Cyclodextrin glucanotransferase production by cell biocatalysts of alkaliphilic bacilli

Cells of obligated alkaliphiles Bacillus pseudalcaliphilus 20RF and Bacillus pseudalcaliphilus 8SB isolated from Bulgarian habitats, producers of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19), were immobilized by three different techniques: on two types of polysulphone membranes; entrapped in agar-gel beads containing magnetite and by nano-particles of silanized magnetite covalently bound on the cell surface. The biocatalysts obtained demonstrated the opportunity for a significantly enhanced CGTase production compared to free cells for a long period of time (10 days semicontinuous cultivation) without impact on their mechanical stability. The cell membrane-biocatalysts exhibited the highest enzyme activity after 240 h repeated batch cultivation and retained 1.3–2.3-fold increase of the CGTase yield compared to free cells at the end of the process. Membrane biocatalysts were applied for a direct cyclodextrin (CD) production. The results obtained demonstrated the possibility of starch conversion into cyclodextrins by immobilized cells without using of crude or purified enzyme. The membrane biocatalysts of both obligated alkaliphiles formed mainly β- and γ-CDs after 6 h enzyme reaction at pH 9.0 of the reaction mixture. Under these conditions, the quantity of γ-CDs was a relative high, to 35–37% of the total CD amount.     

Regioselective glycosylation of hydroquinone to α-arbutin by cyclodextrin glucanotransferase from Thermoanaerobacter sp.

Hydroquinone glycosides were produced by transglycosylation reactions catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. (Toruzyme^® 3.0L). The reactions were carried out in an aqueous system containing hydroquinone (HQ) and maltodextrin as acceptor and donor substrate molecules respectively. The conditions for the synthesis of hydroquinone glucoside (α-arbutin) were 9 mM hydroquinone, maltodextrin (5%, w/v) in 20 mM citrate phosphate buffer, pH 5.5 and 0.025 mg/ml toruzyme at 40 °C for 24 h. The transfer efficiency of hydroquinone glycosylation was 31.8% and 29.2% respectively, when α-cyclodextrin and maltodextrin were employed as donor substrates. The major glycoside product was identified as hydroquinone-1-O-α-d-glucopyranoside (α-arbutin) on the basis of mass spectrometric, nuclear magnetic resonance analysis and component analysis of its enzymatic hydrolysates. The highest molar yield of α-arbutin (21.2%) was obtained when α-cyclodextrin was used as the donor substrate. A two step enzymatic reaction system comprising of CGTase and amyloglucosidase helped to attain a molar yield of 30% for α-arbutin. At room temperature the solubility of α-arbutin in water was determined to be 12.8 g/100 ml which is approximately 1.8 fold higher than that of hydroquinone.     

Nanostructured materials for magnetic biosensing

BackgroundMagnetic nanoparticles (MNPs) are at the leading edge of the field of biomedical applications and magnetic biosensing.MethodsMNPs were fabricated by electrophysical methods of the laser target evaporation (LTE) and spark discharge with electrodynamic acceleration of plasma jumpers (SD). Synthesis of polyacrylamide hydrogel was done in the presence of Fe₂O₃ MNPs in different concentrations obtained by LTE. [FeNi/Ti]₃/Cu/[Ti/FeNi]₃/Ti multilayers for giant magnetoimpedance (GMI) based sensitive elements were prepared by rf-sputtering for testing a biosensor prototype.ResultsIron oxide MNPs, ferrofluids, ferrofluids contacting with biological systems, synthetic ferrogels mimicking natural tissues – are the steps of the discussed in this work development of bionanomaterials. Thorough the structural and magnetic studies of a multilayered sensitive element, MNPs and ferrogels insure the complete characterization of biosensor prototype. The GMI responses were carefully evaluated in initial state and in the presence of ferrogel with known concentration of MNPs. SD MNPs had the smallest 5–8 nm size. This nanomaterial was characterized by large internal strains of the order of 25 × 10^− 3, which can play an important role for the interaction with different biosystems.ConclusionsIron oxide MNPs were fabricated by LTE and SD methods. SD MNPs had the smallest 5–8 nm size and large internal strains of the order of 25 × 10^− 3. Designed GMI biosensor prototype allowed precise evaluation of the stray field of the MNPs present in the ferrogel by evaluating the systematic changes of the GMI in a 20–400 MHz frequency range.General significanceThis work summarizes recent developments in the field of nanomaterials potentially applicable in magnetic biosensing.     

