Concentrated Medium Research Articles

Water-independent effects of dietary restriction in Drosophila

Dietary restriction (DR) is a moderate reduction in food intake, without malnutrition, that extends the healthy life span in many organisms, including Drosophila (1). Although fruit flies have many practical advantages for studying DR, various technical complexities can have large effects on experimental outcomes (1). For DR, it is important that the basic food conditions are optimal. This ensures that increased longevity due to food restriction prolongs a healthy life span rather than returning sick animals to normal health by limiting access to a nutritionally inappropriate diet. We have systematically optimized our conditions for Drosophila DR, eliminated several non-nutritional explanations including water imbalance, and recommend a Brewer's-yeast-based diet (2, 3). Ja et al. (4) report contradictory data. Here, we present additional data in support of our conclusions and point out a flaw in the data concerning the Brewer's yeast diet presented by Ja et al. First, we developed a system that effectively hydrates flies under salt stress (Table 1, experiment 1). Adding 8 g·L−1 NaCl to our standard food [1.0 SYBrewer's (2)] shortened median life span by 24%. Adding to each vial a 200-μl pipette tip filled with water (1% agar) restored normal life span. The rescue was not due to the tip itself because an identical tip filled with dry cotton wool had no effect. Furthermore, addition of a tip containing wet cotton wool also restored normal life span. This was reproducible when the food was made from a different yeast [SYBaker's (2)] with salt added (Table 1, experiment 2). Thus, our technique is effective in delivering water to salt-stressed flies and in rescuing the associated shortening of the life span. Table 1. Providing a water source to flies rescues the life-shortening effect caused by addition of salt (NaCl) to food, but not life-span alterations due to dietary restriction Next, we established that the life-span change associated with DR was not rescued by water addition [Table 1, experiment 3; replicate of our published data (2)].These data demonstrate that DR in Drosophila under our conditions is not due to rescue of hydration stress. These results directly contradict data from an unreplicated experiment by Ja et al. using conditions ostensibly replicating ours, which reported that life-span extension by DR was eliminated by the addition of water, concluding that hydration stress explains DR (figure 2, G–I, in ref. 4). There are two problems with this conclusion. First, Ja et al. used more sugar (100 g·L−1) in their concentrated medium (CM) than did we, and this higher concentration causes a significant reduction in egg laying compared with the level we use (50 g·L−1) (2), perhaps because of water stress. [Furthermore, fly feeding behavior can be dramatically reduced as sugar concentration increases in this range, which may explain why Ja et al. observed lowered feeding in their CM (figure 1F in ref. 4) and we do not.] Second, the data in figure 2, G–I, from Ja et al. (4) demonstrate that water addition shortened DR life span to the level of the CM (Fig. 1). Thus the “rescue” of the DR effect by water addition could also be explained by water shortening the life span of DR flies, rather than increasing the life span of those on CM. Fig. 1. Life-span differences due to DR and water addition. Ja et al. (4) reported significant extension of life span when the concentration of nutrients in a Brewer's yeast diet were diluted (DR vs. CM) and that this difference was eliminated by the addition ... These data presented from our laboratory, both here and elsewhere (2, 3), robustly demonstrate that hydration stress does not explain DR under the conditions that we use. Furthermore, we have discovered that DR in Drosophila is mediated by an amino acid imbalance in the food that is not modified by water addition (3).

SCD4-17b bifunctional protein: extremely broad and potent neutralization of HIV-1 Env pseudotyped viruses from genetically diverse primary isolates.

