Toxicity of anionic and cationic surfactant to Acinetobacter junii in pure culture

The harmful effects of surfactants to the environment are well known. We were interested in investigating their potential toxicity in a pure culture of Acinetobacter junii, a phosphate (P)-accumulating bacterium. Results showed a high acute toxicity of sodium dodecyl sulfate (SDS) and hexadecyltrimethylammonium bromide (HDTMA) against A. junii. The estimated EC₅₀ values of the HDTMA for the inhibition of CFUs in the pure culture of A. junii was 3.27 ± 1.12 × 10⁻⁷ mol L⁻¹ and for the inhibition of the P-uptake rates 2.47 ± 0.51 × 10⁻⁶ mol L⁻¹. For SDS, estimated EC₅₀ values for the inhibition of CFUs in the pure culture of A. junii was 5.00 ± 2.95 × 10⁻⁶ mol L⁻¹ and for the inhibition of the P-uptake rates 3.33 ± 0.96 × 10⁻⁴ mol L⁻¹. The obtained EC₅₀ values in the standardised yeast toxicity test using Saccharomyces cerevisiae were 3.03 ± 0.38 × 10⁻⁴ and 4.33 ± 0.32 × 10⁻⁵ mol L⁻¹ for SDS and HDTMA, respectively. These results emphasized the need to control concentrations of surfactants entering the activated sludge system. The negative effects of these toxicants could greatly decrease populations of P-accumulating bacteria, as well as eukaryotic organisms, inhabiting activated sludge systems, which in turn could result in the decrease of the system efficiency.     

Toxicity of dodecylpyridinium and cetylpyridinium clorides against phosphate-accumulating bacterium

The antibacterial effect of cationic surfactants against the pure culture of phosphate (P)-accumulating bacterium Acinetobacter junii was investigated. The estimated EC₅₀ values of the N-dodecylpyridinium chloride (DPC) for growth inhibition was 1.4±0.5 × 10⁻⁶ mol L⁻¹ and for the inhibition of the P-uptake rates 7.3±2.6 × 10⁻⁵ mol L⁻¹. The estimated EC₅₀ values of the N-cetylpyridinium chloride (CPC) for growth inhibition was 4.9±1.3 × 10⁻⁷ mol L⁻¹ and for the inhibition of the P-uptake rates 7.7±2.9 × 10⁻⁶ mol L⁻¹. This suggests the importance of controlling the amounts of cationic surfactants in influent of the wastewater treatment systems in order to avoid the possible failure of the biological P removal from wastewaters.     

Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo

Summary High yields of human hepatocytes (up to 23×10⁶ viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham’s F12 containing 0.2% bovine serum albumin, 10⁻⁸ M insulin, and 10⁻⁸ M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250±177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50±0.17 nmol glucose·mg⁻¹·min⁻¹) similar to that reported for human liver. Insulin at 10⁻⁸ M activated glycolysis (×1.40) and glycogenesis (×1.34), and glucagon at 10⁻⁹ M stimulated gluconeogenesis (×1.35) and glycogenolysis (×2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, α₁-antitrypsin, α₁-antichymotrypsin, α₁-acid glycoprotein, haptoglobin, α₂-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10⁻⁹ M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol·mg⁻¹ cell protein·min⁻¹, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol·mg⁻¹). The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.     

A primary culture system of adult rat heart cells for the study of toxicologic agents

