Galanthamine production by Leucojum aestivum L. shoot culture in a modified bubble column bioreactor with internal sections

Shoot culture of summer snowflake (Leucojum aestivum L.) was successfully cultivated in an advanced modified glass‐column bioreactor with internal sections for production of Amaryllidaceae alkaloids. The highest amounts of dry biomass (20.8 g/L) and galanthamine (1.7 mg/L) were achieved when shoots were cultured at 22°C and 18 L/(L·h) flow rate of inlet air. At these conditions, the L. aestivum shoot culture possessed mixotrophic‐type nutrition, synthesizing the highest amounts of chlorophyll (0.24 mg/g DW (dry weight) chlorophyll A and 0.13 mg/g DW chlorophyll B). The alkaloids extract of shoot biomass showed high acetylcholinesterase inhibitory activity (IC₅₀ = 4.6 mg). The gas chromatography–mass spectrometry (GC/MS) profiling of biosynthesized alkaloids revealed that galanthamine and related compounds were presented in higher extracellular proportions while lycorine and hemanthamine‐type compounds had higher intracellular proportions. The developed modified bubble‐column bioreactor with internal sections provided conditions ensuring the growth and galanthamine production by L. aestivum shoot culture.     

Multiple sources of carbonic anhydrase activity in pea thylakoids: soluble and membrane-bound forms

Carbonic anhydrase (CA) activity of pea thylakoids, thylakoid membranes enriched with photosystem I (PSI-membranes), or photosystem II (PSII-membranes) as well as both supernatant and pellet after precipitation of thylakoids treated with detergent Triton X-100 were studied. CA activity of thylakoids in the presence of varying concentrations of Triton X-100 had two maxima, at Triton/chlorophyll (triton/Chl) ratios of 0.3 and 1.0. CA activities of PSI-membranes and PSII-membranes had only one maximum each, at Triton/Chl ratio 0.3 or 1.0, respectively. Two CAs with characteristics of the membrane-bound proteins and one CA with characteristics of the soluble proteins were found in the medium after thylakoids were incubated with Triton. One of the first two CAs had mobility in PAAG after native electrophoresis the same as that of CA residing in PSI-membranes, and the other CA had mobility the same as the mobility of CA residing in PSII-membranes, but the latter was different from CA situated in PSII core-complex (Ignatova et al. 2006 Biochemistry (Moscow) 71:525–532). The properties of the “soluble” CA removed from thylakoids were different from the properties of the known soluble CAs of plant cell: apparent molecular mass was about 262 kD and it was three orders more sensitive to the specific CA inhibitor, ethoxyzolamide, than soluble stromal CA. The data are discussed as indicating the presence of, at least, four CAs in pea thylakoids.     

Na/K-ATPase tethers phospholipase C and IP3 receptor into a calcium-regulatory complex

We have shown that the caveolar Na/K-ATPase transmits ouabain signals via multiple signalplexes. To obtain the information on the composition of such complexes, we separated the Na/K-ATPase from the outer medulla of rat kidney into two different fractions by detergent treatment and density gradient centrifugation. Analysis of the light fraction indicated that both PLC-gamma1 and IP3 receptors (isoforms 2 and 3, IP3R2 and IP3R3) were coenriched with the Na/K-ATPase, caveolin-1 and Src. GST pulldown assays revealed that the central loop of the Na/K-ATPase alpha1 subunit interacts with PLC-gamma1, whereas the N-terminus binds IP3R2 and IP3R3, suggesting that the signaling Na/K-ATPase may tether PLC-gamma1 and IP3 receptors together to form a Ca(2+)-regulatory complex. This notion is supported by the following findings. First, both PLC-gamma1 and IP3R2 coimmunoprecipitated with the Na/K-ATPase and ouabain increased this interaction in a dose- and time-dependent manner in LLC-PK1 cells. Depletion of cholesterol abolished the effects of ouabain on this interaction. Second, ouabain induced phosphorylation of PLC-gamma1 at Tyr(783) and activated PLC-gamma1 in a Src-dependent manner, resulting in increased hydrolysis of PIP2. It also stimulated Src-dependent tyrosine phosphorylation of the IP3R2. Finally, ouabain induced Ca(2+) release from the intracellular stores via the activation of IP3 receptors in LLC-PK1 cells. This effect required the ouabain-induced activation of PLC-gamma1. Inhibition of Src or depletion of cholesterol also abolished the effect of ouabain on intracellular Ca(2+).     