BackgroundWe previously described a potent recombinant HIV-1 neutralizing protein, sCD4-17b, composed of soluble CD4 attached via a flexible polypeptide linker to an SCFv of the 17b human monoclonal antibody directed against the highly conserved CD4-induced bridging sheet of gp120 involved in coreceptor binding. The sCD4 moiety of the bifunctional protein binds to gp120 on free virions, thereby enabling the 17b SCFv moiety to bind and block the gp120/coreceptor interaction required for entry. The previous studies using the MAGI-CCR5 assay system indicated that sCD4-17b (in concentrated cell culture medium, or partially purified) potently neutralized several genetically diverse HIIV-1 primary isolates; however, at the concentrations tested it was ineffective against several other strains despite the conservation of binding sites for both CD4 and 17b. To address this puzzle, we designed variants of sCD4-17b with different linker lengths, and tested the neutralizing activities of the immunoaffinity purified proteins over a broader concentration range against a large number of genetically diverse HIV-1 primary isolates, using the TZM-bl Env pseudotype assay system. We also examined the sCD4-17b sensitivities of isogenic viruses generated from different producer cell types.ResultsWe observed that immunoaffinity purified sCD4-17b effectively neutralized HIV-1 pseudotypes, including those from HIV-1 isolates previously found to be relatively insensitive in the MAGI-CCR5 assay. The potencies were equivalent for the original construct and a variant with a longer linker, as observed with both pseudotype particles and infectious virions; by contrast, a construct with a linker too short to enable simultaneous binding of the sCD4 and 17b SCFv moieties was much less effective. sCD4-17b displayed potent neutralizing activity against 100% of nearly 4 dozen HIV-1 primary isolates from diverse genetic subtypes (clades A, B, C, D, F, and circulating recombinant forms AE and AG). The neutralization breadth and potency were superior to what have been reported for the broadly neutralizing monoclonal antibodies IgG b12, 2G12, 2F5, and 4E10. The activity of sCD4-17b was found to be similar against isogenic virus particles from infectious molecular clones derived either directly from the transfected producer cell line or after a single passage through PBMCs; this contrasted with the monoclonal antibodies, which were less potent against the PMBC-passaged viruses.ConclusionsThe results highlight the extremely potent and broad neutralizing activity of sCD4-17b against genetically diverse HIV-1 primary isolates. The bifunctional protein has potential applications for antiviral approaches to combat HIV infection.

Novel anti‐filamin‐A antibody detects a secreted variant of filamin‐A in plasma from patients with breast carcinoma and high‐grade astrocytoma

Identification of tumor-derived proteins in the circulation may allow for early detection of cancer and evaluation of therapeutic responses. To identify circulating tumor-derived proteins, mice were immunized with concentrated culture medium conditioned by human breast cancer cells. Antibodies generated by hybridomas were screened against conditioned media from both normal epithelial cells and tumor cells. Antibody selectively reacting with tumor cell-conditioned media was further characterized. This led to the development of a monoclonal antibody (Alper-p280) that reacts with a newly identified 280-kDa secreted variant of human filamin-A. Circulating filamin-A was detected in patient plasma samples using Alper-p280 in an ELISA assay. Human plasma samples from 134 patients with brain, breast, or ovarian cancer, 15 patients with active arthritis, and 76 healthy controls were analyzed. Filamin-A protein levels in human cell lines and tissues were analyzed by western blotting, immunohistochemistry, and electron and confocal microscopy. Circulating filamin-A was detected in the plasma of 109 of 143 patients with breast cancer and primary brain tumors. Plasma levels of filamin-A showed 89.5% sensitivity (95% confidence interval [CI] = 0.67% to 0.99%) and 97.8% specificity (95% CI = 0.88% to 0.99%) for glioblastoma at a cut-off of 21.0 ng/mL. Plasma levels of filamin-A (>36.0 ng/mL) had 96.7% sensitivity (95% CI = 0.80% to 0.99%) and 67.8% specificity (95% CI = 0.54% to 0.79%) for metastatic breast cancer. Filamin-A levels were increased in malignant breast or brain tissues, but not in normal control tissues. Filamin-A localized to lysosomes in MDA.MB.231 breast cancer cells, but not in normal human mammary epithelial cells, suggesting that filamin-A may undergo cancer-specific processing. Plasma filamin-A appears to be a specific and sensitive marker for patients with high-grade astrocytoma or metastatic breast cancer. Additional novel cancer biomarkers have been identified and are being developed alongside Alper-p280 for use in diagnosis of breast carcinoma and high-grade astrocytoma, and for use in the evaluation of therapeutic responses.