Summary Tricyclic antidepressants (TCAs) are currently used in the treatment of mental depression and nocturnal enuresis. Clinically, these drugs are useful; however, cardiotoxicity can occur even with therapeutic dosages. For example, TCAs are known to alter myocardial function, induce arrhythmias, and produce heart block in individuals with a normal cardiovascular history. The present study was undertaken to establish a culture system of spontaneously contracting adult primary myocardial cells for toxicologic testing and to examine their contractility, morphology, and lactate dehydrogenase release (LDH) after treatment with one of the most cardiotoxic TCAs, amitriptyline. Primary myocardial cell cultures were obtained from approximately 60- to 90-day-old Sprague-Dawley rats. After the cells had been grown in culture for 11 days, they were treated with amitriptyline (1 × 10⁻³, 1 × 10⁻⁴, and 1 × 10⁻⁵ M) for 2 to 24 h. The highest concentration of amitriptyline (1 × 10⁻³ M) completely destroyed the cardiac muscle cells. In addition to moderate and severe vacuole, granule, and pseudopodia formation, all contractile activity was inhibited as early as 2 h after exposure to the intermediate concentration of 1 × 10⁻⁴ M amitriptyline. Significant LDH release did not occur until 8 h after treatment with this intermediate concentration. Even though there was no significant LDH release at all 3 time points tested, there was a 50% decrease in beating activity (154±9 to 77±5 beats/min) and initiation of vacuole formation by 2 h with the lowest concentration of amitriptyline (1 × 10⁻⁵ M). This study presents a new apparatus for the isolation of adult cardiac myocytes for the establishment of primary cell cultures for toxicologic testing. Furthermore, these data demonstrate that amitriptyline induces a concentration- and time-dependent cardiotoxic profile in a model of spontaneously contracting adult cardiac muscle cells in culture.     

Influence of the KDEL signal,DMSO and mannitol on the production of the recombinant antibody 14D9 by long-term <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> cell suspension culture

We have established two transgenic cell suspension culture lines of Nicotiana tabacum that express the catalytic antibody 14D9 as a secretory product (sec-Ab) or as a KDEL-tagged product in the endoplasmic reticulum (Ab-KDEL), respectively. After 3 years of culture, the performance improved to a production level of 0.15 ± 0.03 μg ml⁻¹ on the seventh day of culture for the sec-Ab line and 0.48 ± 0.05 μg ml⁻¹ on the third day for Ab-KDEL line. Analysis of the effect of osmotic stress using mannitol (90 g l⁻¹) as an osmolite revealed that there was a 12-fold increase in antibody yield (1.96 ± 0.20 μg ml⁻¹) on the seventh day of culture in line sec-Ab and a fivefold increase (2.31 ± 0.18 μg ml⁻¹) on the seventh day for line Ab-KDEL. The concentration of the antibody in the culture medium was not significant. Dimethyl sulfoxide used as a permeabilizing agent was not effective in increasing 14D9 yield, but it did cause distinctive cell damage at all concentrations tested.     

Maintenance and expansion of hematopoietic stem/progenitor cells in biomimetic osteoblast niche

In this study, we employed bio-derived bone scaffold and composited with the marrow mesenchymal stem cell induced into osteoblast to replicate a “biomimetic niche.” The CD34⁺ cells or mononuclear cells (MNC) from umbilical cord blood were cultured for 2–5 weeks in the biomimetic niche (3D system) was compared with conventional two dimensional cultures (2D system) without adding cytokine supplement. After 2 weeks in culture, the CD34⁺ cells from umbilical cord blood in the 3D system increased 3.3–4.8 folds when compared with the initial CD34⁺ cells. CD34⁺/CD38⁻ cells accounted for 82–90% of CD34⁺ cells. After 5 weeks, CD34⁺/CD38⁻ cells in the 3D system increased when compared with initial (1.3 ± 0.3 × 10³ vs. 1.0 ± 0.5 × 10⁴, p < 0.05), but were decreased in the 2D system (1.3 ± 0.3 × 10³ vs. 2.5 ± 0.7 × 10², p < 0.05). The CFU progenitors were produced more in the 3D system than in the 2D system (4.6–9.3 folds vs. 1.0–1.5 folds) after 2 weeks in culture, and the colony distribution in the 3D system manifested higher percentage of BFU-E and CFU-GEMM, but in the 2D system was mainly CFU-GM. The LTC-ICs in the 3D system showed 5.2–7.2 folds increase over input at 2 weeks in culture, and maintain the immaturation of hematopoietic progenitor cells (HPCs) over 5 weeks. In conclusion, this new 3D hematopoietic progenitor cell culture system is the first to utilize natural cancellous bone as scaffold with osteoblasts as supporting cells; it is mimicry of natural bone marrow HSC niche. Our primary work has demonstrated it could maintain and expand HSC/HPC in vitro.     

