Conjugation of Arg-Gly-Asp (RGD) Sequence in Copolymer Bearing Sugar Moiety for Insulinoma Cell Line (MIN6) Culture

Copolymers composed of an Arg-Gly-Asp (RGD) sequence for the adhesion molecule and sugar moieties were synthesized for an insulinoma cell (MIN6) culture. MIN6 cells attached on the poly(N-p-vinylbenzyl-D-maltonamide-co-6-(p-vinylbenzamido)-hexanoic acid-g-GRGDS) (p(VMA-co-VBGRGDS))-coated dishes were in a more aggregated form than other polymer-coated surfaces. P(VMA-co-VBGRGDS) also shows faster proliferation of MIN6 cells (about 18% higher) than with p(VLA-co-VBGRGDS). By interaction between cell and matrix, about 80% greater insulin secretion from MIN6 cells was produced with the p(VMA-co-VBGRGDS), and about 50% greater insulin secretion was produced with the poly(N-p-vinylbenzyl-D-lactonamide-co-6-(p-vinylbenzamido)-hexanoic acid-g-GRGDS) (p(VLA-co-VBGRGDS) as compared with unstimulated cells. Moreover, attachment of MIN6 cells treated with RGD monomer was suppressed approximately 50% for the p(VMA-co-VBGRGDS) surface. This result supported the idea that conjugation of adhesion molecules of RGD peptide in p(VMA-co-VBGRGDS) copolymer specifically interact with integrin families on MIN6 cell membrane.     

Conjugation of Arg-Gly-Asp (RGD) sequence in copolymer bearing sugar moiety for insulinoma cell line (MIN6) culture

Copolymers composed of an Arg-Gly-Asp (RGD) sequence for the adhesion molecule and sugar moieties were synthesized for an insulinoma cell (MIN6) culture. MIN6 cells attached on the poly(N-p-vinylbenzyl-D-maltonamide-co-6-(p-vinylbenzamido)-hexanoic acid-g-GRGDS) (p(VMA-co-VBGRGDS))-coated dishes were in a more aggregated form than other polymer-coated surfaces. P(VMA-co-VBGRGDS) also shows faster proliferation of MIN6 cells (about 18% higher) than with p(VLA-co-VBGRGDS). By interaction between cell and matrix, about 80% greater insulin secretion from MIN6 cells was produced with the p(VMA-co-VBGRGDS), and about 50% greater insulin secretion was produced with the poly(N-p-vinylbenzyl-D-lactonamide-co-6-(p-vinylbenzamido)-hexanoic acid-g-GRGDS) (p(VLA-co-VBGRGDS) as compared with unstimulated cells. Moreover, attachment of MIN6 cells treated with RGD monomer was suppressed approximately 50% for the p(VMA-co-VBGRGDS) surface. This result supported the idea that conjugation of adhesion molecules of RGD peptide in p(VMA-co-VBGRGDS) copolymer specifically interact with integrin families on MIN6 cell membrane.     

Esterase activity assay by flow injection analysis (FIA)

An automatic flow injection analysis (FIA) system for on-line determination of esterase activity has been developed. It is based on a colorimetric method using p-nitrophenyl propionate as substrate. The system permits a linear range analysis up to 0.18 U ml^–1, although the range can be extended up to 1 U ml^–1 without external dilution of the sample. The sampling frequency is of 4 samples per h with a relative standard deviation of 0.9%.     

Pulmonary diffusing capacity in reptiles (Relations to temperature and O2-uptake)