TheCryptococcus neoformans cap10 andcap59 mutant strains, affected in glucuronoxylomannan synthesis, differentially activate human dendritic cells

The human pathogen Cryptococcus neoformans causes meningo-encephalitis. The polysaccharide capsule is one of the main virulence factors and consists of two distinct polysaccharides: glucuronoxylomannan and galactoxylomannan. The presence of this polysaccharide capsule was previously shown to interfere with maturation of human dendritic cells (DCs), possibly by shielding cell-wall components from interacting with these host immune cells. Here we show that two mutant strains of C. neoformans, both lacking a visible capsule due to a defect in glucuronoxylomannan synthesis, differentially activate human monocyte-derived DCs. Cells from a cap59 mutant, but not of a cap10 mutant strain, induce maturation of DCs as indicated by an increase in the expression of the costimulatory molecules CD80 and CD86, and production of the cytokines interleukin (IL)-10, IL-12p40 and tumor necrosis factor alpha. Interestingly, cap59 mutant cells reassociated with a concentrated culture medium of wild-type C. neoformans had lost their capacity to induce DC maturation. Summarizing, our data suggest that glucuronoxylomannan confers properties to the capsule that protect the fungus against activation of DCs; however, the presence of intact glucuronoxylomannan is not an absolute requirement to prevent activation of DCs.

PRODUÇÃO IN VITRO DE MUDAS DE Cattleya walkeriana E Cyrtopodium palmifrons A PARTIR DE SEMENTES

Seeds of Cattleya walkeriana and Cyrtopodium palmjfrons ( Orchidaceae ) were aseptically transferred to culture medium Knudson, MS (Murashige& Skoog) and MS supplemented with IAA (l, 15 and 30 mg L -1 ) and kinetine (0.25, 5 and 10 mg L -1 ) for germination. They were kept at room temperature (23 +/- 2°C) under fluorescent light for 30 to 45 days. The seedlings were transferred to pots containing culture medium Knudson and incubated for four months at room temperature and transferred to fresh culture medium until the seedlings attained approximately 1.0 to 2.0 cm size for climatization. The in vitro cultivation of both plant species was considered viable. However the performance of the two orchids differed in relation to protocorms oxidation levels and in vitro growth. The results showed that less concentrated culture medium such as Knudson can be utilized , without the addition of growth regulators for the production of Cattleya walkeriana as well as Cyrtopodium palmifrons . KEY-WORDS: Orchidaceae ; tissue culture; orchid.

Soluble Interleukin (IL)-15Rα Is Generated by Alternative Splicing or Proteolytic Cleavage and Forms Functional Complexes with IL-15

Interleukin 15 (IL-15) is a pleiotropic cytokine that is hardly detectable in biological fluids. Here, we show that IL-15 forms functional heterocomplexes with soluble high affinity IL-15 receptor alpha (IL-15Ralpha) chain in mouse serum and cell-conditioned medium, which prevents IL-15 detection by ELISA. We also demonstrate that two soluble IL-15Ralpha (sIL-15Ralpha) sushi domain isoforms are generated through a novel alternative splicing mechanism within the IL-15Ralpha gene. These isoforms potentiate IL-15 action by promoting the IL-15-mediated proliferation of the CTLL cell line and interferon gamma production by murine NK cells, which suggests a role in IL-15 transpresentation. Conversely, a full-length sIL-15Ralpha ectodomain released by tumor necrosis factor-alpha-converting enzyme (TACE)-dependent proteolysis inhibits IL-15 activity. Thus, a dual mechanism of sIL-15Ralpha generation exists in mice, giving rise to polypeptides with distinct properties, which regulate IL-15 function.