Bioelectrical activity in the heart of the lugworm Arenicola marina

Standard microelectrode technique was used to study electrical activity of the isolated heart of the polychaete annelid, Arenicola marina. Typical pacemaker activity with slow diastolic depolarization was observed in all recordings. The average maximum diastolic potential (−58.4 ± 3.2 mV), the average amplitude of the action potential (28.7 ± 4.7 mV) and the average total duration of the action potential (2,434 ± 430 ms) were determined. There has been no gradient of automaticity observed in our studies, which suggests that all regions of the Arenicola heart could possess pacemaker functions. Acetylcholine (ACh) produced a concentration dependent (5 × 10⁻⁸–5 × 10⁻⁵ M) increase of the beating rate via increase in the rate of the diastolic depolarization. ACh (5 × 10⁻⁵ M) increased beating rate by 2.5-fold compared to the control rate. A stronger action of ACh resulted in depolarization, block of action potential generation and contracture of the heart. The non-hydrolysable ACh analog carbacholine (10⁻⁸–10⁻⁶ M) produced similar effects. All effects of ACh and carbacholine were abolished by 5 × 10⁻⁶ M atropine. d-Tubocurarine (5 × 10⁻⁵ M) did not significantly alter effects of ACh or carbacholine. Epinephrine (10⁻⁸–10⁻⁶ M) caused the slowing of pacemaker activity and marked decrease of action potential duration. 10⁻⁶ M epinephrine produced complete cardiac arrest. The effects of epinephrine were not significantly altered by the β-blocker propranolol (5 × 10⁻⁶ M). The β-agonist isoproterenol (10⁻⁷–10⁻⁵ M) and the α-agonist xylometazoline (10⁻⁶–10⁻⁵ M) did not produce significant effects. Thus, cholinergic effects in the Arenicola heart are likely to be mediated via muscarinic receptors, while the nature of adrenergic effects needs further investigation.     

Optimization of a chemically defined medium for mycelial growth and polysaccharide production by medicinal mushroom <Emphasis Type="Italic">Phellinus igniarius</Emphasis>

A chemically defined medium for mycelial growth and exopolysaccharide (EPS) production by submerged culture of Phellinus igniarius was investigated. The mainly defined medium compositions were optimized by using orthogonal matrix method. The optimal defined medium (per liter) was 40.0 g glucose, 4.0 g. glutamic acid, 4.0 g (NH₄)₂SO₄, and initial pH 6.0. Under the optimal medium, the maximal mycelial biomass and EPS production were 12.33 ± 0.89 and 1.21 ± 0.08 g l⁻¹ at 192 h in shake flask, while the maximal mycelial biomass and EPS production reached 13.86 ± 0.52 and 1.92 ± 0.07 g l⁻¹ at 168 h in 3 l fermenter, respectively. The molecular weights (g mol⁻¹) of four fractions isolated from EPS by gel permeation were about 6.4 × 10⁶, 3.3 × 10⁵, 2.7 × 10⁵ and 2.9 × 10³. This study should be widely applied to other secondary metabolites production from higher fungus in a chemically defined medium and quantitative regulation of the metabolic flux in polysaccharide biosynthesis.     

Isoniazid and rifampicin inhibit allosterically heme binding to albumin and peroxynitrite isomerization by heme–albumin