Summary Pulmonary CO-diffusing capacity (D l _CO), lung volume, pulmonary perfusion and O₂-uptake were measured by non-invasive techniques in the lizardsVaranus exanthematicus andTupinambis teguixin (mean body weight 2.2 kg for both species).The CO-diffusing capacity was at 25–27°C 0.059 mlstpd·kg^–1·min^–1·Torr^–1 inVaranus, which is 47% greater than the value of 0.040 mlstpd·kg^–1·min^–1·Torr^–1 inTupinambis. The lung volume ofVaranus was 36 ml·kg^–1 and that ofTupinambis 20 ml·kg^–1. At 35–37°C the diffusing capacity of lizard lungs are about 25% of those for mammals of comparable size.InVaranus pulmonary CO-diffusing capacity increased with temperature from 0.027 mlstpd·kg^–1·min^–1·Torr^–1 at 17–19 °C to 0.075 mlstpd·kg^–1·min^–1·Torr^–1 at 35–37 °C. This change closely matched a concomitant increase of O₂-uptake. Pulmonary perfusion increased from 27 ml·kg^–1·min^–1 to 55 ml·kg^–1·min^–1 within this temperature range.The study emphasizes that pulmonary diffusing capacity cannot be fully evaluated without information on pulmonary perfusion and O₂-uptake. In reptiles and other ectotherms diffusing capacity must be reported at specified body temperature.     

Demography and population growth of Asplanchna girodi (Rotifera) as a function of prey (Anuraeopsis fissa) density

Laboratory studies on population growth and life table demography of Asplanchna girodi were conducted at 25±1 °c using Anuraeopsis fissa as prey at four (250, 500, 1000 and 2000 ind ml^–1) densities. A prey density of 100 ind ml^–1 per predator per day did not support A. girodi, while at the highest prey concentration, A. girodi reached a peak of 115±7 ind ml^–1. The age distribution of A. girodi indicated that non-adults constituted about 2/3rd of the population at all prey concentrations. A decrease in prey availability resulted in increased mortality of non-adults. At the highest prey density, the rate of population increase (r) was 1.51 d^–1. The significance of estimating mortality in population growth studies is discussed.Life table demography of A. girodi was also studied using the same prey at the same concentrations. None of the survivorship parameters (e.g. mean lifespan and mean survivorship) showed a significant relation to prey density. Net reproductive rate and generation time (but not rate of population increase) were affected by prey abundance.     

Selection of a support for immobilization of a microbial lipase for the hydrolysis of triglycerides

Baterial lipase from Staphylococcus carnosus (pLipMut2) has been immobilized on various supports in order to determine a suitable immobilization technique in terms of activity and stability, when utilized for the hydrolysis of tributyrin. The hydrophobic materials PBA Eupergit and PBA Eupergit 250L prooved to be appropriate supports, when the enzyme was crosslinked with glutaraldehyde after adsorption. No desorption of the immobilized enzyme occured during operation. The pore size of the support has a strong effect on the activity but does not influence stability.The initial activity for immobilized and soluble lipase is found to follow the Arrhenius equation at low temperature, where mass transfer does not affect reaction kinetics. Activation energies for soluble and immobilized lipase were evaluated to be 21.7 kJ mol^–1 and 60.8 kJ mol^–1, respectively.Operational stability was studied in a packed bed recirculation reactor. Thermal desactivation followed first order kinetics with a half-life of 1340 h at 10°C. Model calculations for productivity showed, that optimal temperatures for high productivity are well below the temperature of maximal activity.List of Symbols E _a [kJ mol^–1] activation energy - E _d [kJ mol^–1] activation energy of desactivation - H [–] half-number - k _d [h^–1] desactivation constant - k _d, [h^–1] constant - k _N [–] desactivation constant (number) - N [–] number of runs - p [mol dm^–3] productivity - t [h] time - t _0.5 [h] half-life - T [K] absolute temperature - V [U ml^–1] activity - V(N) [Uml^–1] activity exhibited in the n-th run - V _s,O [U ml^–1] initial activity of supernatant - V _s, [U ml^–1] activity of supernatant after immobilization - V _O [U ml^–1] initial activity - V [U ml^–1] constant - _imm [–] activity yield - [ml ml^–1] ratio of volume of support to volume of supernatant Financial support of this work by the Deutsche Forschungsgemeinschaft (SFB 145, A15) is gratefully acknowledged.     

Callus and suspension cultures for biomass production ofCynara cardunculus (Compositae)

Summary Friable calli, obtained from hypocotyl and leaf segments ofCynara cardunculus, were used for the production of cell suspension cultures in liquid Gamborg B₅ medium supplemented as for callus obtention. The low inoculum cell suspension cultures (6.9×10⁴ cells.ml^–1) presented a td=136.8 hours while those obtained with a high inoculum (15.6×10⁴ cells.ml^–1) reached a td=33.6 hours.     

