 93(8), 3394 - 8 Inhibition of apoptosis by overexpressing Bcl-2 enhances gene amplification by a mechanism independent of aphidicolin pretreatment; Yin DX et al.; To study the effect of apoptosis on gene amplification, we have constructed HeLa S3 cell lines in which the expression of bcl-2 (BCL2) can be controlled by tetracycline in the growth medium . Induction of Bcl-2 expression caused a temporary delay of apoptosis and resulted in roughly a 3-fold increase in the frequency of resistant colonies when cells were selected with trimetrexate . This resistance was due to amplification of the dihydrofolate reductase gene . Cells grown out of the pooled resistant colonies retained the same level of resistance to trimetrexate whether Bcl-2 was induced or repressed, consistent with the theory that Bcl-2 functions by facilitating gene amplification, rather than being the resistance mechanism per se . Pretreating cells with aphidicolin is another method to increase gene amplification frequency . When Bcl-2-expressing cells were pretreated with aphidicolin, the resulting increase in gene amplification frequency was approximately the product of the increases caused by aphidicolin pretreatment or Bcl-2 expression alone, indicating that Bcl-2 increases gene amplification through a mechanism independent of that of aphidicolin pretreatment . These results are consistent with the concept that gene amplification occurs at a higher frequency during drug-induced cell cycle perturbation . Bcl-2 evidently increases the number of selected amplified colonies by prolonging cell survival during the perturbation. Biochem Biophys Res Commun, 1996 Apr 5, 221(1), 129 - 32 Transgenic yeast expressing human cytochrome P450s can serve as a tool in studies of the mechanisms of their induction by various effectors; Kovaleva IE et al.; Transgenic Saccharomyces cerevisiae yeast strains were constructed which express CYP2D6 and CYP3A4 genes under control of an artificial promoter . When added to the growth medium, sparteine, a substrate for CYP2D6, was shown to increase the content of this cytochrome P450 isoform in yeast cells . No such increase was observed when a proteinase-deficient yeast mutant was used as a parent strain . Nifedipine, a substrate for CYP3A4, failed to affect the level of CYP3A4 expression even in wild yeast cells . These results suggest that expression of CYP2D6 in human liver can at least partially be controlled post-transcriptionally by its inductors while for CYP3A4 such a mechanism is hardly possible. Appl Environ Microbiol, 1996 Apr, 62(4), 1265 - 73 Biodegradation of phenols by the alga Ochromonas danica; Semple KT et al.; The eukaryotic alga Ochromonas danica, a nutritionally versatile, mixotrophic chrysophyte, grew on phenol as the sole carbon source in axenic culture and removed the phenol carbon from the growth medium . Respirometric studies confirmed that the enzymes involved in phenol catabolism were inducible and that the alga oxidized phenol; the amount of oxygen consumed per mole of oxidized substrate was approximately 65% of the theoretical value . {U-14C}phenol was completely mineralized, with 65% of the 14C label appearing as 14CO2, approximately 15% remaining in the aqueous medium, and the rest accounted for in the biomass . Analysis of the biomass showed that 14C label had been incorporated into the protein, nucleic acid, and lipid fractions; phenol carbon is thus unequivocally assimilated by the alga . Phenol-grown cultures of O . danica converted phenols to the corresponding catechols, which were further metabolized by the meta-cleavage pathway . This surprising result was rigorously confirmed by taking the working stock culture through a variety of procedures to check that it was axenic and repeating the experiments with algal extracts . This is, as far as is known, the first definitive identification of the meta-cleavage pathway for aromatic ring degradation in a eukaryotic alga, though its incidence in other eukaryotes has been (infrequently) suggested. J Cardiovasc Pharmacol, 1996 Apr, 27(4), 508 - 18 Regulation of protein synthesis by alpha 1-adrenergic receptor subtypes in cultured rabbit aortic vascular smooth muscle cells; Siwik DA et al.; To investigate the role of the sympathetic nervous system in maintenance and remodeling of vascular smooth muscle, we have examined regulation of protein synthesis by alpha 1-adrenergic receptors (alpha 1-AR) in cultured rabbit aortic vascular smooth muscle cells (VSMC) . Stimulation of postconfluent cultures (passages 2-6) in serum-free growth medium with the alpha-AR agonist phenylephrine (PE, 30 microM) increases protein synthesis, measured as {3H}leucine incorporation into protein (146 +/- 5%, p < or = 0.001) and total protein content (117 +/- 2%, p < or = 0.001) . PE treatment does not affect cellular proliferation or {3H}thymidine incorporation into DNA . PE-stimulated protein synthesis is evident within 24 h, sustained over 96 h, concentration-dependent, and saturable . PE-elicited responses are completely inhibited by the alpha 1-AR antagonist prazosin but not by the alpha 2-AR antagonist yohimbine or the beta-AR antagonists propranolol and atenolol; the beta-AR agonist isoproterenol is ineffective . Competition with {3H}prazosin occupancy of alpha 1-AR and agonist-elicited {3H}leucine incorporation by subtype-selective receptor antagonists (WB4101 and 5-methylurapidil, alpha 1A; chloroethylclonidine, alpha 1B) demonstrates that these cells express a majority of alpha 1B-AR (75%) relative to alpha 1A-AR (25%), which elicit protein metabolism in proportion to their relative abundances . These results indicate that alpha 1B-AR predominate in coupling to metabolic responses, in contrast to previous reports that contractile responses in this tissue are preferentially mediated by alpha 1A-AR. Genetics, 1996 Apr, 142(4), 1069 - 82 Functional analysis of the PUT3 transcriptional activator of the proline utilization pathway in Saccharomyces cerevisiae; des Etages SA et al.; Proline can serve as a nitrogen source for the yeast Saccharomyces cerevisiae when preferred sources of nitrogen are absent from the growth medium . PUT3, the activator of the proline utilization pathway, is required for the transcription of the genes encoding the enzymes that convert proline to glutamate . PUT3 is a 979 amino acid protein that constitutively binds a short DNA sequence to the promoters of its target genes, but does not activate their expression in the absence of induction by proline and in the presence of preferred sources of nitrogen . To understand how PUT3 is converted from an inactive to an active state, a dissection of its functional domains has been undertaken . Biochemical and molecular tests, domain swapping experiments, and an analysis of activator-constitutive and activator-defective mutant proteins indicate that PUT3 is dimeric and activates transcription with its negatively charged carboxyterminus, which does not appear to contain a proline-responsive domain . A mutation in the conserved central domain found in many fungal activators interferes with activation without affecting DNA binding protein stability . Intragenic suppressors of the central domain mutation have been isolated and analyzed. J Dermatol Sci, 1996 Apr, 12(1), 64 - 8 Effect of a chemically-synthesized acylglucosylceramide, epidermoside, on normal human keratinocyte differentiation; Uchida Y et al.; Epidemosides (N-(0-linoleoyl)-(1)-hydroxy fatty acyl sphingosyl glucose) are found exclusively in the epidermis not in dermis, and are thought to play important role in forming the mammalian epidermal permeability barrier . A species of epidermoside isolated from guinea pig epidermis and named lipokeratinogenoside has been shown to enhance fetal rat keratinocyte differentiation . In the present investigation, we studied the effects of a chemically synthesized equivalent of human epidermoside on the viability and differentiation of cultured human keratinocytes (HK Cells) . The chemically-synthesized epidermoside was not toxic to cultured HK Cells at concentrations of 0.01 to 10 micrograms/ml . When 10 micrograms/ml of the chemically-synthesized epidermoside was added to keratinocyte growth medium containing 1.2 mM Ca2+, HK Cells showed a 5.6-fold increase of keratin content compared to the vehicle treated control at 144 h of cultivation, and they also displayed morphological changes suggestive of differentiation . A similar increase of cellular keratin content was observed in HK cells treated with tetradecanoyl phorbol-13 myristyl-12 acetate (TPA), an agent known to enhance the differentiation of keratinocytes . Lipokeratinogenoside also increased the keratin content of cultured HK cells . These results suggest that epidermosides have an ability to enhance keratinocyte differentiation . Epidermoside could thus be a key molecule, not only as a constituent of the epidermal permeability barrier, but also as a regulator of keratinocyte differentiation. Yeast, 1996 Apr, 12(5), 457 - 66 The hsp150 delta-carrier confers secretion competence to the rat nerve growth factor receptor ectodomain in Saccharomyces cerevisiae; Simonen M et al.; When the extracellular domain of rat low-affinity nerve growth factor receptor (NGFRe) was synthesized in Saccharomyces cerevisiae with the signal peptide of invertase, NGFRe was translocated to the endoplasmic reticulum (ER) and retained there . However, when NGFRe was fused to the C-terminus of the hsp150 delta-carrier, the hsp150 delta-NGFRe fusion protein was efficiently secreted to the growth medium with no apparent retention in the ER . The NGFRe portion was disulphide-bonded and its single N-glycosylation site was occupied . The hsp150 delta-carrier is an N-terminal signal peptide-containing fragment of a yeast secretory glycoprotein . Hsp150 delta-NGFRe, harvested from the culture medium, inhibited the cross-linking of {125I}NGF to authentic NGFR on the surface of human melanoma cells . Moreover, {125I}NGF could be chemically cross-linked to secretory hsp150 delta-NGFRe, suggesting that the NGFRe portion had adopted a ligand-binding conformation . However, inhibition of the cross-linking by unlabelled NGF was less effective than in the case of the authentic receptor . The hsp150 delta-carrier may have potential in the production of mammalian proteins, which require elaborate folding and disulphide formation in the ER. Biomaterials, 1996 Apr, 17(8), 775 - 80 Chondroitin-6-sulphate incorporated into collagen gels for the growth of human keratinocytes: the effect of cross-linking agents and diamines; Hanthamrongwit M et al.; This study demonstrates the effect of the glycosaminoglycans, hyaluronic acid and chondroitin-6-sulphate (Ch6SO4), diamines and a carbodiimide cross-linking agent on the growth of human epidermal cells on collagen gels . Ch6SO4 incorporated into collagen gels stimulated cell growth rate, but the effect was found to be inconsistent . We found that approximately 50% of the incorporated Ch6SO4 in the gels leached out into the growth medium after the first 3 d in culture, and this is thought to lead to the inconsistent cell growth response . In order to minimize the elution of Ch6SO4 from the gels and thereby maximize its effect on the growth of the keratinocytes, 1-100 micrograms ml-1 Ch6SO4 was added in the medium . The results showed that Ch6SO4 at these concentrations in the medium did not stimulate the cell growth on either plain collagen gels or gels containing 20% Ch6SO4 . As an alternative strategy, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and diamines (putrescine or diaminohexane) were used to immobilize Ch6SO4 onto the collagen gels and to cross-link the gels . The cross-linking process partially prevented the elution of Ch6SO4 from the gels . Interestingly, only putrescine, not diaminohexane, promoted the growth of keratinocytes on the cross-linked plain collagen gels . We proposed to develop an artificial skin substitute containing putrescine as a growth factor for the human epidermal cells. Int J Oral Maxillofac Surg, 1996 Apr, 25(2), 157 - 60 Optimized growth medium for primary culture of human oral keratinocytes; Formanek M et al.; The effect of different media additives in defining optimal growth conditions for primary cultures of human oral keratinocytes was studied . A cocultivation technique with irradiated Swiss-3T3-fibroblasts in 96-well plates enables the comparison of additives for primary keratinocyte cultures derived from one patient . 