Human serum heme–albumin (HSA-heme) displays globin-like properties. Here, the allosteric inhibition of ferric heme [heme-Fe(III)] binding to human serum albumin (HSA) and of ferric HSA–heme [HSA-heme-Fe(III)]-mediated peroxynitrite isomerization by isoniazid and rifampicin is reported. Moreover, the allosteric inhibition of isoniazid and rifampicin binding to HSA by heme-Fe(III) has been investigated. Data were obtained at pH 7.2 and 20.0 °C. The affinity of isoniazid and rifampicin for HSA [K ₀ = (3.9 ± 0.4) × 10⁻⁴ and (1.3 ± 0.1) × 10⁻⁵ M, respectively] decreases by about 1 order of magnitude upon heme-Fe(III) binding to HSA [K _h = (4.3 ± 0.4) × 10⁻³ and (1.2 ± 0.1) × 10⁻⁴ M, respectively]. As expected, the heme-Fe(III) affinity for HSA [H ₀ = (1.9 ± 0.2) × 10⁻⁸ M] decreases by about 1 order of magnitude in the presence of saturating amounts of isoniazid and rifampicin [H _d = (2.1 ± 0.2) × 10⁻⁷ M]. In the absence and presence of CO₂, the values of the second-order rate constant (l _on) for peroxynitrite isomerization by HSA-heme-Fe(III) are 4.1 × 10⁵ and 4.3 × 10⁵ M⁻¹ s⁻¹, respectively. Moreover, isoniazid and rifampicin inhibit dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) in the absence and presence of CO₂. Accordingly, isoniazid and rifampicin impair in a dose-dependent fashion the HSA-heme-Fe(III)-based protection of free l-tyrosine against peroxynitrite-mediated nitration. This behavior has been ascribed to the pivotal role of Tyr150, a residue that either provides a polar environment in Sudlow’s site I (i.e., the binding pocket of isoniazid and rifampicin) or protrudes into the heme-Fe(III) cleft, depending on ligand binding to Sudlow’s site I or to the FA1 pocket, respectively. These results highlight the role of drugs in modulating heme-Fe(III) binding to HSA and HSA-heme-Fe(III) reactivity.     

Non-parametric estimation of thresholds for radiation effects in vertebrate species under chronic low-LET exposures

Databases on effects of chronic low-LET radiation exposure were analyzed by non-parametric statistical methods, to estimate the threshold dose rates above which radiation effects can be expected in vertebrate organisms. Data were grouped under three umbrella endpoints: effects on morbidity, reproduction, and life shortening. The data sets were compiled on a simple ‘yes’ or ‘no’ basis. Each data set included dose rates at which effects were reported without further details about the size or peculiarity of the effects. In total, the data sets include 84 values for endpoint “morbidity”, 77 values for reproduction, and 41 values for life shortening. The dose rates in each set were ranked from low to higher values. The threshold TDR5 for radiation effects of a given umbrella type was estimated as a dose rate below which only a small percentage (5%) of data reported statistically significant radiation effects. The statistical treatment of the data sets was performed using non-parametric order statistics, and the bootstrap method. The resulting thresholds estimated by the order statistics are for morbidity effects 8.1 × 10⁻⁴ Gy day⁻¹ (2.0 × 10⁻⁴–1.0 × 10⁻³), reproduction effects 6.0 × 10⁻⁴ Gy day⁻¹ (4.0 × 10⁻⁴–1.5 × 10⁻³), and life shortening 3.0 × 10⁻³ Gy day⁻¹ (1.0 × 10⁻³–6.0 × 10⁻³), respectively. The bootstrap method gave slightly lower values: 2.1 × 10⁻⁴ Gy day⁻¹ (1.4 × 10⁻⁴–3.2 × 10⁻⁴) (morbidity), 4.1 × 10⁻⁴ Gy day⁻¹ (3.0 × 10⁻⁴–5.7 × 10⁻⁴) (reproduction), and 1.1 × 10⁻³ Gy day⁻¹ (7.9 × 10⁻⁴–1.3 × 10⁻³) (life shortening), respectively. The generic threshold dose rate (based on all umbrella types of effects) was estimated at 1.0 × 10⁻³ Gy day⁻¹.     