The NMDA receptor is coupled to the ERK pathway by a direct interaction between NR2B and RasGRF1

The NMDA subtype of glutamate receptors (NMDAR) at excitatory neuronal synapses plays a key role in synaptic plasticity. The extracellular signal-regulated kinase (ERK1,2 or ERK) pathway is an essential component of NMDAR signal transduction controlling the neuroplasticity underlying memory processes, neuronal development, and refinement of synaptic connections. Here we show that NR2B, but not NR2A or NR1 subunits of the NMDAR, interacts in vivo and in vitro with RasGRF1, a Ca(2+)/calmodulin-dependent Ras-guanine-nucleotide-releasing factor. Specific disruption of this interaction in living neurons abrogates NMDAR-dependent ERK activation. Thus, RasGRF1 serves as NMDAR-dependent regulator of the ERK kinase pathway. The specific association of RasGRF1 with the NR2B subunit and study of ERK activation in neurons with varied content of NR2B suggests that NR2B-containing channels are the dominant activators of the NMDA-dependent ERK pathway.     

Integrative taxonomy provides evidence for the species status of the Ibero‐Maghrebian grass snake Natrix astreptophora

The grass snake (Natrix natrix) is Europe's most widely distributed and, in many regions, most common snake species, with many morphologically defined subspecies. Yet, the taxonomy of grass snakes is relatively little studied and recent work has shown major conflicts between morphologically defined subspecies and phylogeographical differentiation. Using external morphology, osteological characters, and information from 13 microsatellite loci and two mitochondrial markers, we examine differentiation of the subspecies N. n. astreptophora from the North African Maghreb region, the Iberian Peninsula and neighbouring France. According to previous studies, N. n. astreptophora corresponds to a deeply divergent mitochondrial clade and constitutes the sister taxon of all remaining grass snakes. In the French Pyrenees region, there is a contact zone of N. n. astreptophora with another subspecies, N. n. helvetica. Our analyses of microsatellites and mitochondrial DNA reveal that the distribution ranges of the two taxa abut there, but both hybridize only exceptionally. Even though many morphological characters are highly variable and homoplastic in grass snakes, N. n. astreptophora differs consistently from all other grass snakes by its reddish iris coloration and in having significantly fewer ventral scales and another skull morphology. Considering further the virtual absence of gene flow between N. n. astreptophora and N. n. helvetica, and acknowledging the morphological distinctiveness of N. n. astreptophora and its sister group relationship to all remaining subspecies of grass snakes, we conclude that Natrix astreptophora (Seoane, 1884) should be recognized as a distinct species. Further research is needed to explore whether N. astreptophora is polytypic because a single sample of N. astreptophora from Tunisia turned out to be genetically highly distinct from its European conspecifics.     

Thin semitransparent gels containing phenylboronic acid: porosity, optical response and permeability for sugars

Radical copolymerization of acrylamide (Am) (90 mol%) with N-acryloyl-m-aminophenylboronic acid (NAAPBA) (10 mol%) carried out on the surface of glass slides in aqueous solution and in the absence of chemical cross-linkers, resulted in the formation of thin semitransparent gels. The phenylboronic acid (PBA) ligand density was ca. 160 micromol/ml gel. The gels exhibited a macroporous structure and displayed optical response to sucrose, lactose, glucose and fructose in 50 mM sodium phosphate buffer, in the pH range from 6.5 to 7.5. The response was fairly reversible and linearly depended on glucose concentration in the wide concentration range from 1 to 60 mM at pH 7.3. The character of response was explained by the balance of two competing equilibrium processes: binding of glucose to phenylboronate anions and binary hydrophobic interactions of neutral PBA groups. The apparent diffusion coefficient of glucose in the gels was ca. 2.5 x 10(-7) cm(2)/s. A freshly prepared gel can be used daily for at least 1 month without changes in sensitivity. Autoclaving (121 degrees C, 1.2 bar, 10 min) allows for the gels sterilization, which is important for their use as glucose sensors in fermentation processes.     