Changes in the size distribution of goat steroidogenic luteal cells during pregnancy

Experiments were conducted to investigate the size distribution of goat steroidogenic luteal cells throughout pregnancy. Corpora lutea were collected from very early (<6 weeks), early (6–8 weeks), middle (9–14 weeks) or late (15–18 weeks) stages of pregnancy. Luteal tissue was dissociated into single-cell suspension by enzyme treatments. Cells were stained for 3β-hydroxysteroid dehydrogenase (3β-HSD) activity, a marker for steroidogenic cells. The steroidogenic cells covered a wide spectrum of size ranging from 5 to 45 μm in diameter. There was a significant increase in mean cell diameter (P>0.01) as pregnancy progressed. Mean diameter of 3β-HSD positive cells increased from 14.73±0.35 μm in the corpus luteum of very early pregnancy to 24.20±0.45 μm in the corpus luteum of late pregnancy. The ratio of large (>20 μm in diameter) to small (5–20 μm in diameter) luteal cells was 0.28:1.0 in very early pregnancy, with the 7.5–15 μm cell size class being dominant. However, the ratio of large-to-small luteal cells was increased to 1.77:1.0 μm as pregnancy advanced and 25–35 μm cell sizes became predominant. It is likely that small luteal cells could develop into large cells as pregnancy progresses. Development of pregnancy is also associated with an increase in size of steroidogenic luteal cells.     

Zinc induced damage to kidney proximal tubular cells: Studies on chemical speciation leading to a mechanism of damage

This study was carried out to investigate whether zinc can potentiate renal toxicity using monolayer cultures of kidney proximal tubular cells and if so to establish the chemical species and the mechanism involved.MethodsZinc was prepared as the citrate complex at pH 7.4 in phosphate buffered saline. Monolayers of kidney proximal tubular cells under standard cell culture conditions were exposed to zinc concentrations of 0, 5 10, 20, 50 and 100 μmol/L. To assess cellular damage, thiazol blue (MTT) uptake, NAG and LDH release, DAPI staining and Tunel assay were used. Cytoprotective agents: trolox, cysteine, glutathione, ascorbic acid and sodium selenite were used to investigate if the damage was reversible.ResultsIncubation of kidney cells with zinc citrate showed a dose related reduction in cell viability (p < 0.005) associated with cellular uptake of zinc ions. After 24 h incubation with 100 μmol/L Zn citrate, NAG release was not significantly different compared to the control whereas LDH increased 3 fold. DAPI staining showed apoptotic bodies within the cells confirmed by Tunel assay using flow cytometry. Electron microscopy showed significant morphological changes including loss of brush border, vacuolated cytoplasm and condensed nuclei. Trolox almost completely (>85 ± 5%) and sodium selenite partially recovered (40 ± 4%) the viability of cells exposed to Zn but no protection was observed with other cytoprotectants, e.g. glutathione, cysteine or ascorbic acid.In conclusion zinc can induce damage to kidney cells by a mechanism dependent on zinc ions entering the cell, binding to the cell organelles and disrupting cellular processes rather than damage initiated by free radical and ROS production.     

Magnetic susceptibility of iron in malaria-infected red blood cells

During intra-erythrocytic maturation, malaria parasites catabolize up to 80% of cellular haemoglobin. Haem is liberated inside the parasite and converted to haemozoin, preventing haem iron from participating in cell-damaging reactions. Several experimental techniques exploit the relatively large paramagnetic susceptibility of malaria-infected cells as a means of sorting cells or investigating haemoglobin degradation, but the source of the dramatic increase in cellular magnetic susceptibility during parasite growth has not been unequivocally determined. Plasmodium falciparum cultures were enriched using high-gradient magnetic fractionation columns and the magnetic susceptibility of cell contents was directly measured. The forms of haem iron in the erythrocytes were quantified spectroscopically. In the 3D7 laboratory strain, the parasites converted approximately 60% of host cell haemoglobin to haemozoin and this product was the primary source of the increase in cell magnetic susceptibility. Haemozoin iron was found to have a magnetic susceptibility of (11.0 ± 0.9) × 10^? 3 mL mol^? 1. The calculated volumetric magnetic susceptibility (SI units) of the magnetically enriched cells was (1.88 ± 0.60) × 10^? 6 relative to water while that of uninfected cells was not significantly different from water. Magnetic enrichment of parasitised cells can therefore be considered dependent primarily on the magnetic susceptibility of the parasitised cells.     