Effects of reduced diameter of bag cultures on content of essential fatty acids and cell density in a continuous algal production system

Cell density and fatty acid (FA) content of Pavlova lutheri and Chaetoceros muelleri were analysed in a continuous algal production system (250-L bags) with reduced diameter. The cell density and FA content and composition in the algal production system were determined in replicate bags over a period of 5 weeks. The results showed that the cell density and essential FAs increased during the experiment for both species. After 5 weeks the mean cell numbers had increased to 6.0 ± 0.3 × 10⁶ cells mL⁻¹ in the P. lutheri bags and 6.0 ± 0.4 × 10⁶ cells mL⁻¹ in the C. muelleri bags. The content of total FAs increased significantly (p < 0.05) in all of the bags during the experiment. At the end of the experiment the mean total FA content were 2.7 ± 0.3 pg cell⁻¹ in the P. lutheri bags and 1.8 ± 0.1 pg cell⁻¹ in the C. muelleri bags. Maximum total FA content registered was 3.0 pg cell⁻¹ in one of the P. lutheri bags. The content of the essential FAs (ARA, EPA, DHA) increased over time in both of the species. At the end of the experiment the content of EPA (0.6 ± 0.1 pg cell⁻¹) and DHA (0.3 ± 0.0 pg cell⁻¹) were highest in the P. lutheri bags, while ARA (0.1 ± 0.0 pg cell⁻¹) was highest in C. muelleri. EPA and DHA constituted 22% and 11%, respectively, of total FA content in P. lutheri, while ARA constituted 6% of total FA content in C. muelleri. The results from this experiment indicate that flagellates such as P. lutheri perform better in narrow bags with improved light conditions, while diatoms like C. muelleri perform better in wider bags under light limitation. Implications for bivalve hatcheries are discussed.     

Growth-associated biosynthesis of astaxanthin in heterotrophic Chlorella zofingiensis (Chlorophyta)

The green microalga Chlorella zofingiensis can produce the ketocarotenoid astaxanthin under heterotrophic culture conditions. Here we report the growth-associated biosynthesis of astaxanthin in this biotechnologically important alga. With glucose as sole carbon and energy source, C. zofinginesis grew fast in the dark with rapid exhaustion of nitrogen and carbon sources from media, leading to a high specific growth rate (0.034 h⁻¹). Cultures started at a cell concentration of about 3.4 × 10⁹ cells l⁻¹ reached, after 6 days, standing biomass values of 1.6 × 10¹¹ cells or 8.5 g dry weight l⁻¹. Surprisingly, the biosynthesis of astaxanthin was found to start at early exponential phase, independent of cessation of cell division. A general trend was observed that the culture conditions benefiting cell growth also benefited astaxanthin accumulation, indicating that astaxanthin was a growth-associated product in this alga. The maximum cell dry biomass and astaxanthin yield were 11.75 g l⁻¹ and 11.14 mg l⁻¹ (about 1 mg g⁻¹), simultaneously obtained in the fed-batch culture with a combined glucose–nitrate mixture addition, which were the highest ever reported in dark-heterotrophic algal cultures. The possible reasons why dark-heterotrophic C. zofingiensis could produce astaxanthin during the course of cell growth were discussed.     

Ethanol and xylitol production from glucose and xylose at high temperature by <Emphasis Type="Italic">Kluyveromyces</Emphasis> sp. IIPE453

A yeast strain Kluyveromyces sp. IIPE453 (MTCC 5314), isolated from soil samples collected from dumping sites of crushed sugarcane bagasse in Sugar Mill, showed growth and fermentation efficiency at high temperatures ranging from 45°C to 50°C. The yeast strain was able to use a wide range of substrates, such as glucose, xylose, mannose, galactose, arabinose, sucrose, and cellobiose, either for growth or fermentation to ethanol. The strain also showed xylitol production from xylose. In batch fermentation, the strain showed maximum ethanol concentration of 82 ± 0.5 g l⁻¹ (10.4% v/v) on initial glucose concentration of 200 g l⁻¹, and ethanol concentration of 1.75 ± 0.05 g l⁻¹ as well as xylitol concentration of 11.5 ± 0.4 g l⁻¹ on initial xylose concentration of 20 g l⁻¹ at 50°C. The strain was capable of simultaneously using glucose and xylose in a mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹, achieving maximum ethanol concentration of 38 ± 0.5 g l⁻¹ and xylitol concentration of 14.5 ± 0.2 g l⁻¹ in batch fermentation. High stability of the strain was observed in a continuous fermentation by feeding the mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹ by recycling the cells, achieving maximum ethanol concentration of 30.8 ± 6.2 g l⁻¹ and xylitol concentration of 7.35 ± 3.3 g l⁻¹ with ethanol productivity of 3.1 ± 0.6 g l⁻¹ h⁻¹ and xylitol productivity of 0.75 ± 0.35 g l⁻¹ h⁻¹, respectively.     

