Catalpol protects primary cultured cortical neurons induced by Aβ1–42 through a mitochondrial-dependent caspase pathway

It has been reported that catalpol, an iridoid glucoside, isolated from the root of Rehmannia glutinosa, protected cells from damage induced by a variety of toxic stimulus such as LPS, MPP⁺ and rotenone. Here, we further evaluated the effect of catalpol against Aβ_1–42-induced apoptosis in primary cortical neuron cultures. In the present study, the primary cortical neuron culture treated with Aβ_1–42 was severed as cell model of Alzheimer's disease (AD) in vitro. By exposure to Aβ_1–42 (5 μM) for 72 h in cultures, neuronal apoptosis occurred characterized by enhancement of activities of caspases and reactive oxygen species (ROS) as well as Bax increase, loss of mitochondrial membrane potential and cytochrome c release. Pretreatment with catalpol (0.5 mM) for 30 min prior to Aβ_1–42 treatment attenuated neuronal apoptosis not only by reversing intracellular ROS accumulation, Bax level, mitochondrial membrane potential and, cytochrome c release to some extent, but also through regulating the activity and cleavage of caspase-3 and caspase-9. Thus, catalpol protects primary cultured cortical neurons induced by Aβ_1–42 through a mitochondrial-dependent caspase pathway.     

Taxol production in suspension cultures of Taxus baccata

The response of Taxus baccata (PC2) to basic manipulations of culture conditions is described. Suspension cultures of Taxus baccata (PC2) were maintained at 25°C on a modified B5 medium with two-week transfers. Under these conditions, no taxol^® is formed. However, if the cells are left in the same medium for 7 or more additional days, taxol is produced and released (ca. 90%) into the extracellular medium. Levels as high as 13 mg 1^–1 extracellular taxol were achieved in shake flask cultures and taxol was the primary taxane formed representing between 50 and 80% of total taxane in the medium. The cells are sensitive to changes in culture conditions and cultures cycle through periods of high (13 mg 1^–1) and low (<0.1 mg 1^–1) levels of taxol production during extended culture. Picloram was the most effective of the auxins tested with respect to cell growth but it suppressed taxol production. Addition of fructose to moderately-productive cultures (ca. 4 mg 1^–1) improved taxol production, but cultures in a high producing state did not respond. Glucose suppressed taxane production. Two isoprenoids (geraniol and pinene) had a modest effect on taxol production when added to cultures at 10 mg 1^–1.®|Taxol is a registered trademark of Bristol Meyer Squibb for paclitaxel     

Use of swimming speed and egg ratio as predictors of the status of rotifer cultures in aquaculture

This study evaluated the use of egg ratio (eggs rotifer^–1) and swimming speed (mm min^–1) as prediction criteria for production and culture quality in mass cultures of the rotifer Brachionus plicatilis. Egg ratio was determined to be a suitable predictor of rotifer growth and production in the cultures. Low egg ratios (i.e., 0–0.17 eggs rotifer^–1) indicate reduced rotifer population over time (i.e., negative net population growth rates). However, at this time egg ratio dynamics are not suitably understood to predict in advance a sudden population collapse.Swimming speed of reproductive, egg-carrying females in the exponential growth phase was 40–45 mm min^–1. During exponential growth swimming speed was independent of the food used. Lower swimming speeds were obtained in late stationary phase (10–25 mm min^–1) when yeast was used as a food source. Both environmental factors (e.g., accumulating metabolites) and changes in nutritional state of the rotifers may have affected the swimming speed, but environmental factors appear to be the most important. We believe that swimming speed has the potential of becoming an accurate predictor of culture quality in mass cultures of rotifers.     