3H-labeled thymidine uptake showed no growth or growth inhibition with adenine, choleratoxin or transferrin compared to basal medium (Dulbecco's modified Eagle's medium (DMEM) and 10% fetal calf serum) . Among single additives, 5 micrograms/ml hydrocortisone, 5 micrograms/ml insulin, 10 ng/ml EGF, 2 micrograms/ml bovine pituitary extract, and 10(-9) M triiodothyronine showed the greatest capacity to promote keratinocyte growth . With all possible combinations of additives, maximum stimulation was found with a combination of EGF (10 ng/ml), insulin (5 micrograms/ml), and hydrocortisone (5 micrograms/ml); none of the other combinations were more effective . Our data indicate that in short-term cultures (up to 5 days) various media additives described in the literature are not necessarily required in this system of primary culture of human oral keratinocytes. Arch Biochem Biophys, 1996 Apr 1, 328(1), 1 - 8 Heterologous expression and characterization of soybean cytosolic ascorbate peroxidase; Dalton DA et al.; Ascorbate peroxidase is a widespread plant enzyme that catalyzes the removal of potentially harmful H2O2 . This enzyme is particularly important in legume root nodules due to their high potential for generating activated forms of oxygen . A cDNA clone of soybean nodule ascorbate peroxidase was used to construct an expression system in Escherichia coli . The recombinant protein had an N-terminal tag of six consecutive histidine residues to allow for purification by Ni(2+)-agarose affinity chromatography . Large amounts of recombinant peroxidase (about 27% of total soluble protein) were produced but most of the peroxidase was present in the apo-form (without heme) . Addition of delta-aminolevulinic acid to the growth media resulted in an increase in production of holoprotein . Apoprotein was easily converted to the holo-form by in vitro reconstitution with hemin . The reconstituted protein was catalytically, spectrally, and immunologically indistinguishable from native ascorbate peroxidase. J Bacteriol, 1996 Apr, 178(8), 2465 - 8 Two transcription factors, Gln3p and Nil1p, use the same GATAAG sites to activate the expression of GAP1 of Saccharomyces cerevisiae; Stanbrough M et al.; We present an analysis of the DNA region located upstream of GAP1, the structural gene for the general amino acid permease, which contains the sites required for activation of transcription of this gene in response to the nitrogen source of the growth medium . This gene is not expressed in media containing glutamine, and its transcription is activated in response to Gln3p in cells using glutamate as the source of nitrogen and by Nil1p in cells using urea as the source of nitrogen . We show that full response to both activators requires the presence of two GATAAG sites, as well as the presence of auxiliary sites located in the interval between 602 and 453 bp from the translational start site . The fact that both Gln3p and Nil1p utilize GATAAG sites to activate transcription is reflected in the high homology of the zinc finger regions of the two proteins. Exp Parasitol, 1996 Apr, 82(3), 298 - 305 Why is man an unsuitable reservoir for the transmission of Leishmania major? Schlein Y, Jacobson RL. Leishmania major strains cause human cutaneous leishmaniasis in various arid regions in the Old World . Nevertheless, there is apparently no anthroponotic transmission even when the incidence of cases is very high and the involvement of reservoir animals is obligatory . To investigate this phenomenon we compared the development of L . major in Phlebotomus papatasi artificially infected with parasites in human, rabbit, or sand rat blood . The parasites, sandflies, and reservoir rodents came originally from the Jordan Valley . Following meals of promastigotes in human blood, parasites were retained in 48.0% and heavy infections developed in 6.6% of the sandflies . Such meals with sand rat blood resulted in 76.9% infected, including 58.5% heavily infected flies, whereas rabbit blood produced intermediate results . Similar results were obtained when infections were initiated with amastigotes and the infection rates were significantly different . Only flies with heavy infections are considered as potential transmitters of leishmaniasis . The adverse effect of human blood was attributed to the erythrocytes after similar experiments in which sandflies ingested promastigotes either with human erythrocytes and rabbit plasma or rabbit erythrocytes and human plasma . Amastigotes of an Israeli strain of L . major died in medium containing 50% human blood . Also, addition of 20% human blood to growth media of parasites from Israel, Kenya, or Turkemenistan caused mortality of 70 to 80% of the initial inoculum in 24 hr . At that time there was a fivefold increase in the number of Israeli parasites cultured with sand rat blood . These results imply that the vector potential and the chances of transmission are drastically decreased when man is the source of L . major parasites. Plant J, 1996 Apr, 9(4), 477 - 89 Isolation of pl 4.6 extensin peroxidase from tomato cell suspension cultures and identification of Val-Tyr-Lys as putative intermolecular cross-link site; Schnabelrauch LS et al.; Extensins and kindred hydroxyproline-rich glycoproteins occur in dicot cell walls mainly as insoluble integral components that may form an intermolecularly cross-linked network interpenetrated by other polymers . Extensins also occur in muro as a small pool of soluble monomeric precursors to network extensin . These precursors were prepared in milligram quantities by salt elution from the surface of intact cells grown as tomato suspension cultures . Based on an FPLC (Superose-6) gel filtration assay of cross-linked extensin oligomers, a pl 4.6 extensin cross-linking peroxidase isozyme was partially purified from the culture growth medium . Purification involved: volume reduction, ultracentrifugation to remove pectin and co-adsorbed cationic peroxidase, followed by chromatography of anionic extensin peroxidase (pl 4.6) on DEAE-Trisacryl and TSK-gel DEAE-5PW columns . With tomato P1 extensin as substrate and 60 microM H2O2 as co-substrate, at 23 degrees pl 4.6 extensin peroxidase gave a Km of 0.22 mM P1 and a Vmax 0f 70 mumol P1 cross-linked min-1mg-1 enzyme, at the optimum pH 5.5 . Assayed with 12 different extensins from representative monocots, dicots, and gymnosperms, the pl 4.6 isozyme cross-linked highly selectively, indicating two natural groups: cross-linking or CL-extensins and non-cross-linking or NCL-extensins . CL-extensins contained the X-Hyp-Val-Tyr-Lys motif and were also highly glycosylated . However, the simplest motif common to CL-extensins but absent from NCL-extensins was Val-Tyr-Lys . Thus, peroxidative coupling of extensin monomers and resistance of the resultant oligomers to depolymerization by anhydrous HF suggests that the intermolecular cross-link involves tyrosine or lysine. Mutat Res, 1996 Mar 26, 351(1), 3 - 7 Role of E . coli DNA helicase II in a umuCD-dependent Wiegle reactivation; Altshuler ML; Wiegle reactivation is a manifestation of a umuCD-mediated enhancement in the replication of damaged phage DNA (Caillet-Fauquet et al., 1977; Defais et al., 1989; Rajagopalan et al., 1992) . I have obtained Wiegle reactivation of lambda by irradiating the uvrA cells in rich growth medium . This Wiegle reactivation was lost upon transduction of the umuC mutation into the strain . There was also a drastic reduction of Wiegle reactivation in an isogenic strain carrying the uvrA mutation and deletion of the uvrD gene . (uvrD codes for E . coli DNA helicase II) . The effect of the uvrD deletion on Wiegle reactivation can indicate a specific requirement of DNA helicase II for the unwinding of damaged phage DNA during its replication or (and) an inefficient induction of the SOS response in delta uvrD uvrA cells. Proc Natl Acad Sci U S A, 1996 Mar 19, 93(6), 2322 - 7 Induction of cytochrome P4501A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin or indolo(3,2-b)carbazole is associated with oxidative DNA damage; Park JY et al.; Induction of cytochrome P4501A1 (CYP1A1) in the hepatoma Hepa1c1c7 cell line results in an elevation in the excretion rate of 8-oxoguanine (oxo8Gua), a biomarker of oxidative DNA damage and the major repair product of 8-oxo-2'-deoxyguanosine (oxo8dG) residues in DNA . Treatment of this cell line with 2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD), a nonmetabolized environmental contaminant, and indolo(3,2-b)carbazole (ICZ), a metabolite of a natural pesticide found in cruciferous vegetables, is shown to both induce CYP1A1 activity and elevate the excretion rate of oxo8Gua; 7,8-benzoflavone (7,8-BF or alpha-naphthoflavone), an inhibitor of CYP1A1 activity and an antagonist of the aryl hydrocarbon (Ah) receptor, reduced the excretion rate of oxo8Gua . The essential role of Ah-receptor, which mediates the induction of CYP1A1, is shown by the inability of TCDD to induce CYP1A1 and to increase excretion of oxo8Gua in Ah receptor-defective c4 mutant cells . While there was a significant 7.0-fold increase over 2 days in the excretion rate of oxo8Gua into the growth medium of TCDD-treated Hepa1c1c7 cells compared to control, no significant increase was detected in the steady-state level of oxo8dG in the DNA presumably due to efficient DNA repair . Thus, the induction of CYP1A1 appears to lead to a leak of oxygen radicals and consequent oxidative DNA damage that could lead to mutation and cancer. Yeast, 1996 Mar 15, 12(3), 199 - 205 Selection of yeast cells with a higher plasmid copy number in a Saccharomyces cerevisiae autoselection system; Compagno C et al.; Autoselection systems allow the selection of a genetically engineered population independently of the growth medium composition . The structure of a Saccharomyces cerevisiae population transformed with an autoselection plasmid, in which a carbon-source-dependent modulation of the plasmid copy number occurs, was analysed . By means of flow cytometric procedures we tested the cell viability, dynamics of growth and heterologous protein production at single cell level . Such analyses allow the identification and the tracking of a specific cellular sub-population with a higher plasmid copy number which arises after the carbon source shift . The effects of the cellular plasmid distribution on the dynamics of growth are also discussed. Front Biosci, 1996 Mar 01, 1, d19 - 29 Microinjection strategies for the study of mitogenic signaling in mammalian cells; Lamb NJ et al.; First used in the analysis of dynamic changes in cell structure, microneedle microinjection allows in situ study of individual living cells as opposed to large scale metabolic analysis of heterogeneous cell culture . In addition, microinjection also offers the possibility to examine in vivo regulated processes by modulating the intracellular levels and activity of key regulatory proteins and genes in both a specific and controlled manner . A number of different strategies have been developed over the past 5 years to examine the pathways and effectors that are involved in mitogenic signaling as well as in the regulation of gene expression during the proliferative response to growth factors by normal fibroblasts . These strategies include: 1 . Direct in vivo competition for various trans-activating DNA binding activities by microinjection of double-stranded oligonucleotides, microinjection of monospecific antibodies against transcription factors and microinjection of dominant negative mutants of transcription factors based upon their DNA binding domain . 2 . Microinjection of purified enzymes (kinases and phosphatases) or peptides and antibodies that specifically inhibit these activities . 3 . Microinjection of expression plasmids which encode various normal and epitope-tagged regulatory molecules . In many of the experiments described below, c-fos gene expression was monitored as an early marker of mitogenic response . The c-fos gene belongs to a family of genes whose transcription is activated very early after addition of growth factor (1-4) . For in vivo studies, the c-fos promoter offers several unique advantages . Primarily, it is easy to manipulate . In practical terms, when mammalian fibroblasts are made quiescent (by replacing the normal growth media, with growth factors-depleted media) and subsequently activated by re-adding mitogen (growth factors, serum), c-fos RNA expression is restored within 15 minutes and the protein is specifically detected in the nuclei of cells after 90 minutes, but is no longer detectable after 3 hours . Secondly, results obtained with the c-fos promoter are directly applicable to cell growth since expression of c-fos is itself a prerequisite for proliferation as demonstrated by microinjection of anti-fos antibodies which prevented proliferation in mammalian cells (5) . Thirdly, the c-fos promoter is exquisitely sensitive to agents which cause cell stress . In this respect, heat-shock, poor microinjection or microinjection in the presence of heavy metals or chelating agents in the culture media all rapidly stimulate c-fos expression . However, when compared to c-fos expression in the proliferative response, stress mediated c-fos expression is induced both more rapidly and strongly, reverses more slowly (the protein is still detectable after 5-6 hours) and does not result in cell proliferation (unpublished observation) . As such, it provides an excellent internal control for identifying poor treatment and manipulation of cells . Finally, the c-fos promoter is subject to several levels of auto-regulation enabling the analysis of not only components involved in transcriptional activation , but also various aspects of transcriptional down regulation and shut-off. Appl Microbiol Biotechnol, 1996 Mar, 45(1-2), 278 - 85 A method to increase silver biosorption by an industrial strain of Saccharomyces cerevisiae; Simmons P et al.; Ag+ biosorption by an industrial strain of Saccharomyces cerevisiae was