Mechanism of IFN-gamma-induced endocytosis of tight junction proteins: myosin II-dependent vacuolarization of the apical plasma membrane

Disruption of epithelial barrier by proinflammatory cytokines such as IFN-gamma represents a major pathophysiological consequence of intestinal inflammation. We have previously shown that IFN-gamma increases paracellular permeability in model T84 epithelial cells by inducing endocytosis of tight junction (TJ) proteins occludin, JAM-A, and claudin-1. The present study was designed to dissect mechanisms of IFN-gamma-induced endocytosis of epithelial TJ proteins. IFN-gamma treatment of T84 cells resulted in internalization of TJ proteins into large actin-coated vacuoles that originated from the apical plasma membrane and resembled the vacuolar apical compartment (VAC) previously observed in epithelial cells that lose cell polarity. The IFN-gamma dependent formation of VACs required ATPase activity of a myosin II motor but was not dependent on rapid turnover of F-actin. In addition, activated myosin II was observed to colocalize with VACs after IFN-gamma exposure. Pharmacological analyses revealed that formation of VACs and endocytosis of TJ proteins was mediated by Rho-associated kinase (ROCK) but not myosin light chain kinase (MLCK). Furthermore, IFN-gamma treatment resulted in activation of Rho GTPase and induced expressional up-regulation of ROCK. These results, for the first time, suggest that IFN-gamma induces endocytosis of epithelial TJ proteins via RhoA/ROCK-mediated, myosin II-dependent formation of VACs.     

Stress granules: RNP-containing cytoplasmic bodies springing up under stress. The structure and mechanism of organization

In this review recent data describing stress granules are summarized. Stress granules are specific RNA-containing structures in the cytoplasm of living cells which arise under stress conditions (e. g. heat shock, UV irradiation, energy depletion and oxidative stress). It became evident that stress granules accumulate non-canonical 48S initiation complexes and contain mRNA with associated proteins, small ribosomal subunits and some initiation factors. Stress granules are depleted with ternary complex and large ribosomal subunit. It's proposed that eIF2alpha phosphorylation and ternary complex decrease can be a trigger for stress granule formation. Shuttling nuclear and cytoplasmic RNA-binding protein TIA-1 plays a crucial role in this process. It's proposed that TIA-1 forms prion-like aggregates, and these aggregates are scaffolds for other components of stress granules. Cytoskeletal structures facilitate the accumulation of stress granule components in local cytoplasmic sites. Investigation of process of stress granule formation is important for understanding of cell reaction to stress and translation regulation mechanisms.     

Somatic mutagenesis at T-cell receptor locus in inhabitants of radiation polluted regions as a result of the Chernobyl disaster

In the period of 2001-2004, frequency of cells bearing mutations at T-cell receptor (TCR) locus was assessed in 553 inhabitants of radiation polluted regions of the Russian Federation and 154 unexposed control persons. The inhabitants were divided into three groups according to age at the moment of the Chernobyl disaster and 137Cs pollution density: 1) in utero, 37-555 kBq/m2; 2) 0-14 years old, 20-555 kBq/m2; 3) 18 and more years old, highest 137Cs density (185 more than 555 kBq/m2). The most intense changes of the TCR-mutant cell frequency were observed in the group of persons exposed to ionizing radiation in utero. The mean frequency of the mutant cells was higher in the first group than in age-matched control group by about 1.5-fold: 4.0 x 10(-4) vs 2.7 x 10(-4) accordingly (p < 0.0001). Elevation in the mean TCR-mutant cell frequency was less expressed in group of inhabitants aged 0-14 years at the moment of irradiation start: 1.3-fold increase in comparison to age-matched control (3.8 x 10(-4) vs 2.9 x 10(-4), p = 0.0002). It was not found significant differences in mutant cell frequencies between control group and adults consisting in the third group (18 and more years old at the moment of the Chernobyl accident). The changes of the TCR-mutant cell frequency in persons exposed in pre- and postnatal periods differ not only quantitatively, but qualitatively. In the fist case all persons react to irradiation by increasing number of the TCR-mutant cells in some degree. In the second case - only a part of population. Proportion of reacting persons depends on age at the start of irradiation and, perhaps, on dose absorbed. The TCR-mutant frequency was significantly higher in persons with benign tumors of different localizations and nodules in thyroid gland than in persons without this pathology.