Optimizing recombinant protein expression in the T7 system under the control of the proUp promoter

-Galactosidase and streptokinase expression was tested under the control of the T7 promoter in batch and fed-batch cultures. An Escherichia coli host GJ1158, which contained the T7 RNA polymerase gene under the osmo-responsive proUp promoter, was used for expression studies. -Galactosidase expression was enhanced from 26 mg l^–1 to 127 mg l^–1 in batch culture when a combination of sucrose and sorbitol was used instead of salt as an inducer. Similarly in fed-batch cultures 140 mg streptokinase l^–1 was formed with sucrose and sorbitol induction which was higher than that achieved with IPTG induced cultures.     

Modelling the production of nisin by Lactococcus lactis in fed-batch culture

Nisin production in batch culture and fed-batch cultures (sucrose feeding rates were 6, 7, 8, and 10 g l^–1 h^–1, respectively) by Lactococcus lactis subsp. lactis ATCC 11454 was investigated. Nisin production showed primary metabolite kinetics, and could be improved apparently by altering the feeding strategy. The nisin titer reached its maximum, 4,185 IU ml^–1, by constant addition of sucrose at a feeding rate of 7 g l^–1 h^–1; an increase in 58% over that of the batch culture (2,658 IU ml^–1). Nisin biosynthesis was affected strongly by the residual sucrose concentration during the feeding. Finally, a mathematical model was developed to simulate the cell growth, sucrose consumption, lactic acid production and nisin production. The model was able to describe the fermentation process in all cases.     

Excitotoxin-induced changes in transglutaminase during differentiation of cerebellar granule cells

Summary. Excitotoxicity induced by NMDA receptor stimulation is able to increase the activity of many enzymes involved in neuronal cell death. Primary cultures of rat cerebellar granule cells were used to elucidate the role of transglutaminase reaction in the excitotoxic cell response, and to evaluate the role of glutamate receptors in cell survival and degeneration. Granule neurons, maintained in vitro for two weeks, were exposed to NMDA at different stages of differentiation. Following NMDA receptor activation, increases in transglutaminase activity were observed in cell cultures. The levels of enzyme activity were higher in cells at 5 days in vitro than in those at 8–9 or 13–14 days in vitro. Moreover, NMDA exposure up-regulated tTG expression in neurons as young as 5 days in vitro. These cultures also exhibited morphological changes with clear apoptotic features. Results obtained demonstrate that susceptibility of granule cells to excitotoxicity depends on the developmental stage of neurons.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

Attachment of Pneumocystis carinii to Primary Cultures of Rat Alveolar Epithelial Cells

Pneumocystis carinii (PC) is an exclusively extracellular pathogen which causes pneumonia in immunocompromised individuals. Histologic studies have demonstrated that PC organisms attach preferentially to type I alveolar epithelial cells and rarely bind to type II cells. Previous reports have demonstrated that cultured type II cells develop a type I cell-like phenotype and express type I cell surface antigens. The current study examines the attachment of PC organisms to isolated rat type H alveolar epithelial cells as a function of time in culture. PC attachment to isolated type II cells increased as the type II cells differentiated in culture from 2.3 ± 1.2% on Day 2 to 18.4 ± 2.7% by Day 8. Previous studies have indicated a role for fibronectin (Fn) and Fn receptors as mediators of PC attachment. Addition of anti-Fn antibodies decreased attachment of PC to Day 8 type II cells from 19.4 ± 2.5% to 9.4 ± 1.9% (P < 0.01). Addition of antibodies to the α_v and α₅ integrin subunits resulted in significant decreases in PC attachment to Day 8 type II cells. Examination of expression of α_v and α₅ integrins on Day 2 and Day 8 type II cells demonstrated increased expression of both α_v and α₅ integrin subunits on Day 8 type II cells. Overall these data indicate that attachment of PC to isolated rat type II cells increases as the cells differentiate into a type I cell-like phenotype in vitro and correlates with increased expression of Fn-binding integrins on the cell surface of the cultured type II cells.     

Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Signaling and Cell Death in the Immature Central Nervous System after Hypoxia-Ischemia and Inflammation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family. The interaction of TRAIL with death receptor 4 (DR4) and DR5 can trigger apoptotic cell death. The aim of this study was to investigate the role of TRAIL signaling in neonatal hypoxia-ischemia (HI). Using a neonatal mouse model of HI, mRNA, and protein expression of TRAIL, DR5 and the TRAIL decoy receptors osteoprotegerin (OPG), mDcTRAILR1, and mDcTRAILR2 were determined. In vitro, mRNA expression of these genes was measured in primary neurons and oligodendrocyte progenitor cells (OPCs) after inflammatory cytokine (TNF-α/IFN-γ) treatment and/or oxygen and glucose deprivation (OGD). The toxicity of these various paradigms was also measured. The expression of TRAIL, DR5, OPG, and mDcTRAILR2 was significantly increased after HI. In vitro, inflammatory cytokines and OGD treatment significantly induced mRNAs for TRAIL, DR5, OPG, and mDcTRAILR2 in primary neurons and of TRAIL and OPG in OPCs. TRAIL protein was expressed primarily in microglia and astroglia, whereas DR5 co-localized with neurons and OPCs in vivo. OGD enhanced TNF-α/IFN-γ toxicity in both neuronal and OPC cultures. Recombinant TRAIL exerted toxicity alone or in combination with OGD and TNF-α/IFN-γ in primary neurons but not in OPC cultures. The marked increases in the expression of TRAIL and its receptors after cytokine exposure and OGD in primary neurons and OPCs were similar to those found in our animal model of neonatal HI. The toxicity of TRAIL in primary neurons suggests that TRAIL signaling participates in neonatal brain injury after inflammation and HI.     

Biomass production in mixotrophic culture of Euglena gracilis under acidic condition and its growth energetics

When Euglena gracilis was grown in the heterotrophic condition with glucose and (NH₄)₂SO₄ as the carbon and nitrogen source, a high cell yield (4.28–4.48 g l^–1) was obtained and the culture pH decreased to 1.6–2. The biomass production in the heterotrophic culture was compared to those in the autotrophic and mixotrophic cultures. Autotrophic growth was 4.7–6.3% of the heterotrophic one, whereas about 15–19% higher growth was obtained in the mixotrophic culture. Moreover, good production of chlorophyll (39.4 mg l^–1) and carotenoids (13.8 mg l^–1) were attained in the mixotrophic culture, giving the highest fermenter productivity with respect to biomass as well as chlorophyll and carotenoids. Through an energetic analysis in the mixotrophic culture, it was estimated about 25–28% of the total ATP requirement is formed in the photochemical reactions. This resulted in an improved biomass production in the mixotrophic culture of E. gracilis.     

Effects of Ranolazine on Astrocytes and Neurons in Primary Culture

Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10⁻⁷, 10⁻⁶ and 10⁻⁵ M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on pro-inflammatory mediators IL-β and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 β and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents.     

Characteristics of mixotrophic growth of Synechocystis sp. in an enclosed photobioreactor

Synechocystis sp. PCC 6803 was grown in a 2.5 l enclosed photobioreactor on medium with or without glucose. The incident light intensities ranged from 1.5 klux to 7 klux. The highest average specific growth rates of mixotrophic culture and photoautotrophic culture were, respectively, 1.3 h^–1 at a light intensity of 7 klux on 3.2 g l^–1 glucose and 0.3 h^–1 at both light intensities of 5 klux and 7 klux. The highest cell density 2.5 g l ^–1 was obtained at both of light intensities 5 klux and 7 klux on 3.2 g glucose l^–1. Glucose consumption decreased with decreasing light intensity. The energy yields of mixotrophic cultures were 4 to 6 times higher than that of photoautotrophic cultures. Light favored mixotrophic growth of Synechocystis sp. PCC 6803, especially at higher light intensities (5–7 klux).     