investigated . Older (96 h old) biomass had half the biosorption capacity of younger (24 h old) biomass (0.187 and 0.387 mmol Ag+/g dry mass respectively) . Comparisons of cell walls isolated from biomass of either age indicated that chemical composition and Ag+ biosorption capacity varied little over the time span examined and that cell walls from either age of culture had small Ag+ biosorption capacities compared to whole cells of a similar age . Silver-containing precipitates were observed both on the cell wall and within the cell, indicating that intracellular components sorbed Ag+ . The concentration of these precipitates within the cell appeared visually to decrease with age in Ag(+)-exposed cells . Incorporation of L-cysteine into the growth medium resulted in biomass with increased silver biosorption capacities, protein and sulphydryl group content . Increasing the concentration of L-cysteine in the growth medium from 0 to 5.0 mM increased silver biosorption from 0.389 to 0.556 mmol Ag+/g dry mass . Isolated cell walls of biomass grown in supplemented media also showed a possible link between silver biosorption capacities, protein and sulphydryl group content . No precipitates were observed in silver-exposed biomass that had been grown in the presence of 5.0 mM L-cysteine. Appl Microbiol Biotechnol, 1996 Mar, 45(1-2), 267 - 72 Optimizing biodegradation of phenanthrene dissolved in nonaqueous-phase liquids; Birman I et al.; A study was conducted to optimize the biodegradation in soil slurries of phenanthrene initially dissolved in nonaqueous-phase liquids (NAPLs) . The slow rate of degradation of phenanthrene in dibutyl phthalate was increased by addition of phenanthrene-degrading microorganisms to soil slurries containing the NAPL . The rate was further increased and the acclimation phase was shortened if the inoculum was grown in a medium containing the hydrocarbon and the phthalate before addition to the slurries . Composition of the growth medium only shortened the acclimation but had no effect on the rate . Vigorous agitation increased the rate and extent of mineralization of phenanthrene in dibutyl phthalate . The effect of temperature was affected by the presence and identity of the inoculum . Rapid and extensive mineralization of phenanthrene initially present in hexadecane and diesel oil were attained by use of intense agitation of the NAPL/soil slurry and inoculation with microorganisms grown in the presence of the NAPLs, but the influence of these variables was less with other NAPLs . Vigorous agitation and addition of an inoculum 24 h after introduction of a nonionic surfactant enhanced biodegradation of phenanthrene initially in 150 Bright stock oil and dibutyl phthalate . The results suggest improved means for the bioremediation of sites contaminated with NAPLs. Microbiology, 1996 Mar, 142 ( Pt 3), 469 - 75 Alteration in membrane fluidity and lipid composition, and modulation of H(+)-ATPase activity in Saccharomyces cerevisiae caused by decanoic acid; Alexandre H et al.; Decanoic acid, a lipophilic agent, inhibited in vitro the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae grown in YPD medium . Conversely, when decanoic acid (35 microM) was present in the growth medium, the measured H(+)-ATPase activity was four times higher than that of control cells . Km, and pH and orthovanadate sensitivity were the same for the two growth conditions, which indicated that H(+)-ATPase activation was not due to conformational changes in the enzyme . The activation process was not entirely reversible which showed that plasma membrane H(+)-ATPase activation is due to several mechanisms . 1,6-diphenyl-1,3,5-hexatriene anisotropy performed on protoplasts from cells grown in YPD revealed that as decanoic acid concentration was increased, anisotropy significantly decreased, i.e . membrance fluidity increased . Cells grown in media containing decanoic acid exhibited greater membrane fluidity compared with control cells . Furthermore, these cells did not show any fluidifying effect when increased concentrations of decanoic acid were added . Chemical analysis of cell membrane lipid composition revealed a modification in the distribution of the phospholipid fatty acids and sterols in cells grown in the presence of 35 microM decanoic acid compared with control cells . Our results support the view that the plasma membrane H(+)-ATPase activation induced by decanoic acid is correlated with an alteration in membrane lipid constituents. Can J Microbiol, 1996 Mar, 42(3), 227 - 33 Degradation of diclofop-methyl by pure cultures of bacteria isolated from Manitoban soils; Smith-Greenier LL et al.; Pure cultures of Chryseomonas luteola and Sphingomonas paucimobilis isolated from Manitoban soils were able to utilize diclofop-methyl (methyl-2-{4-(2,4-dichlorophenoxy)phenoxy} propanoate) as the sole source of carbon and energy . An actively growing culture of C . luteola completely degraded 1.5 micrograms diclofop-methyl.mL-1 to diclofop acid and 4-(2,4-dichlorophenoxy)phenol within 71 h, as determined by gas chromatographic analysis . The accumulation of these metabolites in the growth medium resulted in the cessation of growth, indicating the organism's inability to degrade phenoxyphenol in the presence of diclofop acid . Sphingomonas paucimobilis mineralized 1.5 micrograms diclofop-methyl.mL-1 to diclofop acid within 54 h . A biphasic growth pattern indicated that this organism was capable of degrading diclofop acid to 4-(2,4-dichlorophenoxy)phenol and 2,4-dichlorophenol and (or) phenol . Neither of the organisms was able to utilize 2,4-dichlorophenol as the sole source of carbon and energy. Vet Microbiol, 1996 Mar, 49(1-2), 21 - 30 Motility and chemotaxis in Serpulina hyodysenteriae; Kennedy MJ et al.; Chemotactic- or motility-regulated mucus association appears to be the predominant mechanism of mucosal association by the causative agent of swine dysentery, Serpulina hyodysenteriae . In the present study, a modification of the Adler capillary assay was used to evaluate the chemotactic responses of S . hyodysenteriae to a variety of potential stimuli . First, however, it became necessary to study factors that influenced motility of the spirochete in vitro, since standard cultivation methods produced motility inferior to that observed for in vivo grown cells . A number of factors were found to influence S . hyodysenteriae motility, but of these growth medium and growth phase appeared to be the most important . The type and even batch of culture medium also were found to have a significant influence on S . hyodysenteriae motility . Optimal motility and chemotaxis for S . hyodysenteriae was observed when the cells were harvested in mid- to late-log phase, and in vivo-like motility could be induced by suspending the cells in physiologic saline . S . hyodysenteriae was strongly attracted to hog gastric mucin, certain concentrations of blood, L-fucose, L-serine and other compounds . Selected sugars and other amino acids did not serve as chemoattractants for S . hyodysenteriae . The chemotactic response of S . hyodysenteriae toward L-fucose and L-serine, constituents of mucin, may be important factors in the affinity of the spirochete for the mucus in the intestinal tract of swine. Genetics, 1996 Mar, 142(3), 737 - 47 Underproduction of the largest subunit of RNA polymerase II causes temperature sensitivity, slow growth, and inositol auxotrophy in Saccharomyces cerevisiae; Archambault J et al.; In the yeast Saccharomyces cerevisiae, mutations in genes encoding subunits of RNA polymerase II (RNAPII) often give rise to a set of pleiotropic phenotypes that includes temperature sensitivity, slow growth and inositol auxotrophy . In this study, we show that these phenotypes can be brought about by a reduction in the intracellular concentration of RNAPII . Underproduction of RNAPII was achieved by expressing the gene (RPO21), encoding the largest subunit of the enzyme, from the LEU2 promoter or a weaker derivative of it, two promoters that can be repressed by the addition of leucine to the growth medium . We found that cells that underproduced RPO21 were unable to derepress fully the expression of a reporter gene under the control of the INO1 UAS . Our results indicate that temperature sensitivity, slow growth and inositol auxotrophy is a set of phenotypes that can be caused by lowering the steady-state amount of RNAPII; these results also lead to the prediction that some of the previously identified RNAPII mutations that confer this same set of phenotypes affect the assembly/stability of the enzyme . We propose a model to explain the hypersensitivity of INO1 transcription to mutations that affect components of the RNAPII transcriptional machinery. Protein Expr Purif, 1996 Mar, 7(2), 220 - 8 Enhanced recovery of a secreted mammalian protein from suspension culture of genetically modified tobacco cells; Magnuson NS et al.; Increasing the level of recovery of mammalian proteins secreted by a genetically modified Nicotiana tabacum was explored in suspension culture . As a model protein system, a mouse monoclonal antibody heavy chain gamma (MAb HC) with an antigen specificity for p-azophenylarsonate was used . Consistent with findings for other plant cell suspension culture systems expressing proteins with mammalian leader sequences, the synthesized mouse MAb HC was secreted through the plasma membrane . In addition, the majority of the MAb HC was also secreted through the cell wall into the growth medium . However, efficient recovery of the protein was only possible when the protein stabilizing agent, polyvinylpyrrolidone (PVP) was present in the plant cell growth medium . The presence of PVP increased the recovered concentration of secreted protein 35-fold from 0.010 to 0.36 micrograms protein/ml culture medium . Biological activity of the approximately 50-kDa MAb HC polypeptide was demonstrated by arsonate affinity matrix binding as determined by Western blot analysis . In addition to antigen binding activity, the secreted protein also exhibited reactivity to protein G, a protein which specifically binds mouse IgG . These findings are important because they demonstrate that culture conditions can significantly influence the concentration of a biologically active foreign protein secreted from plant cells into the media of suspension cultures . The ability to increase the efficiency of mammalian protein production in plant suspension culture systems should provide significant advantage over protein production in intact transgenic plants which require cultivation, harvesting, and expensive extraction procedures to obtain nonsecreted foreign proteins. J Dermatol Sci, 1996 Mar, 11(3), 239 - 49 Ultrastructure of size-regulated hetero-spheroids composed of human keratinocytes and fibroblasts; Butt KI et al.; In order to develop a new in vitro skin model multicellular floating hetero-spheroids were prepared by culturing keratinocytes and fibroblasts on hydrophilic culture dishes coated with type I collagen and a thermo-responsive polymer . Upon decreasing the substratum's temperature to an ambient temperature, the spheroids detached from the substratum and were thereafter maintained in either: Medium I, a medium mixture of keratinocyte growth medium (KGM) and supplemented Dulbecco's modified Eagle's medium (DMEM) at a ratio of 1 to 2; or medium II, KGM for the initial 24 h followed by supplemented DMEM for the remainder of the culture periods . The spheroids displayed a typical pattern of an external rim of keratinocytes with an internal core of fibroblasts . A minute space separated the keratinocytes and fibroblasts . The stratification of cells cultured in medium II was more prominent than that of the cells cultured in medium I . Markers of the advanced stages of keratinization such as keratohyalin granules, membrane coating granules and the cornified envelope were not observed . Interestingly, keratinocytes underwent the same differentiation pathway as non-keratinized stratified epithelia such as esophagus . With consideration to keratinocyte-fibroblast interactions, it may be of interest to incorporate the study of such morphological impairments when investigating the effects of growth factors and their ligands. Eur J Cancer B Oral Oncol, 1996 Mar, 32B(2), 106 - 13 Tenascin: growth and adhesion modulation--extracellular matrix degrading function: an in vitro study; Shrestha P et al.; Tenascin (TN), a recently characterised extracellular matrix protein, largely confined to the process with the development of embryo in areas of epithelial-mesenchymal interactions and in areas where there are morphogenetic movements and tissue patterning, has a highly restricted expression in adult tissues . The expression of TN is enhanced in a variety of human neoplastic lesions . However, function(s) and molecular mechanisms of enhanced expression in neoplastic lesions remain unclear . We employed human tongue carcinoma cells (SCCKN), human salivary gland adenocarcinoma cells (SGT-1), normal mouse embryonic fibroblasts (NIH3T3-3) and K-ras-2 transformed fibroblasts (Cle-H3) in an in vitro study to elucidate the biological roles of TN . In in vitro studies, all the cell lines examined had enhanced secretion of TN in the