Production of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. TS1-1: Optimization of carbon and nitrogen concentration in the feed medium using central composite design

Optimisation of nutrient feeding was developed to overcome the limitation in batch fermentation and to increase the CGTase production from Bacillus sp. TS1-1 in fed batch fermentation. Optimisation of the C/N ratio in the feed stream was conducted in a 5 l fermenter, where feeding was initiated at constant rate of 0.02 h⁻¹. In our initial screening process, the addition of nitrogen source boosted the growth of the microbes, but on the other hand reduced the CGTase production. The amount of tapioca starch and yeast extract was optimised in order to obtain a sufficient growth and thus, increased the CGTase production. Results were analysed using three-dimensional response surface plot, and the optimised values of carbon and nitrogen concentration of 3.30% (w/v) and 0.13% (w/v) were obtained, respectively. CGTase activity increased up to 80.12 U/ml, which is 13.94% higher as compared to batch fermentation (70.32 U/ml). This also led to 14.54% increment of CGTase production in fed batch culture as compared to the production before the optimisation. The CGTase activity obtained was close to the predicted value, which is 78.05 U/ml.     

Nanosecond electric pulses induce DNA breaks in cisplatin-sensitive and -resistant human ovarian cancer cells

Human ovarian cancer cells COC1 and COC1/DDP (cisplatin-resistant subline) were exposed to 6 kV/cm nanosecond electric pulses (nsEP) with a pulse length of 8, 16 or 24 ns. The potential in a subcellular unit was calculated using a multilayer dielectric spherical model, and area under the voltage–time curves (AUC) integrated with a lower limit of 0.2 V. Cell viability was determined, and double-stand and total DNA breaks detected with the neutral and alkaline comet assays. nsEP evoked a higher voltage and AUC in nucleoplasm, and the levels in COC1 cells was just above those in COC1/DDP cells. Comets only appeared in the alkaline assay demonstrating single-stand DNA break. Fewer DNA break (16.51% vs. 35.13% at 24 ns, p = 0.0150) and more survival (22.42% vs. 13.19% at 24 ns, p = 0.0015) occurred in COC1/DDP cells despite an equal electric energy and almost equal cell sizes. 24-ns EP led to higher rates of cell-death and comet. The comet rate correlated with cell-death fraction in either cell line (r = 0.5701, p = 0.0135; r = 0.5110, p = 0.0302). There was no a correlation between the tail length, tail moment or Olive tail moment and cell-death rate. The data showed that response of chemosensitive cells differed from that of chemoresistant cells and DNA damage contributed to percent of cell death.     

Assessment of alterations in X-ray irradiation-induced DNA damage of glioma cells by using proton nuclear magnetic resonance spectroscopy

Glioma is one of the most common types of brain tumors. DNA damage is closely associated with glioma cell apoptosis induced by X-ray irradiation. Alterations of metabolites in glioma can be detected noninvasively by proton nuclear magnetic resonance (1H NMR) spectroscopy. To noninvasively explore the micro mechanism in X-ray irradiation-induced apoptosis, the relationship between metabolites and DNA damage in glioma cells was investigated. Three glioma cell lines (C6, U87 and U251) were randomly designated as control (0 Gy) and treatment groups (1, 5, 10, 15 Gy). After X-ray exposure, each group was separated into four parts: (i) to detect metabolites by 1H NMR spectroscopy; (ii) to make cell colonies; (iii) to detect cell cycle distribution and apoptosis rate by flow cytometry; and (iv) to measure DNA damage by comet assay. The metabolite ratios of lactate/creatine and succinate/creatine decreased (lactate/creatine: C6, 22.17–66.27%; U87, 15.93–44.56%; U251, 26.27–74.48%. succinate/creatine: C6, 14.41–48.35%; U87, 22.03–70.62%; U251, 17.33–60.06%) and choline/creatine increased (C6, 52.22–389.68%; U87, 56.15–82.36%; U251, 31.87–278.62%) in the treatment groups compared with the control group (each P < 0.05), which linearly depended on DNA damage. An increasing dose of X-ray irradiation increased numbers of apoptotic cells (P < 0.01), and the DNA damage parameters were dose-dependent (P < 0.05). The colony-forming rate declined (P < 0.01) and the percentage of cells at G1 stage increased when exposed to 1 Gy X-ray (three cell lines, P < 0.05). Metabolite alterations detected by 1H NMR spectroscopy can be used to determine DNA damage induced by X-ray irradiation. 1H NMR spectroscopy is a noninvasive method to predict DNA damage of glioma cell at the micro level.     