Biotransformation of 2,4,6-Trinitrotoluene (TNT) by the Fungus Fusarium Oxysporum

The fungus Fusarium oxysporum was isolated and identified from the aquatic plant M. aquaticum. The capability of this fungus to transform 2,4,6-trinitrotoluene (TNT) in liquid cultures was investigated. TNT was added to shake flask cultures and transformed into 2-amino-4,6-dinitrotoluene (2-A-DNT), 4-amino-2,6-dinitrotoluene (4-A-DNT), and 2,4-diamino-6-nitrotoluene (2,4-DAT) via 2- and 4-hydroxylamino-dinitrotoluene derivatives, which could be detected as intermediate metabolites. Transformation of TNT, 2-A-DNT, and 4-A-DNT was observed by whole cultures and with isolated mycelium. Cell-free protein extracts from the extracellular, soluble, and membrane-bound fractions were prepared from this fungus and tested for TNT-reducing activity. The concentrated extracellular culture medium was unable to transform TNT; however, low levels of TNT transformation were observed by the membrane fraction in the presence of nicotinamide adenine dinucleotide phosphate in an argon atmosphere. A concentrated extract of soluble enzymes also transformed TNT, but to a lesser extent. When TNT toxicity was studied with this fungus, a 50% decrease in the growth of F. oxysporum mycelium was observed when exposed to 20 mg/L TNT.

Collagenase-2 and -3 Mediate Epidermal Growth Factor Receptor Transactivation by Bradykinin B2Receptor in Kidney Cells

We have previously shown that stimulation of extracellular signal-regulated protein kinase (ERK) by bradykinin (BK) in murine inner medullary collecting duct (mIMCD)-3 cells is mediated by epidermal growth factor receptor (EGFR) transactivation. The mechanism of EGFR transactivation seemed to be novel, because it does not require phospholipase C, Ca(2+), calmodulin, protein kinase C, G alpha(i) subunits, or EGFR-B(2) receptor heterodimerization. In this study, we demonstrated the involvement of matrix metalloproteinases (MMPs) in B(2) receptor-induced EGFR transactivation using their broad-spectrum inhibitors batimastat and N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide (Galardin) (GM-6001). Selective inhibitors for collagenase-2 and -3 (MMP-8 and MMP-13, respectively) blocked BK-induced EGFR phosphorylation and ERK activation, whereas inhibitors for MMP-1, -2, -3, -7, or -9 were without effect. Transfection of mIMCD-3 cells with MMP-8 small interfering RNA (siRNA) resulted in approximately 50% decrease of BK-induced ERK activation. A neutralizing antibody against MMP-13 as well as transfection with MMP-13 siRNA produced a similar effect. Inhibition of both collagenases resulted in approximately 65% decrease of BK-induced ERK activation, supporting roles for both enzymes. Stimulation of mIMCD-3 cells with 10 nM BK increased the activity of collagenases in concentrated culture media within 10 min. Moreover, recombinant MMP-13 and MMP-8, when applied to mIMCD-3 cells for 10 min without BK, stimulated tyrosine phosphorylation of EGFR and caused approximately 250% increase over basal ERK phosphorylation comparable with BK-induced ERK activation. Collagenases-induced ERK activation was inhibited by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478) and thus dependent on EGFR tyrosine kinase activity. This study demonstrates a novel role for collagenase-2 and -3 in signaling of the G(q)-coupled BK B(2) receptor in mIMCD-3 cells.