presence of transforming growth factor-beta in a dose-dependent manner and TN itself was found to possess a growth-enhancing activity . Moreover, studies on adhesion of the cell lines on coated substrates of fibronectin (FN), laminin (LN), tenascin (TN), TN/FN and TN/LN showed that all the cells adhere and spread well on FN and LN . However, on TN they attach poorly and remain rounded . The relative concentrations of TN and FN affected the cellular adhesion and morphology . In SCCKN and SGT-1, but not in NIH3T3 and Cle-He3 fibroblasts, a higher concentration of TN inhibited cellular adhesion on fibronectin, suggesting that cells attach poorly on TN, it may interfere with the action of fibronectin, and the relative concentrations of TN, FN or LN may affect cellular adhesion and morphology which may differ in different cell types . When TN was added in the growth medium of exponentially growing cells, the cells lost their cell to cell contact and were seen to be separating . The presence of these extracellular matrix proteins were further tested to determine whether they could modulate the secretion of proteolytic enzymes responsible for extracellular matrix degradation by tumour cells, when the neoplastic cells but not the non-neoplastic cells grown on FN/TN substrate showed positive immunofluorescence for collagenase . FN, LN or TN alone did not induce collagenase in the tumour cells . If the same is true in vivo, although a number of factors and interactions may implicate the ultimate outcome, the enhanced expression of TN in neoplastic lesions may have potential implications for tumour growth, differentiation, cellular adhesion, invasion and metastasis. J Biomed Mater Res, 1996 Mar, 30(3), 353 - 60 Myoblast seeding in a collagen matrix evaluated in vitro; van Wachem PB et al.; Collagens may be used as biomaterials for soft tissue reconstruction, e.g., the abdominal wall . We previously developed a biocompatible dermal sheep collagen (DSC), which in an abdominal wall reconstruction model showed controlled biodegradation and functioned as a matrix for in-growth of fibroblasts but not of muscle . It was hypothesized that regeneration of muscle via DSC may be possible by seeding of muscle cells . Using a syringe, mouse C2C12 myoblasts were seeded in DSC disks and incubated in methylcellulose-based growth medium, changed at 24 h into differentiation medium . An estimated 85% of the cells were well distributed, especially in the top half of the DSC disks . Some 15% of the cells ended up on top . At 4 h, all cells showed a spherical morphology, sometimes with clear adhesion plaques . At 24 h, cells on the top started to form a "capsule" with well-spread cells . Underneath the capsule, of the remaining 85% of the cells, approximately 30% showed adhesion and spreading on/in between collagen bundles . At day 3 after the addition of differentiation medium, the spread cells showed first indications of myotube formation . At day 7, myotube formation had proceeded, while extracellular matrix, i.e., collagen and elastin, had been deposited . This study shows that myoblast seeding into DSC is feasible, resulting in a reasonable cell distribution and survival of 45% of the cells . The surviving cells are able to differentiate into myotubes and form an extracellular matrix. J Ind Microbiol, 1996 Mar, 16(3), 145 - 54 Review: optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter; Donovan RS et al.; This review examines factors which influence the expression of foreign proteins in Escherichia coli under the transcriptional control of the lac and tac promoters, and discusses conditions for maximizing the production of a foreign protein using this system . Specifically, the influence of IPTG (isopropyl-beta-D-thiogalactoside) concentration, temperature, composition of the growth medium, the point in the growth curve at which cells are induced with either IPTG or lactose, and the duration of the induction phase are discussed. Infect Immun, 1996 Mar, 64(3), 966 - 73 Expression of attaching/effacing activity by enteropathogenic Escherichia coli depends on growth phase, temperature, and protein synthesis upon contact with epithelial cells; Rosenshine I et al.; Enteropathogenic Escherichia coli (EPEC) induces tyrosine phosphorylation of a 90-kDa protein (Hp90) in infected epithelial cells . This in turn facilitates intimate binding of EPEC via the outer membrane protein intimin, effacement of host cell microvilli, cytoskeletal rearrangement, and bacterial uptake . This phenotype has been commonly referred to as attaching/effacing (A/E) . The ability of EPEC to induce A/E lesions was dependent on bacterial growth phase and temperature . Early-logarithmic-phase EPEC grown at 37 degrees C elicits strong A/E activity within minutes after infection of HeLa epithelial cells . EPEC de novo protein syntheses during the first minutes of interaction with the host cell was required to elicit A/E lesions . However, once formed, bacterial viability was not needed to maintain A/E lesions . The type of growth media and partial O2 pressure level do not seem to affect the ability of EPEC to cause A/E lesions . These results indicates that the A/E activity of EPEC is tightly regulated by environmental and host factors. Am J Physiol, 1996 Mar, 270(3 Pt 1), L435 - 45 Effect of cold preservation on pulmonary arterial smooth muscle cells; Hall SM et al.; The efficacy of preservation fluids on the cytoskeleton and contractile function of porcine pulmonary arterial smooth muscle (SM) cells during cooling and rewarming was evaluated, using EuroCollins solution (EC), University of Wisconsin solution (UW), Marshall's solution (MS), and tissue culture growth medium (GM) . Functional studies included passive distensibility and contraction to prostaglandin F2a (PGF2a) in arterial rings and wrinkling of silicone membranes by cooled-rewarmed cultured SM cells . Immunofluorescence measurements were made of actin brightness in cooled arterial rings . Cultured SM monolayers were stained with antibodies to SM alpha-actin, SM myosin, and tubulin . In cooling, all solutions resulted in increased arterial distensibility, whereas EC and MS reduced cell wrinkling . With the use of all solutions, actin cables thinned, myosin filaments dissociated, and microtubules depolymerized . During rewarming, resistance to imposed tension increased in all arterial rings . After GM,ED, and MS preservation, contraction to PGF2a increased . Wrinkling increased and actin-myosin cables shortened after GM and EC; after UW, wrinkling decreased and actin-myosin cables thinned . No recovery occurred after MS . Thus the type of preservation solution influenced contractility during preservation and after rewarming . The absence of spontaneous contraction in cells cooled in UW may be advantageous. J Virol, 1996 Mar, 70(3), 1804 - 9 A novel murine retrovirus identified during testing for helper virus in human gene transfer trials; Miller AD et al.; An important requirement for the use of retroviral vectors in human gene transfer experiments is the avoidance of human exposure to replication-competent (helper) retroviruses . To meet this requirement, we used a sensitive marker rescue assay for helper virus to screen vector-transduced cells prior to reinfusion into patients . This assay utilized Mus dunni cells harboring a retroviral vector that can be rescued by helper retroviruses . The assay indicated the presence of helper virus in medium exposed to hematopoietic cells from all patients tested, including six patients with various cancers and one patient with Gaucher's disease, whether or not the patient cells had been exposed to retroviral vectors . All of the helper viruses were in a single interference group . We have now shown that treatment of the M . dunni marker rescue assay cells with 5-iodo-2'-deoxyuridine or hydrocortisone can activate production of an apparently identical helper virus, which we have named M . dunni endogenous virus (MDEV) . Thus, production of virus in the assays of patient materials was likely due to exposure of the marker rescue assay cells to the hydrocortisone present in the hematopoietic cell growth medium . MDEV does not belong to any of the known murine leukemia virus groups by interference analysis, and we have called the new group multitropic because of the wide range of cells from different species that MDEV can infect. J Cell Physiol, 1996 Mar, 166(3), 585 - 92 Cytokine expression by human marrow-derived mesenchymal progenitor cells in vitro: effects of dexamethasone and IL-1 alpha; Haynesworth SE et al.; We previously reported the purification, culture-expansion, and osteogenic differentiation potential of mesenchymal progenitor cells (MPCs) derived from human bone marrow . As a first step to establishing the phenotypic characteristics of MPCs, we reported on the identification of unique cell surface proteins which were detected with monoclonal antibodies . In this study, the phenotypic characterization of human marrow-derived MPCs is further established through the identification of a cytokine expression profile under standardized growth medium conditions and in the presence of regulators of the osteogenic and stromal cell lineages, dexamethasone and interleukin-1 alpha (IL-1 alpha), respectively . Constitutively expressed cytokines in this growth phase include G-CSF, SCF, LIF, M-CSF, IL-6, and IL-11, while GM-CSF, IL-3, TGF-beta 2 and OSM were not detected in the growth medium . Exposure of cells in growth medium to dexamethasone resulted in a decrease in the expression of LIF, IL-6, and IL-11 . These cytokines have been reported to exert influence on the differentiation of cells derived from the bone marrow stroma through target cell receptors that utilize gp130-associated signal transduction pathways . Dexamethasone had no effect on the other cytokines expressed under growth medium conditions and was not observed to increase the expression of any of the cytokines measured in this study . In contrast, IL-1 alpha increased the expression of G-CSF, M-CSF, LIF, IL-6 and IL-11 and induced the expression of GM-CSF . IL-1 alpha had no effect on SCF expression and was not observed to decrease the production of any of the cytokines assayed . These data indicate that MPCs exhibit a distinct cytokine expression profile . We interpret this cytokine profile to suggest that MPCs serve specific supportive functions in the microenvironment of bone marrow . MPCs provide inductive and regulatory information which are consistent with the ability to support hematopoiesis, and also supply autocrine, paracrine, and juxtacrine factors that influence the cells of the marrow microenvironment itself . In addition, the cytokine profiles expressed by MPCs, in response to dexamethasone and IL-1 alpha, identify specific cytokines whose levels of expression change as MPCs differentiate or modulate their phenotype during osteogenic or stromagenic lineage entrance/progression. J Biol Chem, 1996 Feb 16, 271(7), 3581 - 9 Regulatory elements that control transcription activation and unsaturated fatty acid-mediated repression of the Saccharomyces cerevisiae OLE1 gene; Choi JY et al.; In Saccharomyces cerevisiae, unsaturated fatty acids are formed from saturated acyl-CoA precursors by Ole1p, a delta-9 fatty acid desaturase . OLE1 mRNA levels are differentially regulated by the addition of saturated or unsaturated fatty acids to the growth medium . One component of this regulation system involves the control of OLE1 transcription . Saturated fatty acids induce a 1.6-fold increase in transcription activity, whereas a large family of unsaturated fatty acids repress OLE1 transcription as much as 60-fold . A deletion analysis of OLE1 promoter::lacZ fusion reporter genes identified a 111-base pair (bp) fatty acid-regulated (FAR) region approximately 580 bp upstream of the start codon that is essential for transcription activation and unsaturated fatty acid repression . Deletion of an 88-bp sequence within that region resulted in a complete loss in transcription activation and unsaturated fatty acid regulation . The 111-bp FAR element strongly activates transcription and confers unsaturated fatty acid regulation on a heterologous CYC1 promoter test plasmid . Essential elements required for unsaturated fatty acid repression of OLE1 were found in the 5 and 3 region of the 111-bp sequence . The FAR element-mediated activation and fatty acid repression of transcription was found to be closely tied to fatty acyl-CoA metabolism . Two fatty acid activation genes, FAA1 and FAA4, were found to be essential for unsaturated fatty acid repression of OLE1 through the FAR sequences . Disruption of either gene results in reduced levels of unsaturated fatty acid repression; disruption of both genes completely blocks the regulatory response . Acyl-CoA binding protein (ACBP) plays a role in determining the level of FAR element activated transcription . Disruption of the ACBP gene causes a >5-fold activation of OLE1 transcription and a similar increase in OLE1 mRNA levels . Unsaturated fatty acid repression of OLE1 transcription, however, is not affected by the disrupted ACBP gene . These studies show that promoter elements responsible for unsaturated fatty acid-mediated transcription