Hyaluronic acid interaction with chitosan-conjugated magnetite particles and its purification

The polyelectrolyte complex (PEC) effect between hyaluronic acid (HA) and chitosan was explored to recover HA from fermentation broth. Chitosan was conjugated with the magnetic nanoparticles by co-precipitation method to facilitate its recovery. The magnetic chitosan particles (chitosan–magnetite) have an average size about 5 μm and point of zero charge (PZC) around 6.5. pH lower than PZC favored the HA capture. About 39 mg of HA was captured per gram of particles at pH 6. Nearly quantitative release of captured HA was achieved at pH 8. Although HA could not be directly isolated from Streptococcus zoopedemics fermentation broth by manipulating pH between 6 and 8, HA free of contaminant protein could be purified from the crude ethanol precipitate using chitosan–magnetite.     

Effect of dithiocarbamate pesticide zineb and its commercial formulation,azzurro: IV. DNA damage and repair kinetics assessed by single cell gel electrophoresis (SCGE) assay on Chinese hamster ovary (CHO) cells

Single cell gel electrophoresis (SCGE) was used to analyse dithiocarbamate zineb- and the zineb-containing technical formulation azzurro-induced DNA damage and repair in CHO cells. Cells were treated with zineb (50.0 μg/ml) or azzurro (100.0 μg/ml) for 80 min, washed and reincubated in pesticide-free medium for 0–12 h until SCGE. Viability of treated cells (0 h) did not differ from control remaining unchanged up to 6 h of incubation. After 12 h, viability decreased up to 70 and 54% in zineb- and azzurro-treated cultures, respectively. SCGE revealed at 0 h the absence of undamaged cells and an increase of slightly damaged and damaged cells in zineb-treated cultures or by an increase in damaged cells in azzurro-treated cultures. For both chemicals, a time-dependent repair of pesticide-induced DNA damage within a 0–12 h post-treatment incubation period was observed. Overall, damaged cells decreased as a function of the repair time for both pesticides while the slightly damaged cells decreased as a function of the repair time of zineb-induced DNA damage. Concomitantly, a time-dependent increase of undamaged cells was observed within the 0.5–12 h repair time for both pesticides. At 12 h after treatment, no differences in the frequencies of undamaged, slightly damaged and damaged cells were found between both zineb- or azzurro-treated cultures and control values as well as between zineb- and azzurro-treated cells. Immediately after exposure, nuclear DNA from zineb and azzurro-treated cells were larger and wider than nuclear DNA from untreated cells. When damaged cells were allowed to repair, a time-dependent decrease of the amount of free DNA migrating fragments was observed committed only to damaged cells but not in slightly or undamaged cells. On the other hand, no time-dependent alteration on nuclear DNA width within the 0–12 h repair period was observed.     