State-Dependent Block of Na<Superscript>+</Superscript> Channels by Articaine Via the Local Anesthetic Receptor

Articaine is widely used as a local anesthetic (LA) in dentistry, but little is known regarding its blocking actions on Na⁺ channels. We therefore examined the state-dependent block of articaine first in rat skeletal muscle rNav1.4 Na⁺ channels expressed in Hek293t cells. Articaine exhibited a weak block of resting rNav1.4 Na⁺ channels at −140 mV with a 50% inhibitory concentration (IC₅₀) of 378 ± 26 μM (n = 5). The affinity was higher for inactivated Na⁺ channels measured at −70 mV with an IC₅₀ value of 40.6 ± 2.7 μM (n = 5). The open-channel block by articaine was measured using inactivation-deficient rNav1.4 Na⁺ channels with an IC₅₀ value of 15.8 ± 1.5 μM (n = 5). Receptor mapping demonstrated that articaine interacted strongly with a D4S6 phenylalanine residue, which is known to form a part of the LA receptor. Thus the block of rNav1.4 Na⁺ channels by articaine is via the conserved LA receptor in a highly state-dependent manner, with a ranking order of open (23.9×) > inactivated (9.3×) > resting (1×) state. Finally, the open-channel block by articaine was likewise measured in inactivation-deficient hNav1.7 and rNav1.8 Na⁺ channels, with IC₅₀ values of 8.8 ± 0.1 and 22.0 ± 0.5 μM, respectively (n = 5), indicating that the high-affinity open-channel block by articaine is indeed preserved in neuronal Na⁺ channel isoforms.     

Growth and carrageenan quality of <Emphasis Type="Italic">Kappaphycus striatum</Emphasis> var. <Emphasis Type="Italic">sacol</Emphasis> grown at different stocking densities,duration of culture and depth

Kappaphycus striatum var. sacol was grown in two separate studies: (1) at two stocking densities, and (2) at four different depths, each for three different durations of culture (30, 45 and 60 days) in order to determine the growth rate of the seaweed and evaluate the carrageenan content and its molecular weight. The results demonstrated that stocking density, duration of culture and depth significantly (P < 0.01) affected the growth rate, carrageenan content and molecular weight of K. striatum var. sacol. Decreasing growth rate was observed at both stocking densities and at four depths as duration of culture increased. A lower stocking density (500 g m⁻¹line⁻¹) showed a higher growth rate for the shortest durations, i.e. 30 days, as compared to those grown at a higher density. Likewise, decreasing growth rate was observed as depth increased, except at 50 cm after 60 days of culture. A 45-day culture period produced the highest molecular weight at both stocking densities (500 g m⁻¹line⁻¹ = 1,079.5 ± 31.8 kDa, 1,000 g m⁻¹line⁻¹ = 1,167 ± 270.6 kDa). ‘Sacol’ grown for 30 days at 50 cm (1,178 kDa) to 100 cm (1,200 kDa) depth showed the highest values of molecular weight of carrageenan extracted. The results suggested that K. striatum var. sacol is best grown at a stocking density of 500 g m⁻¹line⁻¹, at a depth of 50–100 cm, and for a duration of 30 days in order to provide the highest growth rate, carrageenan content and molecular weight.     

Relevance of bacterioplankton abundance and production in the oligotrophic equatorial Indian Ocean