repression are tightly linked to OLE1 activation sequences and that OLE1 transcription levels are closely tied to acyl-CoA metabolism. J Biol Chem, 1996 Feb 9, 271(6), 3238 - 46 Transcriptional regulation of alpha1,3-galactosyltransferase in embryonal carcinoma cells by retinoic acid . Masking of Lewis X antigens by alpha-galactosylation; Cho SK et al.; Treatment of mouse teratocarcinoma F9 cells with all-trans-retinoic acid (RA) causes a 9-fold increase in steady-state levels of mRNA for UDP-Gal:beta-D-Gal alpha1,3-galactosyltransferase (alpha1,3GT) beginning at 36 h . Enzyme activity rises in a similar fashion, which also parallels the induction of laminin and type IV collagen . Nuclear run-on assays indicate that this increase in alpha1,3GT in RA-treated F9 cells, like that of type IV collagen, is transcriptionally regulated . Differentiation also results in increased secretion of soluble alpha1,3GT activity into the growth media . The major alpha-galactosylated glycoprotein present in the media of RA-treated F9 cells, but not of untreated cells, was identified as laminin . Differentiation of F9 cells is accompanied by an increase in alpha-galactosylation of membrane glycoproteins and a decrease in expression of the stage-specific embryonic antigen, SSEA-1 (also known as the Lewis X antigen or LeX), which has the structure Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-R . However, flow cytometric analyses with specific antibodies and lectins, following treatment of cells with alpha-galactosidase, demonstrate that differentiated cells contain LeX antigens that are masked by alpha-galactosylation . Thus, RA induces alpha1,3GT at the transcriptional level, resulting in major alterations in the surface phenotype of the cells and masking of LeX antigens. FEBS Lett, 1996 Feb 5, 379(3), 279 - 85 TPA and cycloheximide modulate the activation of NF-kappa B and the induction and stability of nitric oxide synthase transcript in primary neonatal rat hepatocytes; Menegazzi M et al.; 12-O-Tetradecanoylphorbol 13-acetate (TPA) elicited a transient increase in the transcription of the inducible nitric oxide synthase (iNOS) gene coupled with a shortening of the half-life of its mRNA in primary neonatal rat hepatocytes . These effects of TPA were preceded by a surge in nuclear translocation of the transcription factor NF-kappa B, and followed by a mounting accumulation of NO-2 in the growth medium . Even cycloheximide (CHX) added by itself elicited an early, sustained activation of NF-kappa B followed by an intense induction of iNOS gene expression, irrespective of what degree of protein synthesis inhibition was brought about by the several concentrations tested . When given together, TPA and CHX exerted additive effects on hepatocellular iNOS mRNA levels . These results suggest the likelihood of an ordered sequence of events by which an activated NF-kappa B mediates the induction of iNOS gene expression in TPA- and/or CHX-treated primary hepatocytes. J Biol Chem, 1996 Feb 2, 271(5), 2627 - 33 Progesterone inhibits cholesterol biosynthesis in cultured cells . Accumulation of cholesterol precursors; Metherall JE et al.; Cells acquire cholesterol through endogenous synthesis and through receptor-mediated uptake of cholesterol-rich low density lipoprotein (LDL) . Esterification of LDL-derived cholesterol is catalyzed by acyl-CoA:cholesterol acyltransferase (ACAT) in the endoplasmic reticulum (ER) . Progesterone inhibits esterification, and, although the mechanism of inhibition is not completely understood, this inhibition results from progesterone's ability to inhibit the activity of multiple drug resistance (MDR) P-glycoproteins (P . DeBry and J . E . Metherall, submitted for publication) . In the current manuscript, we demonstrate that progesterone inhibits cholesterol biosynthesis resulting in the accumulation of a number of sterol precursors . In Chinese hamster ovary (CHO) cells, high concentrations (100 microM) of progesterone completely blocked cholesterol production, resulting in the accumulation of lanosterol and a lanosterol precursor . Lower concentrations (40 microM) of progesterone cause plasma membrane accumulation of several sterol products . The majority of these sterols are precursors of cholesterol since they were efficiently converted to cholesterol upon removal of progesterone from the culture medium . Although very high concentrations (> 200 microM) of progesterone killed CHO cells, their growth was restored by the addition of cholesterol to the growth medium, indicating that progesterone toxicity resulted from cholesterol auxotrophy . The effect of progesterone was not unique to CHO cells; progesterone also inhibited cholesterol biosynthesis in all human cell lines tested . These observations suggest that a common progesterone-sensitive pathway is involved in both cholesterol biosynthesis and the processing of LDL-derived cholesterol. Microbiology, 1996 Feb, 142 ( Pt 2), 347 - 57 The sigA gene encoding the major sigma factor of RNA polymerase from the marine cyanobacterium Synechococcus sp . strain PCC 7002: cloning and characterization; Caslake LF et al.; The gene encoding the principal sigma factor from Synechococcus sp . strain PCC 7002 was isolated and characterized . The Synechococcus sp . strain PCC 7002 sigA gene encodes a protein of 375 amino acids (43 center dot 7 kDa) that is required for viability under normal growth conditions . The SigA protein was overproduced in Escherichia coli and the purified protein was used to raise polyclonal antiserum in rabbits . This antiserum was used in immunoblot analyses of partially purified RNA polymerase from Synechococcus sp . strain PR6000 . The probable in vivo translational start site was identified by a comparison of amino acid sequencing results obtained with SigA proteins overproduced in E . coli with immunoblot analyses of SigA protein in crude preparations of RNA polymerase from the cyanobacterium . The sigA gene is encoded on a transcript of 1700 bases that initiates 496 nucleotides upstream from the probable in vivo translational start site . The abundance of sigA transcripts decreases rapidly after the removal of combined nitrogen from the growth medium. Res Commun Mol Pathol Pharmacol, 1996 Feb, 91(2), 245 - 8 Augmentation of epirubicin cytotoxicity by cycloheximide; Furusawa S et al.; The effect of cycloheximide (CH) on the cytotoxic activity of the anthracycline antibiotic epirubicin (EPI) was examined in cell cultures of murine leukemia doxorubicin (DOX)-sensitive P388 and -resistant P388 cells . The addition of CH (0.002 mu g/ml) to the growth medium markedly enhanced the EPI-induced cytotoxic effect in sensitive cells as well as that observed in resistant cells . CH, however, did not affect the intracellular content of EPI . The inhibition of DNA synthesis in leukemia cells was remarkable with the combination of EPI and CH compared with each drug alone . These results suggest that the combination of EPI and CH appears to be useful in killing mouse leukemia cells. J Clin Microbiol, 1996 Feb, 34(2), 385 - 8 Axenic growth and drug sensitivity studies of Balamuthia mandrillaris, an agent of amebic meningoencephalitis in humans and other animals; Schuster FL et al.; A cell-free growth medium for the opportunistic pathogenic ameba Balamuthia mandrillaris is presented . This represents an advance over the use of monkey kidney cells for growth of the amebas and can be helpful in isolation of these amebas from brain tissue from cases in which amebic meningoencephalitis is a diagnostic possibility, as well as for biochemical and molecular biological studies . Three isolates of Balamuthia have been cultured in this medium . The cell-free growth system was also used to screen cultures for sensitivity to a variety of antimicrobial agents . Of the various drugs tested, pentamidine isethionate was most effective against amebas (ca . 90% inhibition after 6 days of exposure), but the drug was amebastatic and not amebacidal in the axenic system at the highest concentration tested (10 micrograms/ml). Am J Physiol, 1996 Feb, 270(2 Pt 1), C514 - 21 Interferon-gamma induces persistent depletion of internal Ca2+ stores in a human salivary gland cell line; Wu AJ et al.; Interferon-gamma (IFN-gamma), in the presence of tumor necrosis factor-alpha (TNF-alpha), decreases proliferation of a human salivary gland ductal cell line, HSG (Wu, A., R . Kurrasch, J . Katz, P . Fox, B . Baum, and J . Atkinson . J . Cell . Physiol . 161:217-226, 1994) . We examined the possible effects of these cytokines (1,000 U/ml IFN-gamma +/- 20 U/ml TNF-alpha for 7 days) on Ca2+ mobilization in HSG cells . In HSG cells, fetal bovine serum (10%) or carbachol (100 microM) stimulated rapid increases in cytosolic Ca2+ concentration ({Ca2+}i), apparently mobilized from different thapsigargin-sensitive intracellular Ca2+ stores . Serum induced a proliferative effect on HSG cells, which was suppressed (> 90%) by treatment with IFN-gamma +/- TNF-alpha, but not with TNF-alpha alone . Serum-, carbachol-, and thapsigargin-stimulated {Ca2+}i elevations were reduced by 90, 60, and > 65%, respectively, in cells treated with IFN-gamma +/- TNF-alpha and 30, 45, and 45%, respectively, in cells treated with TNF-alpha . Removal of the cytokines from the growth medium induced recovery of both cell proliferation and Ca2+ mobilization responses within 7 days . Treatment of HSG cells with thapsigargin (0.02-2 nM) induced a dose-dependent decrease in cell proliferation . Additionally, acute treatment (< 10 min) of cells with IFN-gamma did not affect {Ca2+}i or alter carbachol-, thapsigargin-, or serum-induced changes in {Ca2+}i . These data demonstrate that prolonged treatment of HSG cells with IFN-gamma +/- TNF-alpha leads to a persistent depletion of intracellular Ca2+ stores . We suggest that this may have a role in cell growth. Ecotoxicol Environ Saf, 1996 Feb, 33(1), 38 - 43 Accumulation and physiological and biochemical effects of cadmium in a simple aquatic food chain; Devi M et al.; The toxicity of cadmium with regard to the vegetative reproduction of duckweed, Lemna gibba, grown in sterile culture, was determined . The EC50 was found to be 800 ppb . Duckweed grown in 2.24 ppm cadmium (supplied as cadmium nitrate) for 7 days accumulated 98.5% of the available cadmium from the growth medium . Plants that had been grown for 7 days in 2.24 ppm cadmium and control plants were fed to red swamp crayfish, Procambarus clarkii, for 14 days . The concentrations of cadmium were measured in hepatopancreata and muscles of crayfish on Day 0 and in crayfish fed duckweed grown in cadmium for 14 days . Accumulation of this metal in hepatopancreata increased 26-fold, i.e., 176.80 ppb on Day 0 to 4657.56 ppb on Day 14, and in muscles almost 7-fold, i.e., 6.75 ppb on Day 0 to 46.28 ppb on Day 14 . Crayfish fed cadmium-containing duckweed demonstrated inhibition (55% after 14 days of feeding) of acetylcholinesterase activity in their central nervous tissue compared to crayfish fed cadmium-free duckweed . The ovarian index and total lipids content in the ovaries of crayfish fed cadmium-containing duckweed demonstrated significant increases on Day 14. Anticancer Drugs, 1996 Feb, 7(2), 204 - 12 In vivo/in vitro studies on the effects of cyclophosphamide on growth and differentiation of hamster palate; Shah RM et al.; A study was undertaken to examine the effects of cyclophosphamide (CP) on growth and differentiation of palatal tissues . An in vivo/in vitro approach was designed to analyze (1) whether the damage caused by in vivo administration of CP in the developing palate can be altered in vitro, and (2) to determine the effects of CP on the synthesis of collagen and glycosaminoglycan (GAG), which are essential for proper palate development . In addition, effects of vitamin B1 and/or B6 on in vivo modulation of CP teratogenicity was evaluated . Pregnant hamsters were given 30 mg/kg CP or 1 ml saline on day 10 of gestation . Control and CP-treated embryonic palates were dissected on day 11 of gestation and incubated in vitro in the presence or absence of CP . In order to allow metabolic activation of CP in vitro, either a slice of hamster liver or microsomal S9 fraction of liver was added to the culture medium . To study collagen and GAG synthesis, palates were obtained between days 10 and 13 of gestation, and incubated in growth medium supplemented with {14C}proline or {3H}glucosamine, as appropriate . The rates of collagen and GAG synthesis were determined . The results showed that, in the controls, the presence of a liver slice or S9 fraction in the culture medium had no effects on in vitro closure of palate . In vivo CP exposed palates did not fuse in vitro . When drug was given in vitro, or both in vivo and in vitro, palatal closure did not occur . CP reduced synthesis of both collagen and GAG in the vertically developing palate . The drug-treated shelves reoriented only after the rates of collagen and GAG synthesis were restored to the levels comparable to the control counterparts . Co-administration of vitamin B1 and B6 did not interfere with the teratogenicity of CP . It was suggested that CP treatment affected DNA synthesis and injured growing cells, which in turn reduced