Skeletal muscle pericyte subtypes differ in their differentiation potential

Neural progenitor cells have been proposed as a therapy for central nervous system disorders, including neurodegenerative diseases and trauma injuries, however their accessibility is a major limitation. We recently isolated Tuj1 + cells from skeletal muscle culture of Nestin–GFP transgenic mice however whether they form functional neurons in the brain is not yet known. Additionally, their isolation from nontransgenic species and identification of their ancestors is unknown. This gap of knowledge precludes us from studying their role as a valuable alternative to neural progenitors. Here, we identified two pericyte subtypes, type-1 and type-2, using a double transgenic Nestin–GFP/NG2–DsRed mouse and demonstrated that Nestin–GFP +/Tuj1 + cells derive from type-2 Nestin–GFP +/NG2–DsRed +/CD146 + pericytes located in the skeletal muscle interstitium. These cells are bipotential as they generate either Tuj1 + cells when cultured with muscle cells or become “classical” α-SMA + pericytes when cultured alone. In contrast, type-1 Nestin–GFP ?/NG2–DsRed +/CD146 + pericytes generate α-SMA + pericytes but not Tuj1 + cells. Interestingly, type-2 pericyte derived Tuj1 + cells retain some pericytic markers (CD146 +/PDGFRβ +/NG2 +). Given the potential application of Nestin–GFP +/NG2–DsRed +/Tuj1 + cells for cell therapy, we found a surface marker, the nerve growth factor receptor, which is expressed exclusively in these cells and can be used to identify and isolate them from mixed cell populations in nontransgenic species for clinical purposes.     

Solid-phase extraction of tanshinones from Salvia Miltiorrhiza Bunge using ionic liquid-modified silica sorbents

New ionic liquid-modified silica sorbents were developed by the surface chemical modification of the commercial silica using synthesized ionic liquids. The obtained ionic liquid-modified particles were successfully used as a special sorbent in solid-phase extraction process to isolation of cryptotanshinone, tanshinone I and tanshinone IIA from Salvia Miltiorrhiza Bunge. Different washing and elution solvents such as water, methanol and methanol–acetic acid (90/10, v/v) were evaluated. A comparison of ionic liquid-modified silica cartridges and traditional silica cartridge show that higher recovery was observed using ionic liquid-modified silica sorbents. A quantitative analysis was conducted by high-performance liquid chromatography using a C₁₈ column (5 μm, 150 mm × 4.6 mm) with methanol–water (78:22, v/v, and containing 0.5% acetic acid) as a mobile phase. Good linearity was obtained from 0.5 × 10^?4 to 0.5 mg/mL (r² > 0.999) with the relative standard deviations less than 4.8%.     

Enzymatic synthesis of piceid glycosides by cyclodextrin glucanotransferase

Novel glycosides of piceid (3,4′-5-trihydroxy stilbene 3-O-β-d-glucoside) were produced by the transglycosylation reactions of cyclodextrin glucanotransferase (CGTase) from Bacillus macerans, with piceid (PicG₁) and maltodextrin as the acceptor and donor substrates, respectively. The reactions were performed at 40 °C with 2.56 mM piceid (0.1% w/v) and maltodextrin (5% w/v) in 0.02 M citrate phosphate buffer, pH 6.0 containing 5% (v/v) methanol for 6 h. Glucose, maltose, sucrose, maltotriose and α-cyclodextrin (α-CD) were also used to analyze their ability to function as donor substrates, for the glycosylation of piceid. Among the different donor substrates used, the maximum transfer efficiency (TE) of glycosylation of piceid was observed for α-cyclodextrin (78.9%) followed by maltodextrin (72.1%). The partially purified piceid glycoside products (PicG₂ and PicG₃) were identified by mass spectrometry.     

Domain replacement to elucidate the role of B domain in CGTase thermostability and activity

The B domain of CGTase has been generally accepted as a domain involved in thermostability. However, limited work has been performed in which entire B domain is substituted with the thermostable counterpart. Using overlap extension PCR, we replaced the B domain of a variant of CGTase Bacillus sp. G1 by six other B domains from thermostable CGTases. Likely due to distortion in the substrate-binding cleft adjacent to the active site, variants with the domain replacements from Thermoanaerobacter, Thermococcus, Thermococcus kodakarensis, Anaerobranca gottschalkii and Pyrococcus furiosus completely lost their catalytic function. A mutant designated Cgt_ET1 with a domain replacement from a Bacillus stearopthermophilus ET1 CGTase was the only variant that retained activity after domain exchange. Both the parental enzyme and the mutant Cgt_ET1 had an identical optimum temperature at 60 °C. The activity half-life was 22 min for the parental CGTase, whereas a marked increase to 57 min was observed for the mutant. Further mutagenesis on Cgt_ET1 was performed at residue 188 by replacing a Phe residue with Tyr. The mutant Cgt_ET1_F188Y displayed a decreased activity half-life of 28 min. Both mutants exhibited a better cyclodextrin-forming ability and a faster turnover rate (k_cat) than the parental CGTase.     