Bacterioplankton abundance and production, chlorophyll a (Chl a) concentrations and primary production (PP) were measured from the equatorial Indian Ocean (EIO) during northeast (NEM), southwest (SWM) and spring intermonsoon (SpIM) seasons from 1°N to 5°S along 83°E. The average bacterial abundance was 0.52 ± 0.29, 0.62 ± 0.33 and 0.46 ± 0.19 (× 10⁸ cells l⁻¹), respectively during NEM, SWM and SpIM in the top 100 m. In the deep waters (200 m and below), the bacterial counts averaged ∼0.35 ± 0.14 × 10⁸ cells l⁻¹ in SWM and 0.39 ± 0.16 × 10⁸ cells l⁻¹ in SpIM. The 0–120 m column integrated bacterial production (BP) ranged from 19 to 115 and from 10 to 51 mg C m⁻² d⁻¹ during NEM and SWM, respectively. Compared with many open ocean locations, bacterial abundance and production in this region are lower. The bacterial carbon production, however, is notably higher than that of phytoplankton PP (BP:PP ratio 102% in SWM and 188% in NEM). With perpetually low PP (NEM: 20, SWM: 18 and SpIM: 12 mg C m⁻² d⁻¹) and Chl a concentration (NEM: 16.5, SWM: 15.0 and SpIM: 20.9 mg m⁻²), the observed bacterial abundance and production are pivotal in the trophodynamics of the EIO. Efficient assimilation and mineralization of available organics by bacteria in the euphotic zone might serve a dual role in the ultra-oligotrophic regions including EIO. Thus, bacteria probably sustain microheterotrophs (micro- and meso-zooplankton) through microbial loop. Further, rapid mineralization by bacteria will make essential nutrients available to autotrophs.     

The influence of differentiation-inducing agents on plasma membrane surface characteristics of THP-1 cells

Undifferentiated THP-1 cells from Cell Culture Collection of the Institute of Cytology, RAS (St. Petersburg), are characterized by weak expression of Toll-like receptor-4 (TLR4) on the cell surface (up to 2%) and by almost undetectable expression of CD14 and CD11b receptors. Differentiation agent phorbol-12-myristate-13-acetate independently of its concentration (2 × 10⁻⁷ M or 10⁻⁸ M) and incubation time (24 or 48 h) did not initiate CD11b surface expression and did not change the parameter S_app (0.605 ± 0.005 at 37°C) reflecting the cell membrane viscosity. Differentiation of THP-1 cells induced by another differentiation agent, 1α,25-dihydroxyvitamin D₃, caused expression of CD14 (up to 70–80%) and CD11b (up to 15–20%) receptors, again without changes in plasma membrane viscosity. The rate constants of the reduction of 5- and 16-doxyl-stearic acids by THP-1 cells were in the range of 6–8 × 10⁻³ s⁻¹ at 37°C. During cell differentiation significant changes in cell electrophoretic mobility (EM, μm s⁻¹ V⁻¹ cm) were observed. Mean value of EM for undifferentiated THP-1 cells was −1.332 ± 0.011, whereas for phorbol-12-myristate-13-acetate- and 1α,25-dihydroxyvitamin D₃-treated cells it was −1.432 ± 0.030 and −1.212 ± 0.016, respectively.     

The viability to a wall shear stress and propagation of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> in the intensive membrane bioreactor

Bifidobacterium longum grew at 65 L pilot scale of the membrane bioreactor (MBR), externally fitted with ceramic membrane (0.7 m²). Cell mass at the MBR reached 22.18 g L⁻¹ as dry cell weight in 12 h, which is 8.44 times higher than cell mass attained at the vial culture. The growth rate in the vial culture was μ = 0.385 h⁻ and at the batch culture was μ = 1.13 h⁻ in the exponential period and μ = 0.31 h⁻¹ in the stationary period. In the fed-batch mode was μ = 1.102 h⁻¹ for 6 h with inoculation and declined to μ = 0.456 h⁻¹ with feeding of feed medium. The growth rate at the MBR was μ = 0.134 h⁻¹. The number of viable cells was 6.01 × 10¹² cfu L⁻¹ at the batch culture, but increased to 1.15 × 10¹⁴ cfu L⁻¹ at the MBR culture. The specific growth rate of viable cell number (colony-forming units per liter, per hour) improved by 6.01 times from the batch to the MBR culture. The wall shear stress mainly generated by the pump, and the membrane incorporated into the MBR was controlled during the cultivation at the MBR. The viability of B. longum declined to under 10% in the first 2 weeks of the 4-week stability test (40°C) as B. longum was exposed to over wall shear stress 713 Pa, but the viability improved to 30–40% in wall shear stress of 260 Pa or STR culture. The loss in the cell viability can be saved by managing with wall shear stress during the cultivation at the MBR.     