the synthesis of GAG and collagen and affected the expansion of shelf volume to delay the reorientation of the palatal shelves . Furthermore, it appears that in vivo treatment with CP changes the programming of palatal tissues to prevent the fusion process in vivo, which could not be altered in vitro. Plant Mol Biol, 1996 Feb, 30(3), 467 - 78 Over-production of the D1:2 protein makes Synechococcus cells more tolerant to photoinhibition of photosystem II; Soitamo AJ et al.; Over-expression of the psbAIII gene encoding for the D1 protein (form II; D1:2) of the photosystem II reaction centre in the Synechococcus sp . PCC 7942 was studied using a tac promoter and the lacIQ system . Over-expression was induced with 40 microgram/ml IPTG in the growth medium for either 6 or 12 h at growth irradiance (50 mumol photons m-2 s-1) . This treatment doubled the amount of psbAII/III mRNA and the D1:2 protein in membranes but decreased the amount of psbAI messages and the D1:1 protein . The total amount of both heterodimeric reaction centre proteins, D1 and D2, remained constant under growth light conditions, indicating that the number of PSII centres in the membranes was not affected, only the form of the D1 protein was changed from D1:1 to D1:2 in most centres . When the cells were photoinhibited either at 500 or 1000 mumol photons m-2 s-1, in the presence or absence of the protein synthesis inhibitor lincomycin, the D1:2 protein remained at a higher level in cells in which over-expression had been induced by IPTG . These cells were also less prone to photoinhibition of PSII . It is suggested that the tolerance of cells to photoinhibition increases when most PSII reaction centres contain the D1:2 protein at the beginning of high irradiance . This tolerance is further strengthened by maintaining psbAIII gene over-expression during the photoinhibitory treatment. Curr Genet, 1996 Feb, 29(3), 227 - 33 Secretion of Trichoderma reesei beta-glucosidase by Saccharomyces cerevisiae; Cummings C et al.; An intronless form of the bgl1 gene encoding an extracellular beta-glucosidase from Trichoderma reesei was expressed in the yeast Saccharomyces cerevisiae under the control of the yeast GAL1 promoter . Transformation of a yeast strain with this vector resulted in transformants that produce and secrete active beta-glucosidase into the growth medium . Additionally, active recombinant beta-glucosidase protein was shown to be localized predominantly in the periplasmic space by using a p-nitrophenyl beta-D-glycoside hydrolysis assay against fractionated yeast cells . The apparent size of the recombinant enzyme was 10-15 kDa larger than that of the native form . Treatment of the recombinant beta-glucosidase with endoglycosidase-H indicated the apparent increase in size was due to N-linked glycosylation. Implant Dent, 1996 Winter, 5(4), 264 - 71 In vitro mineralization and implant calcium phosphate-hydroxyapatite crystallinity; Morgan J et al.; Biological dissolution of implant calcium phosphate coatings release local concentrations of divalent ions, which may influence mineralization . The objective of this study was to determine the effects of calcium phosphate release from coated commercially pure titanium discs using a bone-like cell culture bioassay . Sandblasted discs were prepared with or without hydroxyapatite crystallinities (50, 75, and 90 percent) . Samples of each coating were randomly assigned and either preincubated for 24 hours with media or not before the addition of cells (2200/ mm2) . Cultures were grown for 72 hours in culture medium containing 0.5 microCi/mL45 Ca . After rinsing, the remaining calcium phosphate surface was dissolved and counted . Three independent trials were performed . Results indicated proliferation was not altered as a function of crystallinity (P > 0.05) among any of the groups . However, a significant (P < 0.01) inverse relationship was found for biologically mediated mineralization as a function of calcium phosphate crystallinity . Low crystalline surfaces (nominally 50 percent) had the highest level of mineralization, with 75 percent crystalline surfaces being intermediate and 90 percent crystalline samples having the lowest amount of relative mineral formation . Mineralization only occurred on sandblasted commercially pure titanium upon supplementation of the growth medium with an organophosphate (beta-glycerophosphate), although this was less than on culture plastic . The results suggest calcium phosphate dissolution, as a function of implant coating crystallinity, can alter biological mineralization and may be one means in which enhanced mineral formation occurs around calcium phosphate-coated dental implants. Mycopathologia, 1996, 135(2), 109 - 13 Factors affecting growth and urease production by Trichophyton spp; Mahmoud AL et al.; Among Trichophyton spp . examined for urease production, T . rubrum was negative, whereas T . mentagrophytes appeared to be the most active species . Urease was not detected in cell-free culture fluids of the tested fungi . The endocellular urease of the test fungi was essentially constitutive . Moreover, addition of urea to the growth medium of these organisms markedly inhibited their mycelial biomass and ureolytic yield . Environmental factors showed variable effects on the test fungi and there was no correlation between mycelial growth and urease activity of these fungi. Rocz Panstw Zakl Hig, 1996, 47(3), 325 - 32 {Factors modulating the sensitivity of bacterial spores to steam under pressure}; Krzywicka H et al.; The sensitivity of spores B . subtilis and B . stearothermophilus to steam under pressure depended on the growth medium and duration of cultivation . B . subtilis and B . stearothermophilus produced the largest number of spores on medium with Mn and yeast extract . However the spores grown on the medium were not the most resistant . The resistant spores was growing up with the age of cultures . The highest level of resistance was obtained in the case of the medium with Ca, after 7-10 days of cultivation . The sporicidal effect of steam under pressure depended on the number of spores on the test. Jpn J Ophthalmol, 1996, 40(3), 367 - 70 Susceptibility of cultured rabbit corneal epithelial cells to various herpes simplex virus isolates; Mori Y et al.; Rabbit corneal epithelial cells were cultured in rabbit corneal epithelial growth medium containing 0.03 mM/L Ca2+ without serum . Their susceptibility to herpes simplex virus (HSV) was compared with that of vero cells . Six clinical isolates obtained from the cornea, three from the oral mucosa, and two from the facial skin were examined . The 50% tissue culture infectious dose (TCID)50 of each isolated strain was calculated for both types of cells . Corneal epithelial cells appeared to be more susceptible to all strains isolated from the cornea or oral mucosa, while corneal epithelial cells and vero cells appeared to be equally susceptible to the two isolates from the skin . Thus, isolates derived from herpetic keratitis will grow better in corneal epithelial cells, suggesting that rabbit corneal epithelial cells are more suitable for isolating viruses from the cornea. Morfologiia, 1996, 110(4), 64 - 70 {The morphological differentiation of murine neuroblastoma NIE-115 cells}; Kostenko MA et al.; Characters and kinesis of mouse neuroblastoma (MN) cells morphological differentiation affected by non-serum, hypo- and hyperosmotic media and salt solution were studied . The nature of morphological differentiation was found not to depend on the inductors, while kinesis varies significantly . Morphological differentiation speed is directly proportional to the extent of non-specific action and probability of the following cell death . on the other hand, the number of irreversibly differentiated cells is inversely proportional to the action strength . For the induction of morphological differentiation a minimal nutrition media do not containing serum is sufficient and a common growth media changed once in 3-5 days according to how it acidifies is better to use for the prolonged maintenance of it . Universality of neuronal morphological differentiation is under discussion according tot he data obtained from MN cells and cultured mollusk isolated neurons. Bull Cancer Radiother, 1996, 83 Suppl, 201s - 6s Boron neutron capture irradiation: setting up a clinical programme in Nice; Pignol JP et al.; Neutron capture irradiation aims to selectively destroy tumor cells using 10B(n,alpha)7Li nuclear reactions produced within themselves . Following the capture reaction, an alpha particle and a, 7Li ion are emitted . Carrying an energy of 2.79 MeV, they destroy all molecular structures along their path close to 10 microns . These captures, used exclusively with a 'slow' neutron irradiation, provide a neutron capture therapy (BNCT) . If they are used in addition to a fast neutron beam irradiation, they provide a neutron capture potentiation (NCP) . The Centre Antoine-Lacassagne in Nice is actively involved in the European Demonstration Project for BNCT of grade IV glioblastomas (GBM) after surgical excision and BSH administration . Taking into account the preliminary results obtained in Japan, work on an 'epithermal' neutron target compatible with various cyclotron beams is in progress to facilitate further developments of this technique . For NCP, thermalized neutron yield has been measured in phantoms irradiated in the fast neutron beam of the biomedical cyclotron in Nice . A thermal peak appears after 5 cm depth in the tissues, delayed after the fast neutron peak at 1.8 cm depth . Thus, a physical overdosage of 10% may be obtained if 100 ppm of 10B are assumed in the tissues . Our results using CAL 58 GBM cell line demonstrate a dose modification factor (DMF) of 1.19 when 100 ppm of boric acid are added to the growth medium . Thus for the particles, issued from neutron capture, a biological efficiency at least twice that of fast neutrons can be derived . These results, compared with historical data on fast neutron irradiation of glioblastoma, suggest that a therapeutic window may be obtained for GBM. Dev Neurosci, 1996, 18(5-6), 397 - 404 Modulation of oxygen-radical-scavenging enzymes by oxidative stress in primary cultures of rat astroglial cells; Pinteaux E et al.; Cytosolic and mitochondrial alterations induced by exposure of rat astroglial primary cultures to reactive oxygen species (ROS) generated by a xanthine/xanthine oxidase (X/XO) mixture or by lipopolysaccharide (LPS) have been investigated biochemically and immunochemically . In the presence of ROS generated by X/XO, a significant decrease in Cu,Zn superoxide dismutase (Cu,Zn-SOD) and in glutamine synthetase (GS) activity was observed whereas mitochondrial Mn-SOD activity and enzyme protein levels were significantly enhanced . Similar effects on GS, Cu,Zn- and Mn-SOD activities were observed by glucose/glucose oxidase treatment of the cells . Addition of LPS to the cell growth medium also specifically induces Mn-SOD synthesis but was without effect on Cu,Zn-SOD . It is suggested that in all these tested situations, hydrogen peroxide could represent a specific inducer of the observed phenomenon and it may therefore be considered as an intracellular messenger involved in the regulation of some aspects of astroglial oxidative metabolism, particularly the defence against ROS. Microbios, 1996, 85(342), 35 - 44 In vitro susceptibility of Mycobacterium leprae to oxygen-mediated damage; Dhople AM; In order to evaluate factors responsible for the failure of Mycobacterium leprae to multiply in cell-free cultures in vitro studies were undertaken to determine the possible poisoning of the organism by hydroxide and superoxide radicals produced in the growth medium . The superoxide dismutase activity was very low, 10% of the levels found in armadillo cells, while measured activity of catalase and glutathione peroxidase was negligible . Susceptibility of M . leprae to hydrogen peroxide was enhanced by potassium iodide but not by lactoperoxidase . The addition of high amounts of catalase completely prevented hydrogen peroxide-mediated killing of M . leprae . Superoxide generated by the action of xanthine oxidase on xanthine was lethal to M . leprae, but superoxide dismutase added to the reaction mixture gave significant protection . Thus superoxide radicals may be a major cause for the sudden termination of growth of M . leprae in primary cultures and also for failure of subcultures. Drugs Exp Clin Res, 1996, 22(3-5), 219 - 23 Estimation of direct influence of Ukrain preparation on influenza viruses and the bacteria E . coli and S . aureus; Ciebiada I et al.; Ukrain is a semisynthetic drug with immunomodulatory properties derived from Chelidonium majus L . alkaloids and thiophosphoric acid . It acts selectively in a lytic way on cancer cells . Its protective properties have been shown in mice infected by influenza viruses . In this paper, the studies made on the estimation of the direct activity of Ukrain preparation on viruses and bacteria E . coli and S . aureus are described . Viruses of different haemagglutination titres were incubated with different concentrations of the preparation during period of 1, 2 and 24 h . Afterwards the samples