Evaluation of Tris–citric acid,skim milk and sodium citrate extenders for liquid storage of Punjab Urial (Ovis vignei punjabiensis) spermatozoa

The Punjab Urial (Ovis vignei punjabiensis) is an endangered subspecie of ovidae, distributed as small scattered populations in the forest belt of the Himalayan foothills of Pakistan and in the areas enclosed by the Indus and the Jhelum rivers. The present study was conducted to evaluate the liquid storage of Punjab Urial spermatozoa in different extenders for use in future in situ conservation activities. Semen was collected by electro-ejaculation from three captive Punjab Urial rams. Suitable ejaculates of individual animals were pooled and divided into three aliquots for dilution with the experimental extenders (Tris–citric acid, skim milk and sodium citrate) at 37 °C. Extended semen was cooled from 37 °C to 5 °C in 2 h, and stored for three days at 5 °C. Sperm motility (%), viability (%; live/dead), acrosome integrity (%) and plasma membrane integrity (%) were assessed on days 1, 2 and 3 of storage. On day 1, sperm motility, viability as well as acrosome and plasma membrane integrity were similar (p > 0.05) in all three experimental extenders. On day 2, sperm motility, viability, acrosome and plasma membrane integrity were higher (p < 0.05) in Tris–citric acid extender compared to sodium citrate based extender. On day 3 of storage, the values of motility, viability and acrosome integrity were higher (p < 0.05) in Tris–citric acid extender than in skim milk and sodium citrate based extenders. In conclusion, Tris–citric acid extender appears to be a better option compared with skim milk and sodium citrate extenders for liquid storage of Punjab Urial semen.     

Small molecule tolfenamic acid and dietary spice curcumin treatment enhances antiproliferative effect in pancreatic cancer cells via suppressing Sp1, disrupting NF-kB translocation to nucleus and cell cycle phase distribution

Combination of dietary/herbal spice curcumin (Cur) and COX inhibitors has been tested for improving therapeutic efficacy in pancreatic cancer (PC). The objective of this study was to identify agent with low toxicity and COX-independent mechanism to induce PC cell growth inhibition when used along with Cur. Anticancer NSAID, tolfenamic acid (TA) and Cur combination were evaluated using PC cell lines. L3.6pl and MIA PaCa-2 cells were treated with Cur (5–25 μM) or TA (25–100 μM) or combination of Cur (7.5 μM) and TA (50 μM). Cell viability was measured at 24–72 h posttreatment using CellTiter-Glo kit. While both agents showed a steady/consistent effect, Cur + TA caused higher growth inhibition. Antiproliferative effect was compared with COX inhibitors, Ibuprofen and Celebrex. Cardiotoxicity was assessed using cordiomyocytes (H9C2). The expression of Sp proteins, survivin and apoptotic markers (western blot), caspase 3/7 (caspase-Glo kit), Annexin-V staining (flow cytometry), reactive oxygen species (ROS) and cell cycle phase distribution (flow cytometry) was measured. Cells were treated with TNF-α, and NF-kB translocation from cytoplasm to nucleus was evaluated (immunofluorescence). When compared to individual agents, combination of Cur + TA caused significant increase in apoptotic markers, ROS levels and inhibited NF-kB translocation to nucleus. TA caused cell cycle arrest in G₀/G₁, and the combination treatment showed mostly DNA synthesis phase arrest. These results suggest that combination of Cur + TA is less toxic and effectively enhance the therapeutic efficacy in PC cells via COX-independent mechanisms.     