Specific combinations of human serum albumin introns direct high level expression of albumin in transfected COS cells and in the milk of transgenic mice

A new series of expression vectors, each comprised of the -lactoglobulin (BLG) promoter driving one of a variety of human serum albumin (HSA) minigenes or the entire gene, were evaluated for their ability to direct expression of HSAin vitro in COS tissue culture cells and into the milk of transgenic mice. Vectors directed a hierarchy of expression levelsin vitro, dependent upon the specific complement of HSA introns included. HSA introns acted in a synergistic manner. In addition, minigenes comprised of specific subsets of introns were more efficacious than the entire HSA gene with all of its introns. Transgenic mice expressed as much as 10 mg ml^–1 of HSA in their milk. Vectors comprised of specific intron subsets directed levels at 1 mg ml^–1 or greater in the milk of 20% of generated transgenics. A statistical correlation between the expression level trendin vitro with the trend of expressionin vivo (% which express) at detectable levels (p=0.0015) and at the level of greater than 0.1 mg ml^–1 (p=0.0156) was demonstrated. A weak correlation existed (p=0.0526) atin vivo levels of 1 mg ml^–1 or greater. These new vectors are expected to direct the production of high levels of HSA in the milk of a large percentage of generated transgenic dairy animals.     

Effect of Aeolosoma sp. (Aphanoneura: Aeolosomatidae) on the Population Dynamics of Selected Cladoceran Species

Although oligochaete worms naturally coexist with cladocerans in many shallow freshwater ponds and lakes, their influence on the latter is not well established. In this work we studied the effect of Aeolosoma sp. on the population growth of Alona rectangula, Ceriodaphnia dubia, Daphnia pulex, Macrothrix triserialis and Moina macrocopa. Population growth studies were conducted at one algal food density (1 × 10⁶cells ml^–1 of Chlorella vulgaris). The experimental design was similar for all five cladoceran species, where we used 100 ml capacity transparent jars containing 50 ml of EPA medium with the desired algal density and three replicates for each treatment. The test medium was changed daily and fresh algal food was added. The initial density of each of the cladoceran species in the population growth studies was 0.4 ind ml^–1 while that of the worms 1.0 ind ml^–1. Following inoculation, we estimated daily the number of cladocerans and the worms for duration of 21 days. Regardless of the presence of worms, Moina macrocopa and Macrothrix triserialis showed rapid population growth while A. rectangula took more than 2 weeks to reach peak abundances. With the exception of M. triserialis, all the other our cladoceran species declined in the presence of Aeolosoma sp. The lowest peak population density (about 1 ind ml^–1) was observed for M. triserialisin controls. The remaining species had peak densities of about 3–5 ind ml^–1. The rates of population increase per day varied from 0.03 to 0.19 depending on the cladoceran taxa and the treatment. In general we found that pelagic taxa were more adversely affected by the presence of the worms than were the littoral cladocerans.     

The role of phototrophic sulfur bacteria as food for meiobenthic harpacticoid copepods inhabiting eutrophic coastal lagoons

Laboratory experiments were undertaken using Amonardia normani and Schizopera cf. compacta, two meiobenthic harpacticoid copepods commonly found in coastal lagoons. The first experiments were designed to determine if the phototrophic sulfur bacteria Chromatium gracile can be ingested by these copepods and at what concentrations. Egestion rate was used as an index of feeding rate. The response of the egestion rate, expressed in numbers of faecal pellets produced by copepod per day, as a function of bacterial concentration followed the functional model. A. normani attained constant feeding rates from the bacterial concentration of 1 × 10⁷ cells ml^–1 (5 µg C ml^–1) onwards, S. cf. compacta attained constant feeding rates from 2.6 × 10⁷ cells ml^–1 (13 µg C ml^–1) onwards. The faecal pellet volume changed significantly (p<0.05) between food concentrations for A. normani but not for S. cf. compacta (p>0.05). In order to investigate the effect of the phototrophic bacterial diet on the population dynamics of A. normani three groups of nauplii were maintained at 2 × 10⁷ cells ml^–1 and observed every day. The mortality of these nauplii was very high compared to those maintained on a diatom diet (Nitzschia constricta); only in one of the groups did some copepodites develop but no adults were ever observed. Adults fed on bacteria did not have different (p>0.05) survival rates compared to those fed on diatoms, nevertheless, the number of nauplii produced was significantly less (p<0.05) on the bacterial diet. These results lead us to suggest that although the phototrophic sulfur bacteria (Chromatium gracile) can be ingested by both copepod species it cannot sustain the full development of the A. normani population. Thus, a bloom of phototrophic sulfur bacteria does not seem to be a favourable situation for opportunistic benthic copepods to colonize eutrophic coastal lagoons after a dystrophic crisis.     