In vitro Decline in Plant Cultures: Detection of a Legion of Covert Bacteria as the Cause for Degeneration of Long-term Micropropagated Triploid Watermelon Cultures

In vitro cultures of triploid seedless watermelon cv. Arka Manik displayed a decline in shoot and root growth after 4–5 years of active culturing. Visibly clean cultures upon indexing on enriched media showed covert bacteria, and a significant improvement in proliferation and rooting in response to surface sterilization. The bacteria however survived endophytically. Low pH and reduced clarity of agar gelled medium were found to mask the expression of bacteria in the tissue culture medium. Gentamycin, streptomycin or broad-spectrum bactericide cefazolin provided as a 2 ml overlay in the medium in factorial combination at 0 or 50 mg l^–1 resulted in selective suppression of some bacteria depending on the treatment and eight bacterial clones comprising of four Gram-positive (Bacillus spp.) and four-Gram negative (3 × Pseudomonas spp. and 1 × Aeromonas sp.) strains were isolated from the cultures. Provision of 50 mg l^–1 gentamycin in 2 ml overlay in the multiplication or rooting medium coupled with occasional decontamination of cultures helped in circumventing the decline problem. The plants established in the field after 6 years of active in vitro culturing appeared normal and fertile suggesting the feasibility of keeping cultures for long periods, thus saving time and other resources. Freeing the cultures from covert bacteria was complicated by the presence of different bacterial types and this will be addressed later.     

Production of antithrombotic hirudin in <Emphasis Type="Italic">GAL1</Emphasis>-disrupted <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A gratuitous strain was developed by disrupting the GAL1 gene (galactokinase) of recombinant Saccharomyces cerevisiae harboring the antithrombotic hirudin gene in the chromosome under the control of the GAL10 promoter. A series of glucose-limited fed-batch cultures were carried out to examine the effects of glucose supply on hirudin expression in the gratuitous strain. Controlled feeding of glucose successfully supported both cell growth and hirudin expression in the gratuitous strain. The optimum fed-batch culture done by feeding glucose at a rate of 0.3 g h^–1 produced a maximum hirudin concentration of 62.1 mg l^–1, which corresponded to a 4.5-fold increase when compared with a simple batch culture done with the same strain.     

Anaerobic and aerobic continuous cultures of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>: comparison of plasmid stability and<Emphasis Type="Italic"> EXG1</Emphasis> gene expression

Two bioreactor continuous cultures, at anaerobic and aerobic conditions, were carried out using a recombinant Saccharomyces cerevisiae strain that over-expresses the homologous gene EXG1. This recombinant system was used to study the effect of dissolved oxygen concentration on plasmid stability and gene over-expression. Bioreactor cultures were operated at two dilution rates (0.14 and 0.03 h^–1) to investigate the effect of other process parameters on EXG1 expression. Both cultures suffered severe plasmid instability during the first 16 generations. Segregational plasmid loss rate for the aerobic culture was two-fold that of the anaerobic operation. In spite of this fact, exo--glucanase activity at aerobic conditions was 12-fold that of the anaerobic culture. This maximal activity (30 U ml^–1) was attained at the lowest dilution rate when biomass reached its greatest value and glucose concentration was zero.     

Cell suspension cultures ofOnonis natrix L. subspeciesramosissima: Establishment and culture conditions

Summary Explants from petioles, folioles or hypocotyls ofOnonis natrix have been used for calli initiation. Hypocotyls inoculated on MS medium supplemented with 2% sucrose and 0.5 mg.1^–1 2,4-D / 1 mg.1^–1 Kin showed to be the best primary explant. Cell suspension cultures were established in MS basal medium supplemented with 2% sucrose, 0.5 mg.1^–1 NAA or 2,4-D and 1 mg.1^–1 Kin. Different subculturing periods, inoculum density, hormonal supplementation and sucrose concentration were assayed in order to obtain the best culture growth conditions. The optimal conditions were achieved with cultures initiated with 40 g.1^–1 of initial inoculum, growing in MS basal medium supplemented with 4% sucrose, 0.5 mg.1^–1 NAA and 1 mg.1^–1 Kin subcultured every twelve days. Under these experimental conditions, the cultures showed a doubling time of 36.3 hours.     