Manganese species from human serum,cerebrospinal fluid analyzed by size exclusion chromatography-, capillary electrophoresis coupled to inductively coupled plasma mass spectrometry

Manganese (Mn) at high concentrations can have adverse effects on health, mainly because of its toxicity to the central nervous system. Health impacts of Mn are known mostly from occupational health studies, but the exact mechanisms how Mn, being bound to transferrin (TF) in the blood, enters the brain – are unknown. Mn speciation at the neural barriers can help to obtain more information about the pathways and carriers. This paper summarizes investigations on the size distribution of Mn carriers (e.g. proteins, peptides, carbonic acids) in serum before the neural barriers and in cerebrospinal fluid (CSF) behind them as a first characterization step of the Mn carriers being involved in moving Mn across the neural barriers. Further identification of Mn-species in CSF was successfully achieved by CZE–inductively coupled plasma (ICP)–dynamic reaction cell (DRC)–mass spectrometry (MS). Serum samples showed Mn mean concentrations of 1.7±0.8 μg L⁻¹. The size distribution of Mn-carriers showed a main peak in the TF/albumin size fitting to the known physiological ligands. However, also an increasing Mn peak at 700 Da with increasing total Mn concentration was seen. Samples of CSF showed Mn mean concentrations of 2.6 μg L⁻¹=48 nM. In CSF Mn was found to be mostly bound to low-molecular-mass (LMM)-Mn carriers in the range of 640–680 Da. This is similar to the LMM compound in serum and to Mn–citrate complexes suggested to be present in body fluids. Citrate concentration was 573 μM, thus being in huge excess compared to Mn. CSF was further analyzed by CZE–ICP–DRC–MS. Several Mn-species were monitored and mostly identified. The most abundant Mn-species was Mn–citrate at a concentration of around 0.7 μg Mn L⁻¹.     

Endothelial actin and cell stiffness is modulated by substrate stiffness in 2D and 3D

There is a growing appreciation of the profound effects that passive mechanical properties, especially the stiffness of the local environment, can have on cellular functions. Many experiments are conducted in a 2D geometry (i.e., cells grown on top of substrates of varying stiffness), which is a simplification of the 3D environment often experienced by cells in vivo. To determine how matrix dimensionality might modulate the effect of matrix stiffness on actin and cell stiffness, endothelial cells were cultured on top of and within substrates of various stiffnesses. Endothelial cells were cultured within compliant (1.0–1.5 mg/ml, 124±8 to 202±27 Pa) and stiff (3.0 mg/ml, 502±48 Pa) type-I collagen gels. Cells elongated and formed microvascular-like networks in both sets of gels as seen in previous studies. Cells in stiffer gels exhibited more pronounced stress fibers and ～1.5-fold greater staining for actin. As actin is a major determinant of a cell's mechanical properties, we hypothesized that cells in stiff gels will themselves be stiffer. To test this hypothesis, cells were isolated from the gels and their stiffness was assessed using micropipette aspiration. Cells isolated from relatively compliant gels were 1.9-fold more compliant than cells isolated from relatively stiff gels (p<0.05). Similarly, cells cultured on top of 1700 Pa polyacrylamide gels were 2.0-fold more compliant that those cultured on 9000 Pa (p<0.05). These data demonstrate that extracellular substrate stiffness regulates endothelial stiffness in both three- and two-dimensional environments, though the range of stiffnesses that cells respond to vary significantly in different environments.     

Isolation and characterization of tetrahydrofuran-degrading Rhodococcus aetherivorans strain M8

We isolated tetrahydrofuran (THF)-degrading bacteria from waste sludge obtained from a chemical factory in Japan. The isolate designated as strain M8 was identified as Rhodococcus aetherivorans by sequence analysis of its 16S rRNA gene. It grew in a medium containing THF as the sole source of carbon and energy, and its optimal growth pH range and temperature were 6–9 and 37 °C, respectively. Strain M8 grew even in the presence of 35 mM THF. For its growth, this bacterium used 1,4-butanediol and γ-butyrolactone, which are supposed to be metabolites of THF. To elucidate the pathway involved in THF metabolism in strain M8, the resting cell reaction was performed, and the metabolites of THF were analyzed. In the resting cell reaction, 5 mM THF was completely degraded within 5 h. Cells were harvested at 2, 3, and 4 h after the initiation of the reaction; the intermediates accumulated in the cells were extracted using methanol and were derivatized using phenyl boronate. Gas chromatography–mass spectrometry (GC–MS) analysis of the derivatized products showed 4-hydroxybutyrate accumulating in the resting cells. This result suggests that R. aetherivorans strain M8 degrades THF via the oxidation pathway.