Separation and simultaneous determination of nalidixic acid, hydroxynalidixic acid and carboxynalidixic acid in serum and urine by micellar electrokinetic capillary chromatography

Separation in capillary electrophoresis is governed by various factors, including buffer type, buffer concentration, pH, temperature, voltage and micelles. Through proper adjustment of these parameters, nalidixic acid and its two major metabolites, 7-hydroxynalidixic and 7-carboxynalidixic, could be separated by micellar electrokinetic capillary chromatography using an electrophoretic electrolyte consisting of 50 mM borate buffer (pH 9) containing 25 mM sodium dodecyl sulphate and 10% acetonitrile. A linear relationship between concentration and peak area for each compound was obtained in the concentration range 0.15–100 μg ml⁻¹, with a correlation coefficient greater than 0.999 and detection limits in the 0.2–0.7 ng ml⁻¹ range. Intra- and inter-day precision values of about 0.8–1.2% RSD (n=11) and 1.3–2.0% RSD (n=30), respectively, were obtained. The method has been applied to the analysis of nalidixic acid and its two major metabolites in serum and urine with limits of sensitivity lower than 0.8 ng ml⁻¹.     

Bacterial response to extracellular dissolved organic carbon released from healthy and senescent Fragilaria crotonensis (Bacillariophyceae) in experimental systems

Responses of bacteria to dissolved organic carbon (DOC) released from healthy and senescent Fragilaria crotonensis (Bacillariophyceae) were examined in experimental systems. The alga released DOC actively, although the concentration fluctuated greatly in both the axenic (the alga alone) and the mixed (the alga plus the enriched bacteria) cultures. In the control (the bacteria alone) cultures, both DOC concentration and bacterial density were low and almost constant throughout the experiment: 5.0 mg C 1^–1 and 1.1 × 10⁵ cells ml^–1, respectively. In the mixed cultures, bacterial growth was negligible during the exponential growth phase of the alga, but rapid proliferation of the bacteria occurred after the onset of the stationary growth phase. As the bacterial population grew, the density of senescent algal cells also increased. When the bacteria were fed on the DOC from healthy algae, their growth rate was relatively low (0.44 d^–1), but the maximum cell density was high (6.4 × 10⁵ cells ml^–1). Conversely, when the bacteria fed on the DOC of senescent algae, they grew at a relatively high rate (0.51 d^–1), but the maximum cell density was low (2.8 × 10⁵ cells ml^–1). These results suggest that DOCs released from dominant phytoplankton species in different physiological states affect the biomass and activity of bacteria.     

Plant regeneration through direct somatic embryogenesis from protoplasts of banana (Musa spp.)

Summary We report the isolation and regeneration of protoplasts from an embryogenic banana (Musa spp.) cell suspension culture initiated from in vitro proliferating meristems. A high yielding isolation method (up to 6×10⁷ protoplasts.ml^–1 packed cells) is discussed. Optimal regeneration, with more than 50% of the protoplasts showing initial cell division, occurred when high inoculation densities (10⁶ protoplasts.ml^–1) or nurse cultures were applied. Under these conditions, the frequency of microcolony formation was 20–40%. These microcolonies developed directly, without an intervening callus phase, into somatic embryos which later germinated and formed plantlets.     