N-terminal peptide of Rhizopus oryzae lipase is important for its catalytic properties

In a culture medium, the Rhizopus oryzae strain produces only one form of lipase, ROL32. When the concentrated culture medium was stored at 0°C during several months or kept at 6°C during a few days, we noticed the appearance of a second shorter form of ROL32 lacking its N-terminal 28 amino acid (ROL29). ROL29 was purified to homogeneity and its 21 N-terminal amino acid residues were found to be identical to the 29–49 sequence of ROL32. The cleavage of the N-terminal peptide reduced the specific activity of ROL29 by 50% using either triolein or tributyrin as substrates. In order to explain this decrease of the specific activity of ROL29, we measured its critical surface pressure of penetration into phosphatidyl choline from egg yolk films which was found to be 10mN/m, in contrast to a value of 23mN/m found in ROL32. A kinetic study on the surface pressure dependency, stereoselectivity and regioselectivity of ROL29 was performed using the three dicaprin isomers spread as monomolecular films at the air–water interface. Our results showed that in contrast to ROL32, ROL29 presented a preference for the distal ester groups of one diglyceride isomer (1,3-sn-dicaprin). Furthermore, ROL32 was markedly more stereoselective than ROL29 for the sn-3 position of the 2,3-sn-enantiomer of dicaprin. A structural explanation of the enhanced penetration capacity as well as the catalytic activity of ROL32 was proposed by molecular modeling. We concluded that the N-terminal peptide of ROL32 can play an important role in the specific activity, the regioselectivity, the stereoselectivity and the binding of the enzyme to its substrate.

A Study of Microbiologically Induced Corrosion by Sulfate-Reducing Bacteria on Carbon Steel Using Hydrogen Permeation

Abstract The mechanism of microbiologically influenced corrosion (MIC) on carbon steel (CS) by the bacteria Desulfovibrio desulfuricans subs. desulfuricans was studied using hydrogen permeation, open-circuit potential, and cathodic polarization techniques, in a concentrated culture medium containing bacteria cells (107 cell/mL) and ferrous ions (300 mg/L) designed to simulate a condition common in systems for the secondary recovery of crude oil, characterized by highly contaminated microenvironments that severely corrode iron alloys in a short time period. This research project was carried out using several 24-h experiments to define initial stages of the corrosive process under the conditions indicated. The results evidenced a hydrogen permeation current peak of about 12 μA correlated with a minimum open-circuit potential of −780 mV vs saturated calomel electrode (SCE), 400 min after inoculation. Next, the permeation current decreased abruptly to its base line and the potential increased, stabilizing at ...

Temperature-induced change of variant surface antigen expression in Paramecium involves antigen release into the culture medium with considerable delay between transcription and surface expression.

We analyzed temperature-induced changes of variant surface antigen (vsAg) expression in Paramecium primaurelia, using immuno-techniques and mRNA determinations. Upon a 23 degrees C to 33 degrees C shift, the old vsAg, type 156G, remains on the cell surface for a time, when already mRNA for the new form, 156D, is expressed. A considerable amount of 156D-specific mRNA is formed 45-48 h after the temperature shift, while 156D surface expression reaches maximal levels only after >72 h. A new aspect of these experiments is that, during this transition, the old vsAg is steadily released in high-molecular-weight form into the culture medium, as found by dot blot and Western blot analysis of concentrated culture medium. The new vsAg form is first inserted into the somatic cell membrane, before it spreads also into cilia. In the reverse transition, 33 degrees C to 23 degrees C, the adaptation on the level of transcription and surface expression is considerably faster. While we had previously shown, under steady-state conditions (constant temperature), the occurrence of a degradation pathway by endocytotic and phagocytotic ingestion of vsAg this may proceed in parallel to the steady release of old vsAg from the cell surface into the medium. Altogether these combined processes may facilitate the installation of the new vsAg type.

Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre-elafin (trappin-2) expressed in Pichia pastoris.