Establishment of Physalis minima hairy roots culture for the production of physalins

This report describes the technique used to induce the hairy roots in Physalis minima (Linn.). Different types of explants obtained from in vitro germinated seedlings were aseptically co-cultivated with A. rhizogenesstrain LBA9402 in different media. Root growth and production of physalins were investigated in various basal media grown under dark and light conditions, and compared to that of normal root cultures. Transformed hairy root cultures grew rapidly and reach stationary phase after 15 days on a B5 medium. HPLC analysis of extracts of hairy root cultures showed that the maximum content of physalin B and F was 1.82 and 4.15 mg g^–1 DW, respectively, when grown under dark conditions. Normal root cultures produced higher physalin B (1.60–1.62 mg g^–1 DW) and F (3.30–3.75 mg g^–1 DW) under the same culture conditions. Physalin F synthesis in light-grown root cultures was reduced significantly.     

Mass cultivation of the nitrogen-fixing cyanobacteriumGloeotrichia natans,indigenous to rice-fields

Gloeotrichia natans, a nitrogen fixing cyanobacterium common in rice fields in the Philippines, was used for studies to establish key features of its physiology and potential production in outdoor cultures. Under optimal growth conditions (38 °C, pH 8.0, no carbon enrichment) the specific growth rate of rice-field isolate was 0.076 h^–1. The pH of the medium (between 6.5 and 9.0) did not influence the growth rate, but it did affect phycobiliprotein content, as reflected by a change in colour. At pH 7.0 the culture was green-brown, with phycobiliproteins constituting up to 10% of the total protein, while at pH 9.0 the culture was brownish-black and the pigment content was as high as 28% of the total protein. In outdoor cultures the specific growth rate was related directly to cell density in the range of 0.7–1.5 g dry weight 1^–1 at a rate of stirring of 30 rpm, and inversely related to cell density at half this rate. At a stirring of 30 rpm, daily production of outdoor cultures harvested to maintain cell densities of 0.7, 1.15 andw 1.5 g 1^–1 were 14.7, 17.1 and 18.1 g m^–2 d^t, respectively. This rate of production was maintained for more than 45 days. Phycobiliprotein content in the culture kept at a density of 1.5 g 1^–1 reached 14% of the total biomass.     

Heterotrophic growth of Thiobacillus acidophilus in batch and chemostat cultures

Heterotrophic growth of the facultatively chemolithoautotrophic acidophile Thiobacillus acidophilus was studied in batch cultures and in carbon-limited chemostat cultures. The spectrum of carbon sources supporting heterotrophic growth in batch cultures was limited to a number of sugars and some other simple organic compounds. In addition to ammonium salts and urea, a number of amino acids could be used as nitrogen sources. Pyruvate served as a sole source of carbon and energy in chemostat cultures, but not in batch cultures. Apparently the low residual concentrations in the steady-state chemostat cultures prevented substrate inhibition that already was observed at 150 M pyruvate. Molar growth yields of T. acidophilus in heterotrophic chemostat cultures were low. The Y _max and maintenance coefficient of T. acidophilus grown under glucose limitation were 69 g biomass · mol^–1 and 0.10 mmol · g^–1 · h^–1, respectively. Neither the Y _max nor the maintenance coefficient of glucose-limited chemostat cultures changed when the culture pH was increased from 3.0 to 4.3. This indicates that in T. acidophilus the maintenance of a large pH gradient is not a major energy-requiring process. Significant activities of ribulose-1,5-bisphosphate carboxylase were retained during heterotrophic growth on a variety of carbon sources, even under conditions of substrate excess. Also thiosulphate- and tetrathionate-oxidising activities were expressed under heterotrophic growth conditions.