were collected and used for the infection of the allantoic cavity obtained from 10-day-old hen embryos . A second method was based on the introduction of the Ukrain preparation into the allantoic cavity of embryos before infection with influenza viruses and after the infection of embryos . In both the described methods, the embryos were incubated within 48 hours . Then the presence of influenza viruses in allantoic fluid was estimated using a haemagglutination reaction with 30% hen blood cells . The influence of the preparation on hen embryo was also studied . In order to estimate the antibacterial activity the following procedure was used . To the preparation diluted with the growth medium from 500 micrograms/ml to 1 microgram/ml a definite amount of the bacteria S . aureus or E . coli was added, and after 24 and 48 h of incubation at 37 degrees C the results were read off . In the second method, the bacteria were added to 1, 10, 100 and 500 micrograms of the preparation in 1.0 ml of 0.85% NaCl, and after 1, 2 and 24 of incubation at room temperature the samples were collected and inoculated on solid Mueller-Hinton medium . The presence of bacterial growth or medium turbidity after 24 and 48 h of incubation was taken as a positive result . Our studies have revealed that the above mentioned preparation does not exert any negative influence on hen embryos that could make it difficult to estimate replication of influenza viruses . This preparation did not show any direct influence on the inactivation of influenza viruses and the bacteria E . coli and S . aureus. Cell Motil Cytoskeleton, 1996, 35(2), 94 - 9 Cell cycle-dependent regulation of cellular ATP concentration, and depolymerization of the interphase microtubular network induced by elevated cellular ATP concentration in whole fibroblasts; Marcussen M et al.; In the present work, evidence is presented indicating that an increased cellular ATP concentration during mitosis may, in conjunction with other factors {Verde et al., 1990: Nature 343:233-238; Andersen et al., 1994: J Cell Biol . 127:1289-1299}, induce depolymerization of the interphase microtubular network in cultured fibroblasts . It is shown here that the cellular ATP concentration varies through the cell cycle, reaching a peak at G2M- and minimum at late G1/early S-phase . Furthermore, we have found, using indirect immunofluorescent staining with an antitubulin antibody, that depolymerization of the interphase microtubular network may be induced by increasing the intracellular ATP concentration in cultured fibroblasts from 2.2 mM to 4.1 mM . This may be obtained through addition of adenosine and P1 to the growth medium . Our results indicate that this effect of adenosine and Pi is not mediated via adenosine receptors, but through an elevated cellular ATP concentration . ATP is suggested to act through a concentration-dependent effect on the exchangeable GTP site on tubulin, and not through the action of protein kinases or microtubule-associated proteins. Ann Clin Lab Sci, 1996 Jan-Feb, 26(1), 18 - 30 Actions and interactions of nickel and magnesium on the transformation response of transformed cells in culture; Hass BS et al.; Nickel (Ni) and magnesium (Mg) exert separate and interacting effects on cells: Ni is toxic while Mg enhances the transformation response of transformed cells and protects from heavy metal-induced toxicity . Transformed rat liver epithelial cells were used in the soft agar (SA) assay to measure the effect of Ni and/or Mg on the expression of anchorage independence . Cells were exposed to +/- Ni and +/- Mg in a single passage of growth medium (GM) prior to assay in SA . The cells were then treated with +/- Ni and +/- Mg in the SA resulting in a 4 x 4 treatment matrix yielding 16 Ni/Mg combinations . Nickel was expected to decrease the transformation frequency (TF) and did so in 6 of the 16 cases . Magnesium was expected to enhance the TF independently of Ni; Mg increased TF values in 7 of 16 cases . The Ni-Mg interaction occurred in 11 of 16 cases . In general, Mg and Ni effects were observed more in GM than is SA . It is not evident from this study why the Ni, Mg, and Ni-Mg effects are not observed universally, but it is evident that metal-metal interactions are not simply defined or analyzed in biological systems . A refined factorial design may be useful in further separating such interactions . From the point of view of the implementation of the SA assay, in which test substances are typically dose previous to the implementation of putting the exposed cells in SA, it is clear that assay results can be markedly altered by the presence of the test compound in the soft agar. Free Radic Biol Med, 1996, 21(2), 173 - 9 Cytotoxicity of phosphatidylcholine hydroperoxides is exerted through decomposition of fatty acid hydroperoxide moiety; Kaneko T et al.; The cytotoxicity of phosphatidylcholine hydroperoxides synthesized regioselectively and stereoselectively was examined in human umbilical vein endothelial cells . Phosphatidylcholine hydroperoxides were readily incorporated into cells, after which the contents declined gradually . Phosphatidylcholine with an arachidonic acid hydroperoxide residue was toxic to cells, while phosphatidylcholine with a linoleic acid hydroperoxide residue had no effect . The toxicity of phosphatidylcholine with arachidonic acid hydroperoxide disappeared after the reduction of the hydroperoxide by triphenylphosphine . Phosphatidylcholine with arachidonic acid hydroperoxide that had decomposed partially upon standing at room temperature was much more toxic than the pure hydroperoxide . 4-Hydroxynonenal, known widely as a toxic secondary product in the lipid peroxidation of polyunsaturated fatty acids, was detected in the decomposition mixture of phosphatidylcholine hydroperoxide . Phosphatidylcholine hydroperoxide incorporated into cells did not show toxicity when the hydroperoxide-containing medium was changed to growth medium after a short time . On the other hand, the phosphatidylcholine hydroperoxide was toxic in cells treated with the lipophilic free radical generator, 2,2'-azobis(2,4-dimethylvaleronitrile) . In addition, cells were damaged by long-term treatment in medium containing phosphatidylcholine hydroperoxide and its decomposition products . These results suggest that the toxicity of phosphatidylcholine hydroperoxides is exerted by toxic compounds arising from the decomposition of the hydroperoxide moiety. J Cell Biochem Suppl, 1996, 24, 257 - 68 Expression in human lung cancer cell lines of genes of prohormone processing and the neuroendocrine phenotype; Vos MD et al.; Lung tumor cells and cell lines, principally the histologically classified small cell lung cancer, are characterized by the expression of neuroendocrine (NE) features including AADC (aromatic amino acid decarboxylase, previously called DOPA decarboxylase) and the production of many peptide hormones . The general mechanisms by which most aspects of the NE phenotype affect the clinical behavior of lung tumor cells are unknown, but it is well recognized that peptide hormones can have systemic effects (paraneoplastic syndromes) and several have been shown to be autocrine growth factors for cancer cells . In order to determine the relationship between expression of different aspects of the NE phenotype in lung cancer cell lines, we have compared expression of a gene required for biosynthesis of some active peptide hormones (PAM, peptidylglycine alpha-amidating monooxygenase) to the gene for AADC in 32 lung cancer cell lines . Expression of these genes was quantified by both steady state Northern blot analysis and radiochemical enzymatic activity measurements . To ensure a range of expression of NE markers, non-small cell lung cancer (NSCLC) cell lines were chosen to include several which had previously been shown to express NE markers, and several small cell lung cancer (SCLC) cell lines with previous low levels of AADC were included . PAM enzyme activity and Northern blot analysis showed a two to three log variation in levels of expression in both the small cell and non-small cell lines . A smaller range was found for AADC expression . Using the highly sensitive PAM enzyme assays, all cell lines were found to express detectable PAM . PAM activities were secreted into the growth medium of all cell lines . There was no simple correlation apparent between AADC and PAM gene expression in the lung cancer cell lines . However, classic small cell lines demonstrated high levels of expression of both PAM and AADC genes, as did the carcinoid subset of the NSCLC lines . NSCLC lines expressed levels of PAM mRNA and enzyme activities equivalent to those of SCLC but had infrequent expression of AADC (principally only carcinoid NSCLC expressed AADC) . These data demonstrate that separate aspects of the NE phenotype can be differentially expressed in lung cancer histological sub-types . Expression of PAM enzymes in all sub-types of lung cancer suggests that peptide prohormone activation may be a common mechanism for autocrine growth stimulation even in non-Ne NSCLC cell lines, or may reflect maintenance in cell lines of a common pathway of lung tumor promotion. Free Radic Biol Med, 1996, 20(3), 319 - 29 Depletion of cellular iron by bps and ascorbate: effect on toxicity of adriamycin; Nyayapati S et al.; A new method was developed that reduces the intracellular iron content of cells grown in serum-containing culture without involving the significant uptake of iron-chelating agents into cells . Negatively charged bathophenanthrolinedisulfonate (BPS), together with ascorbate, caused cells to lose much of their cellular iron without causing much depression in HL-60 or H9c2 (2-1) cell proliferation over a 48-h period . When added to serum supplemented RPMI-1640 culture media, BPS and ascorbate efficiently reduced and competed for iron in Fe(III) transferrin to form Fe(II)(BPS)3 . The reaction also occurred with purified human iron-transferrin . When cells were incubated with growth medium containing serum that had been treated with BPS and ascorbate for 24 h, little or no BPS2- or Fe(II)(BPS)(4-)3 entered the cells, according to direct measurements and in agreement with the highly unfavorable 1-octanol/water partition coefficients for these molecules . However, iron was mobilized out of both cell types . After 24 h incubation of cells in this medium, there was no change in the activities of catalase and superoxide dismutase, or in the concentration of glutathione . Glutathione peroxidase was elevated 9% . Using HL-60 and H9c2 (2-1) cells made iron deficient with BPS and ascorbate, HL-60 cells grown in defined-growth media in the absence of iron-pyridoxal isonicotinoyl hydrazone, or Euglena gracilis cells maintained in a defined medium that was rigorously depleted of iron, it was shown that the cytotoxicity of adriamycin is markedly dependent on the presence of iron in each type of cell . Similar results were obtained when HL-60 cells were grown in RPMI-1640 culture medium and serum that had been incubated for 24 h in BPS and ascorbate and then chromatographed over a Bio-Rad desalting column to remove small molecules including BPS, ascorbate, and Fe(II)(BPS)3. Avian Dis, 1996 Jan-Mar, 40(1), 78 - 87 Interferon modulation of Marek's disease virus genome expression in chicken cell lines; Volpini LM et al.; Lines of chicken lymphoblastoid cells were established from local lesions induced by simultaneous injection of Marek's disease virus and various stimulants of T-cell activation . Lines developed with regular medium had relatively high mean rates of spontaneous expression of viral internal antigen (6.2%) . In contrast, lines developed and maintained with conditioned medium generated by mixed-lymphocyte reaction had a 62-fold reduction in the mean rate of viral internal antigen expression (0.1%) . The expression rate could be modulated by the removal or re-addition of conditioned medium to the growth medium . Down regulation involved proteins classified as immediate-early (a 14-kDa polypeptide), early (a 38-kDa phosphoprotein), and late (glycoprotein B homologue) antigens, indicating that the block is very early in virus replication . Once initiated in a given cell, replication apparently proceeded unimpeded . Interferon was determined to be largely responsible for the suppressive activity of the conditioned medium, although involvement of other cytokines could not be ruled out . Also, chicken interferon from other sources, including recombinant interferon, was able to similarly suppress viral antigen expression. Eur J Biochem, 1995 Dec 15, 234(3), 732 - 6 Osmo-expression and fast two-step purification of Escherichia coli translation termination factor RF-3; Mortensen KK et al.; The gene for the translation termination factor RF-3 in Escherichia coli has recently been cloned and sequenced . Only small amounts of the protein have been purified until now, not sufficient for detailed investigation of the structure and function of this factor . For such studies, we have developed an overexpression system and a purification procedure suitable for large quantities of RF-3 . The gene prfC was cloned into the osmo-inducible plasmid pOSEX3 and subsequently transformed into