Elafin and its precursor, trappin-2 or pre-elafin, are specific endogenous inhibitors of human neutrophil elastase and proteinase 3 but not of cathepsin G. Both inhibitors belong, together with secretory leukocyte protease inhibitor, to the chelonianin family of canonical protease inhibitors of serine proteases. A cDNA coding either elafin or its precursor, trappin-2, was fused in frame with yeast alpha-factor cDNA and expressed in the Pichia pastoris yeast expression system. Full-length elafin or full-length trappin-2 were secreted into the culture medium with high yield, indicating correct processing of the fusion proteins by the yeast KEX2 signal peptidase. Both recombinant inhibitors were purified to homogeneity from concentrated culture medium by one-step cationic exchange chromatography and characterized by N-terminal amino acid sequencing, Western blot and kinetic studies. Both recombinant elafin and trappin-2 were found to be fast-acting inhibitors of pancreatic elastase, neutrophil elastase and proteinase 3 with k(ass) values of 2-4 x 10(6) m(-1).s(-1), while dissociation rate constants k(diss) were found to be in the 10(-4) s(-1) range, indicating low reversibility of the complexes. The equilibrium dissociation constant K(i) for the interaction of both recombinant inhibitors with their target enzymes was either directly measured for pancreatic elastase or calculated from k(ass) and k(diss) values for neutrophil elastase and proteinase 3. K(i) values were found to be in the 10(-10) molar range and virtually identical for both inhibitors. Based on the kinetic parameters determined here, it may be concluded that both recombinant elafin and trappin-2 may act as potent anti-inflammatory molecules and may be of therapeutic potential in the treatment of various inflammatory lung diseases.

Matrix Metalloproteinases 2 and 9 Mediate Epidermal Growth Factor Receptor Transactivation by Gonadotropin-releasing Hormone

Rapid engagement of the extracellular signal-regulated kinase (ERK) cascade via the Gq/11-coupled GnRH receptor (GnRHR) is mediated by transactivation of the epidermal growth factor receptor (EGFR). Here we show that the cross-talk between GnRHR and EGFR in gonadotropic cells is accomplished via gelatinases A and B (matrix metalloproteinases (MMPs) 2 and 9), identifying gelatinases as the first distinct members of the MMP family mediating EGFR transactivation by G protein-coupled receptors. Using a specific MMP2 and MMP9 inhibitor, Ro28-2653, GnRH-dependent EGFR transactivation was abrogated. Proving the specificity of the effect, transient transfection of alphaT3-1 cells with ribozymes directed against MMP2 or MMP9 specifically blocked EGFR tyrosine phosphorylation in response to GnRH stimulation. GnRH challenge of alphaT3-1 cells furthered the release of active MMP2 and MMP9 and increased their gelatinolytic activities within 5 min. Rapid release of activated MMP2 or MMP9 was inhibited by ribozyme-targeted down-regulation of MT1-MMP or MMP2, respectively. We found that GnRH-induced Src, Ras, and ERK activation were also gelatinase-dependent. Thus, gelatinase-induced EGFR transactivation was required to engage the extracellular-signal regulated kinase cascade. Activation of c-Jun N-terminal kinase and p38 MAPK by GnRH was unaffected by EGFR or gelatinase inhibition that, however, suppressed GnRH induction of c-Jun and c-Fos. Our findings suggest a novel role for gelatinases in the endocrine regulation of pituitary gonadotropes.

Expression, purification and characterization of the monomeric and dimeric forms of soluble bovine endothelin converting enzyme-1a.

In this study, the catalytic domain of bovine endothelin converting enzyme-1a (ECE-1a) was cloned into a baculovirus transfer vector behind the human alkaline phosphatase signal sequence. The recombinant baculovirus was then used to infect High Five(TM) insect cells in suspension culture. Both the monomeric (85 kDa) and dimeric (170 kDa) forms of soluble ECE-1a were purified to electrophoretic homogeneity from concentrated culture media following sequential concanavalin A, SP-Sepharose, Mono Q and gel filtration column chromatography. Typically, approximately 11 mg of ECE-1a monomer and 6 mg of dimer were obtained from l litre of culture medium. No interconversion of the two forms was detected after purification. Both forms of ECE-1a had a pH optimum of 7.0, were maximally stimulated by NaCl at a concentration of 500 mM, and were inhibited to the same extent by metalloprotease inhibitors such as phosphoramidon and EDTA. However, in kinetic studies using big endothelin-1 (ET-1) as a substrate, the K(m) and k(cat) values for the monomer were 2.2 microM and 1.6 min(-1) respectively, while those of the dimer were 1.4 microM and 4.9 min(-1) respectively. These results show that, although the two forms of ECE-1a behave similarly in many aspects, the dimeric enzyme is more efficient in catalysing the conversion of big ET-1 to ET-1. The present protocol can be utilized to prepare large quantities of both forms of ECE-1a for further biochemical and structural characterization.