Comparative study of Cr(VI) uptake and reduction in industrial effluent by Ochrobactrum intermedium and Brevibacterium sp.

Two chromium-resistant bacterial strains, CrT-1 and CrT-13, tolerant up to 40mg K₂CrO₄ ml^–1 on nutrient agar, 25mgml^–1 in nutrient broth, and up to 10mgml^–1 in acetate-minimal media, were identified as Ochrobactrum intermedium and Brevibacterium sp., respectively, on the basis of 16S rRNA gene sequencing. Uptake of chromate was greater in living cells than in heat-killed on dried cells. CrT-1 reduced 82%, 28% and 16% of Cr(VI) at 100, 500, and 1000gml^–1 after 24h while CrT-13 reduced 41%, 14% and 9%. Other heavy metals at low concentrations did not affect these reductions. At 150 and 300gml^–1 in an industrial effluent sample Cr(VI) was reduced by 87% and 71%, respectively, with CrT-1 and by 68% and 47% with CrT-13.Revisions requested 17 May 2004; Revisions received 2 July 2004     

Kinetics of heart rate and catecholamines during exercise in humans The effect of heart denervation

To elucidate the role of factors other than the nervous system in heart rate (f _c) control during exercise, the kinetics off _c and plasma catecholamine concentrations were studied in ten heart transplant recipients during and after 10-min cycle ergometer exercise at 50 W. Thef _c did not increase at the beginning of the exercise for about 60 s. Then in the eight subjects who completed the exercise it increased following an exponential kinetic with a mean time constant of 210 (SEM 22) s. The two other subjects were exhausted after 5 and 8 min of exercise during whichf _c increased linearly. At the cessation of the exercise,f _c remained unchanged for about 50 s and then decreased exponentially with a time constant which was unchanged from that at the beginning of exercise. In the group of eight subjects plasma noradrenaline concentration ([NA]) increased after 30 s to a mean value above resting of 547 (SEM 124) pg · ml^–1, showing a tendency to a plateau, while adrenaline concentration ([A]) did not increase significantly. In the two subjects who became exhausted an almost linear increase in [NA] occurred up to about 1,300 pg · ml^–1 coupled with a significant increase in [A]. During recovery an immediate decrease in [NA] was observed towards resting values. The values of thef _c increase above resting levels determined at the time of blood collection were linearly related with [NA] increments both at the beginning and end of exercise with a similar slope, i.e. about 2.5 beats · min^–1 per 100 pg · ml^–1 of [NA] change. These findings would seem to suggest that in the absence of heart innervation the increase inf _c depends on plasma [NA].     

Activity Measurements of Planktonic Microbial and Microfouling Communities in a Eutrophic Estuary

[³H]thymidine incorporation, the rate of reduction of iodonitrotetrazolium violet (INT) to INT formazan normalized to DNA, and the ratio of ATP to DNA were adapted to measure the activity of attached and unattached microbial assemblages of Bayboro Harbor, Fla. Activity measurements by [³H]thymidine incorporation were made of cells attached to polystyrene culture dishes, in unfiltered water samples, and in the <1-μm-filtered fraction. In most cases, the activity of attached cells was greater than that of unattached cells either in unfiltered water samples or in the <1-μm fraction. The calculated thymidine incorporation rates for cells in the >1-μm fraction were higher than those for cells either in unfiltered water or in the <1-μm-filtered fraction. By the rate of reduction of INT to INT formazan normalized to DNA and by ATP-to-DNA ratios, attached cells were also more active than cells in unfiltered water samples. These results indicate that the microenvironment afforded by attachment is a more beneficial habitat for microbial growth. Reasons for greater activity by natural populations of attached bacteria are discussed.     

Transport and consumption rate of O2 in alginate gel beads entrapping hepatocytes

Experiments carried out with hepatocytes entrapped in alginate gel particles with a cell density of about 10⁷ cells ml^–1 showed an oxygen consumption rate of about 2.2×10^–16 mol cell^–1 s^–1. Under these conditions, no inner anoxic core is present in 1.8 mm diameter beads. From these data, the maximum bead size consistent with non-critical O₂ concentration at the bead center has been evaluated for different cell densities and O₂ concentrations in the medium.     