A novel animal-component-free medium for rabies virus production in Vero cells grown on Cytodex 1 microcarriers in a stirred bioreactor

Vero cells growth and rabies production in IPT-AF medium, a property animal-component-free medium are described in this work. Kinetics of cell growth and rabies virus (strain LP 2061) production were first conducted in spinner flasks. Over eight independent experiments, Vero cell growth in IPT-AF medium, on 2 g/l Cytodex 1 was consistent. An average Cd (cell division number) of 3.3 ± 0.4 and a specific growth rate μ of 0.017 ± 0.006 h⁻¹ were achieved. Such performances were comparable to those obtained in serum-containing medium (MEM + 10% FCS). Rabies virus production on Vero cells in IPT-AF medium was also optimised in spinner flasks. The effects of multiplicity of infection (MOI), regulation of glucose level at 1 g/l and cell washing step, were investigated. The highest virus titer was achieved when the cells were infected at an MOI of 0.1; this level was equal to 10⁷ FFU/ml. The step of medium exchange before cell infection can be omitted; nevertheless in this case glucose level should be maintained at 1 g/l to avoid a decrease of specific virus productivity. Process optimisation in a 2-l stirred bioreactor pointed out that the aeration mode was the prominent parameter that affected cell growth in IPT-AF medium and on Cytodex 1 microcarriers. An acceptable level of cell density (cell density level of 1.5 × 10⁶ cells/ml) was achieved when cells were grown in batch mode and using headspace aeration. Nevertheless, this aeration mode is not optimal for large-scale culture. The addition of Pluronic F68 at 0.1% at 24 h post inoculation as well as the switch from surface aeration mode to the sparged mode, 2 days after the start of the culture, had markedly improved cell growth performance. A cell density level of 5.5 × 10⁶ cells/ml was reached when cells were grown in a 2-l bioreactor, on 3 g/l Cytodex 1 in IPT-AF medium and using the recirculation culture mode. Cell infection at an MOI of 0.1 and using perfused culture, resulted in a maximal virus titer of 3.5 × 10⁷ FFU/ml. The activity of the pooled inactivated rabies virus harvests showed a protective activity that meets WHO requirements.     

Change in ammonia-oxidizing microorganisms in enriched nitrifying activated sludge

In this study, sludge was taken from a municipal wastewater treatment plant that contained a nearly equal number of archaeal amoA genes (5.70 × 10⁶ ± 3.30 × 10⁵ copies mg sludge⁻¹) to bacterial amoA genes (8.60 × 10⁶ ± 7.64 × 10⁵ copies mg sludge⁻¹) and enriched in three continuous-flow reactors receiving an inorganic medium containing different ammonium concentrations: 2, 10, and 30 mM NH₄⁺–N (28, 140, and 420 mg N l⁻¹). The abundance and communities of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in enriched nitrifying activated sludge (NAS) were monitored at days 60 and 360 of the operation. Early on, between day 0 and day 60 of reactor operation, comparative abundance of AOA amoA genes to AOB amoA genes varied among the reactors depending on the ammonium levels found in the reactors. As compared to the seed sludge, the number of AOA amoA genes was unchanged in the reactor with lower ammonium level (0.06 ± 0.04 mgN l⁻¹), while in the reactors with higher ammonium levels (0.51 ± 0.33 and 0.25 ± 0.10 mgN l⁻¹), the numbers of AOA amoA genes were deteriorated. By day 360, AOA disappeared from the ammonia-oxidizing consortiums in all reactors. The majority of the AOA sequences from all NASs at each sampling period fell into a single AOA cluster, however, suggesting that the ammonium did not affect the AOA communities under this operational condition. This result is contradictory to the case of AOB, where the communities varied significantly among the NASs. AOB with a high affinity for ammonia were present in the reactors with lower ammonium levels, whereas AOB with a low affinity to ammonia existed in the reactors with higher ammonium levels.