Production of full-length human pre-elafin, an elastase specific inhibitor, from yeast requires the absence of a functional yapsin 1 (Yps1p) endoprotease.

Pre-elafin, also known as trappin-2, is an elastase-specific inhibitor that belongs to the trappin gene family. A chimeric gene encoding polyhistidine-tagged human pre-elafin fused to the yeast alpha-factor precursor was expressed in Saccharomyces cerevisiae. The chimera was engineered to keep a single copy of the mature alpha-factor peptide. This enabled the use of a simple bioassay (mating assay) to assess the relative efficiency of both the expression and the secretion of the recombinant molecule. We found that pre-elafin is processed both in vivo and in vitro by yapsin 1, the yeast aspartyl endoprotease encoded by YPS1. Cleavage by yapsin 1 occurred C-terminal to a subset of single lysine residues. Expression in a yapsin 1-deficient yeast strain was an indispensable condition to allow the efficient production of full-length human pre-elafin. The recombinant inhibitor was purified from concentrated culture medium by ammonium sulfate precipitation, affinity purification on a Ni(2+) resin, and cation exchange chromatography. Recombinant human pre-elafin was fully active and showed the same inhibitory profile toward different serine proteases to that reported for mature elafin.

Development of a rapid and sensitive bioassay device using human cells immobilized in macroporous microcarriers for the on-site evaluation of environmental waters.

We developed a novel disposable bioassay device based on the fluorescein isothiocyanate-labelled low-density lipoprotein-uptake activity of human hepatoblastoma Hep G2 cells. The cells were cultured in porous microcarriers at a high cell density and packed in a filter tip that has a hydrophobic membrane. Upon evaluation of water samples, the culture medium was decanted by pipetting it down with a micropipet, and the samples were then introduced to the cell-immobilizing part of the tip only by pipetting them up after mixing them with x10 concentrated culture medium. The new device enabled us to detect almost the same toxicity levels of river water within 2 h of exposure as those detected by a conventional 48-h cell-survival assay. This is the first bioassay device for the rapid on-site evaluation of environmental waters using cultured human cells, and therefore promising for water-quality management based on risk to humans.

Immobilized 4-aminophenyl 1-thio-β- d-galactofuranoside as a matrix for affinity purification of an exo-β- d-galactofuranosidase

An alternative and fast method for the purification of an exo-β- d-galactofuranosidase has been developed using a 4-aminophenyl 1-thio-β- d-galactofuranoside affinity chromatography system and specific elution with 10 mM d-galactono-1,4-lactone in a salt gradient. A concentrated culture medium from Penicillium fellutanum was chromatographed on DEAE–Sepharose CL 6B followed by chromatography on the affinity column, yielding two separate peaks of enzyme activity when elution was performed with 10 mM d-galactono-1,4-lactone in a 100–500 mM NaCl salt gradient. Both peaks behaved as a single 70 kDa protein, as detected by SDS-PAGE. Antibodies elicited against a mixture of the single bands excised from the gel were capable of immunoprecipitating 0.2 units out of 0.26 total units of the enzyme from a crude extract. The glycoprotein nature of the exo-β- d-galactofuranosidase was ascertained through binding to Concanavalin A–Sepharose as well as by specific reaction with Schiff reagent in Western blots. The purified enzyme has an optimum acidic pH (between 3 and 6), and K m and V max values of 0.311 mM and 17 μmol h −1 μg −1 respectively, when 4-nitrophenyl β- d-galactofuranoside was employed as the substrate.