the E . coli strain MKH13 . The expression of prfC in this plasmid, which is under the control of the osmotic pressure in the growth medium, leads to a level of RF-3 more than 100-times higher than that in wild-type cells . Using a new two-step FPLC protein purification procedure consisting of ion-exchange chromatography on Q-Sepharose FF and S-Sepharose HP, we obtain 220 mg pure RF-3 from 10 g overproducing cells, corresponding to 55 mg RF-3/l medium . The identity of the purified protein was confirmed by matrix-assisted laser desorption/ionisation mass spectrometry of tryptolytic fragments and by N-terminal amino acid sequencing . The activity of the purified factor was tested in vitro by measuring the stimulation of RF-2 dependent formylmethionine release from a ribosomal termination complex and the binding capacity of GTP and GDP . All assays showed that the purified RF-3 was highly active with a specific activity of approximately 2000 units/mg. Int J Cancer, 1995 Dec 11, 63(6), 855 - 62 On the relationship between the probenecid-sensitive transport of daunorubicin or calcein and the glutathione status of cells overexpressing the multidrug resistance-associated protein (MRP); Versantvoort CH et al.; Cells exposed to calcein acetoxymethyl ester (calcein AM) in the growth medium become fluorescent following cleavage of calcein AM by cellular esterases to produce the fluorescent derivative calcein . It has previously been shown by others that multidrug resistant cells which overexpress P-glycoprotein accumulate much less fluorescent calcein than the corresponding parental cells . We have now examined the transport of calcein in multidrug resistant cells which overexpress an alternative transporter, the multidrug resistance-associated protein (MRP) . Accumulation of calcein fluorescence was greatly reduced in the MRP-overexpressing human lung cancer cell lines COR-L23/R and MOR/R compared with their parental lines . Energy depletion resulted in a considerably increased accumulation in the resistant lines . Treatment of resistant cells with buthionine sulfoximine (BSO), which depletes cellular glutathione (GSH), did not affect calcein accumulation, in marked contrast to our previous results for daunorubicin or the fluorescent probe rhodamine 123 . Genistein, verapamil, cyclosporin A and ouabain were also each able to modify, to some extent, accumulation of daunorubicin, whilst having essentially no effect on calcein accumulation . However, the organic anion transport inhibitor probenecid was able to increase accumulation of both calcein and daunorubicin in the resistant cells . Genistein and verapamil treatment preferentially reduced the GSH content of resistant cells, whilst probenecid did not . However, probenecid caused a clear decrease in release of GSH from resistant cells into the medium. Ecotoxicol Environ Saf, 1995 Dec, 32(3), 209 - 14 Formation of organic cadmium by marine microorganisms; Yannai S et al.; This research was designed with a view to finding out whether or not there is a microbial or spontaneous transformation of inorganic cadmium into a highly toxic organic derivative of this metal by microorganism obtained from the Eastern Mediterranean marine environment . Sterile and nonsterile marine bottom sediment samples were incubated at ambient temperature for different time intervals, under aerobic and anaerobic conditions, with different concentrations of CdCl2, and the medium, the atmosphere in the incubation flasks, and the sediments were assayed for their organic cadmium contents . The results were: (a) There was microbial growth in all systems containing up to 250 micrograms cadmium per milliliter of growth medium; however, with higher concentrations a complete growth-inhibition was observed . (b) Organic cadmium was found in the bottom sediments, at all cadmium levels permitting considerable microbial growth, but not in the systems' atmospheres or in the liquid growth media . (c) Under aerobic conditions higher levels of organic cadmium were found than in the anaerobic systems . (d) The microorganisms occurring in the different experimental systems were isolated and identified . The predominating species in all systems were bacteria. Poult Sci, 1995 Dec, 74(12), 1970 - 6 Avian hematopoiesis in response to avian cytokines; Nicolas-Bolnet C et al.; The objective of this study was to examine the hematopoietic cell proliferation and differentiation potential of growth factors produced by chicken macrophages . Bone marrow (BM) cells (25 x 10(3)) from newly hatched B15B15 K-strain Leghorn chicks were seeded in .5 mL serum-free semi-solid culture supplemented with 10% (vol/vol) of a conditioned medium (CM) from a chicken macrophage cell line, MQ-NCSU . The conditioned medium was obtained by culturing MQ-NCSU cells either in LM-HAHN (CMI) or RPMI-1640 (CMII) growth medium . The control cultures contained only LM-HAHN or RPMI medium . Bone marrow cells in the presence of CMI differentiated predominately into granulocyte colonies (Experiment 1 = 84 +/- 9.2; Experiment 2 = 105 +/- 5) . No colonies were observed in the control cultures . Stimulation of MQ-NCSU cells with lipopolysaccharide (LPS) produced a CM that differentiated BM cells predominantly into macrophage colonies (122 +/- 16.3 in CMI and 92 +/- 5.6 in CMII) . These data suggest that MQ-NCSU cells spontaneously secrete a factor with the potential to promote granulocyte differentiation . However, upon stimulation with LPS, the factor secreted had macrophage colony stimulation potential (M-CSF), which was similar in activity when compared with the activity of recombinant chicken myelomonocytic growth factor (r-cMGF) . Another CM from chicken fibroblasts (FCM) was tested on BM cells from K-strain Leghorns and Arbor Acres x Arbor Acres broiler chicks . Data from three experiments showed that 25 x 10(3) BM cells from K-strain chicken yielded more macrophage and granulocytes colonies (82 +/- 14) than those from broilers (56 +/- 12) . This study suggests that avian cytokines exhibit progenitor cell differentiation potential and that this activity is dependent upon the source of cytokines and their targets. Chem Res Toxicol, 1995 Dec, 8(8), 1046 - 53 1H NMR spectroscopic studies on the reactions of haloalkylamines with bicarbonate ions: formation of N-carbamates and 2-oxazolidones in cell culture media and blood plasma; Anthony ML et al.; 1H NMR spectroscopic methods have been applied to compare the in vitro reactivity of the renal papillary nephrotoxin 2-bromoethanamine (BEA) with those of selected halide-substituted nephrotoxic analogues, 2-chloroethanamine (CEA), 2-fluoroethanamine (FEA), and 1-phenyl-2-iodoethanamine (PIEA) . The primary 1H NMR-detectable transformation during a 24 h incubation of confluent Madin Darby canine kidney (MDCK) cells with BEA, CEA, and FEA (at concentrations up to the IC50 determined by neutral red uptake) was the appearance in cell culture media of 2-oxazolidone (OX) . Additional novel signals assigned as FEA carbamate (N-carbamoyl-2-fluoroethanamine) were observed in media collected following incubation of cells with FEA . We propose that N-carbamate intermediates are formed from the spontaneous reaction of these haloalkylamines with HCO(3-)-buffered growth media and that OX is formed from the carbamate via elimination of the hydrogen halide . Further 1H NMR experiments, conducted for up to 8 h at 25 degrees C on 5 mM solutions of BEA, CEA, and FEA in 2H2O containing a 20-fold excess of HCO3- at pH 7.6, demonstrated a time-dependent decrease in the concentration of the free haloalkylamines accompanied by the production of N-carbamate intermediates and OX . Under these pseudo-first-order reaction conditions, the formation of OX from BEA was complete within approximately 6 h . In similar reaction conditions OX formation from CEA (24 h after initiation) had reached 54% of its final equilibrium concentration . Equivalent experiments demonstrated that PIEA was almost completely converted to 4-phenyl-2-oxazolidinone (PHOX) within 2 h . These observations reveal the strong disposition of this series of haloalkylamines toward reaction with HCO3- and indicate that the compounds in this family may exist only transiently as free amines in vivo, where there will virtually always be excess HCO3- . The physiological relevance of the in vitro findings is further indicated by the NMR-detectable conversion of BEA to OX and also an alkylating aziridine (AZ) moiety in rat plasma containing BEA . The ability to form carbamoylated species and OX (or PHOX) may mediate the toxicity of this series of haloalkylamines and hence is potentially of considerable significance. J Neurosci Res, 1995 Dec, 42(5), 674 - 83 Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus; Brewer GJ; Two fundamental questions about neuron cell culture were addressed . Can one serum-free medium that was developed for optimum growth of hippocampal neurons support the growth of neurons from other regions of the brain? Is the region specific state of differentiation maintained in culture? To answer these questions, we isolated neurons from six other rat brain regions, placed them in culture in B27/Neurobasal defined medium, and analyzed their morphology and growth dependence on cell density after 4 days in culture . Neuronal identity was confirmed by immunostaining with antibodies to neurofilament 200 . Neurons from each brain region maintained distinctive morphologies in culture in the virtual absence of glia . Cells isolated from embryonic day 18 cerebral cortex by digestion with papain showed the same high survival as hippocampal neurons, e.g., 70% survival for cells plated at 160/mm2 . At this age and density, neurons from the septum showed slightly lower survival, 45% . Survival of dentate granule neurons from postnatal day four brains was 30-40%, significantly lower, and relatively independent of plating density . This suggests an absence of dependence on trophic factors or contact for dentate granule neurons . Growth of cerebellar granule neurons isolated from postnatal day 7, 8, or 9 brains in B27/Neurobasal was compared to growth in BME/10% serum . Viability in serum-free medium at 4 days was much better than that in serum, did not require KCl elevated to 25 mM, and occurred without substantial growth of glia . Cerebellar granule neurons plated at 1,280 cells/mm2 were maintained in culture for three weeks with 17% of the original cell density surviving . Survival of cells isolated from embryonic day 18 substantia nigra was 50% at 160 cells/mm2 after 4 days, similar to that of striatum, but slightly less than hippocampal neuron survival . The dopaminergic phenotype of the substantia nigral neurons was maintained over 2 weeks in culture as judged by immunoreactivity with antibodies to tyrosine hydroxylase . During this time, immunoreactivity was found in the processes as they grew out from the soma . Together, these studies suggest that B27/Neurobasal will be a useful medium for maintaining the differentiated growth of neurons from many brain regions . Potential applications of a common growth medium for different neurons are discussed. Protein Sci, 1995 Dec, 4(12), 2573 - 7 The effects of filtration on protein nucleation in different growth media; Hirschler J et al.; Filtration effects of turkey egg white lysozyme solution (TEWL) prior to subjecting it to crystallization conditions are investigated . Filtering TEWL solution and crystallizing it in ungelled media significantly decreased the number of conditions yielding crystals . This decrease dependent on the membrane cut-off used for filtration . From this, the postulated factors aiding in nucleation are estimated to be 0.17 microns in diameter . The existence of these factors was verified by the procedure of reversed filtration: filtered solutions passed through their inverted filter membrane a second time lead to improved crystallization results . The effect of aging of the TEWL solution prior to subjecting it to ungelled crystallization conditions was also verified . We did not find any time-dependent change in the size or the number of crystals per drop . Repeating the filtration experiments in agarose-gelled crystallization media showed that the influence of filtration on the crystallization outcome was significantly diminished . Far better crystallization results were obtained compared to ungelled media . However, there is a certain aging effect linked to filtration in gelled media . Different crystallization results were obtained depending on whether filtration was performed before or after aging and subsequent crystallization . This suggests a secondary time-dependent effect. Appl Microbiol Biotechnol, 1995 Dec, 44(1-2), 147 - 56 Efficient expression and secretion of Aspergillus niger RH5344 polygalacturonase in Saccharomyces cerevisiae; Lang C et al.; An Aspergillus niger endopolygalacturonase (EC 3.2.1.15) cDNA was expressed in the yeast Saccharomyces cerevisiae . Secretion of the protein into the growth medium was efficiently directed by the fungal leader sequence, and processing occurred at the same site as in Aspergillus . The expression level was significantly enhanced by using a "short" version of the yeast ADHI promoter . An additional increase in the yi