Interactions of Transthyretin (TTR) and Retinol-Binding Protein (RBP) in the Uptake of Retinol by Primary Rat Hepatocytes

The mechanism by which cells take up retinol from retinol-binding protein (RBP) and the role of the RBP–transthyretin (TTR) complex remain unclear. Here we report on retinol uptake through the RBP–TTR complex by primary cultured rat hepatocytes (parenchymal cells, PC) and nonparenchymal cells (NPC) following incubation with [³H]retinol–RBP or the [³H]retinol–RBP–TTR complex under several conditions. The cellular accumulation of retinol was time and temperature dependent in both PC and NPC. Analysis by HPLC showed that the incorporated [³H]retinol in NPC was mainly converted to retinyl ester, although in PC it remained mainly as unesterified retinol. However, the amount of retinol taken up from the RBP–TTR complex was nearly twofold greater than that from RBP alone. The uptake of [³H]retinol from protein-bound retinol was inhibited by an excess of either retinol–RBP or retinol–RBP–TTR complex. Moreover, retinol uptake through the RBP–TTR complex was inhibited by an excess of free TTR. From these results we postulate that TTR may take part as a positive regulator in the delivery of RBP-bound retinol from plasma, possibly by a membrane receptor, and that retinol uptake takes place preferentially from the RBP–TTR complex into both PC and NPC. The uptake of [³H]retinol (2 μM) by PC was saturated, whereas uptake by NPC was not. These results indicate that the physiological importance of TTR in retinol delivery may be especially important to vitamin A-storing stellate (Ito) cells in the NPC fraction.     

Ecological problems of Lake Ladoga: causes and solutions

We studied the outcome of competition between a large (Brachionus calyciflorus) and a small (Anuraeopsis fissa) rotifer species at five algal (Scenedesmus acutus) concentrations (0.5 × 10⁶ to 40.5 × 10⁶ cells ml^–1) and with varying initial densities in mixed populations (100 to 0% of B. calcyciflorus or A. fissa), the combined initial biomass being 0.2 µg ml^–1 in all test jars. Experiments were conducted at 28 ± 1 °C.Regardless of food concentration, B. calcyciflorus showed a greater increase in biomass than A. fissa, peak densities (mean ± standard error) at the lowest food concentration in the controls being 1.34 ± 0.31 µg dry weight ml^–1 and 0.82 ± 0.08 dry weight ml^–1, respectively. At the lower food concentrations, A. fissa displaced B. calyciflorus and vice versa at the higher food concentrations. At the intermediate food concentrations of 4.5 × 10⁶ cells ml^–1, B. calyciflorus outcompeted A. fissa only if its initial population density was three times higher. The rates of population growth in controls varied from 0.792 ± 0.06 d^–1 to 1.492 ± 0.13 d^–1 for B. calyciflorus and 0.445 ± 0.04 to 0.885 ± 0.01 for A. fissa depending on food level. When both species were introduced together, low food levels favoured higher abundance of A. fissa than B. calyciflorus, suggesting, in nature, it is likely that small Anuraeopsis colonize oligotrophic water bodies more successfully than larger Brachionus. The results also suggest that the outcome of competition depends not only on the size of the competing species and food availability but also on their colonizing density.     

A review of the hydrobiology of Lake Khubsugul (Mongolia)

Lake Khubsugul phytoplankton is dominated by Diatoms and Chlorococcales. Its algal flora is rather peculiar, but lacks Baikalian endemics.Primary production ranges from 2 to 5 mg C m^–3 d^–1. Total bacteria in the open water is 150–200 × 10³ cells ml^–1.Predominant in numbers and biomass throughout the year are two pelagial species of Copepoda — the endemic Mixodiaptomus kozhovi Step., and Cyclops abyssorum Sars.The bottom fauna consists of cold stenothermic invertebrates, mostly Chironomidae. In biomass, they rank only third, however, after Gammaridae and Mollusca. The average zoobenthos biomass of the lake is 5.5g m^–2.