Different interactions of phycoerythrin- and phycocyanin-rich Synechococcus spp. with diazotrophic bacteria from the picoplankton of Lake Constance

The picoplankton (PP) from the pelagic zone of Lake Constance comprises photoautotrophic picocyanobacteria of the Synechococcus type and heterotrophs including nitrogen-fixing purple bacteria. Phycocyanin (PC)-rich and phycoerythrin (PE)-rich Synechococcus spp. isolated from this ecosystem responded differently to N-deprivation. The PC-rich strains BO8801, BO8805 and BO8806 exhibited a rapid decrease of phycobiliprotein to chlorophyll-a ratio. Concomittantly, an accumulation of cellular carbohydrates as well as enhanced concentrations of released extracellular polysaccharides were observed. Addition of the concentrated cell-free culture medium from the N-deprived PC-rich strain BO8806 increased the light-induced nitrogenase activity of picoplankton fractions enriched in heterotrophic bacteria including diazotrophic ones from the pelagic zone (HPP). In contrast, the PE-rich strains BO8807, BO8808 and BO8809 did not respond to N-deprivation in pigment composition and no significant accumulation of polysaccharides was observed. Accordingly, no support of bacterial light-induced nitrogenase activity was found when concentrated culture medium of the N-deprived PE-rich isolate BO8807 was applied. This indicates that, different from the PE-rich isolates, the PC-rich strains released compounds which were metabolized by diazotrophic phototrophs present in the HPP. Weekly examination during one year of picoplankton fractions of Lake Constance for the capability to reduce acetylene revealed the occurrence of an enhanced light-induced nitrogenase activity profile concurrent with the spring bloom of picocyanobacteria. These findings suggest that PC-rich picocyanobacteria may contribute to the C-supply of diazotrophic bacteria in the natural environment.

PROTEIN RETENTION IN THE ENDOPLASMIC RETICULUM OF INSECT CELLS IS NOT COMPROMISED BY BACULOVIRUS INFECTION

High level expression of the major auxin-binding protein (ABP1) from maize ( Zea maysL.) has been used to demonstrate that the machinery for retaining proteins in the endoplasmic reticulum (ER) of insect cells functions efficiently throughout the baculovirus infection cycle. Immuno-localization showed wild-type ABP1 (ABP1-KDEL) to be targeted to the lumen of the ER, in accordance with its signal peptide and carboxyterminal KDEL ER-retention signal. The protein accumulated in dilations of the ER, and none was detected at the cell surface. Immunoblotting of concentrated culture medium confirmed that ABP1-KDEL was not secreted at a detectable level. In contrast, when the carboxyterminus was mutated to KEQL, secretion of the baculovirus-expressed protein was readily detected. Immunolocalization and immunoblotting demonstrated that a high proportion of the ABP1-KEQL protein was secreted at the cell surface and into the culture medium. The data demonstrate that the ER of insect cells has a great capacity to retain proteins and that this property is largely unaffected by the cellular disruption caused by baculovirus replication.

Purification and characterization of an extracellular alpha-amylase from Clostridium perfringens type A.

An alpha-amylase (EC 3.2.1.1) secreted by Clostridium perfringens NCTC 8679 type A was purified to homogeneity and characterized. It was isolated from concentrated cell-free culture medium by ion-exchange and gel permeation chromatography. The enzyme exhibited maximal activity at pH 6.5 and 30 degrees C without the presence of calcium. The pI of the enzyme was 4.75. The estimated molecular weight of the purified enzyme was 76 kDa. The purified enzyme was inactivated between 35 and 40 degrees C, which increased to between 45 and 50 degrees C in the presence of calcium (5 mM). The purified enzyme produced a mixture of oligosaccharides as major end products of starch hydrolysis, indicating alpha-amylase activity.