Ruptured fission yeast walls

Distributions of rupture sites of fission yeast cells ruptured by glass beads have been related to a new morphometric analysis. As shown previously (Johnson et al.,Cell Biophysics, 1995), ruptures were not randomly distributed nor was their distribution dictated by geometry, rather, ruptures at the extensile end were related to cell length just as the rate of extension is related to cell length. The extension patterns of early log, mid-log, late log, and stationary phase cells from suspension cultures were found to approximate the linear growth patterns of Kubitschek and Clay (1986). The median length of cells was found to decline through the log phase in an unbalanced manner.     

Contribution to toxicity assessment of <Emphasis Type="BoldItalic">Dinophysis acuminata</Emphasis> (Dinophyceae)

Blooms of Dinophysis in French coastal waters are implicated in most bans on marketing commercial bivalves. However, the relation between Dinophysis cell density and shellfish toxicity is not always consistent. Discrepancies may be due to the simple fact that it is nearly impossible to compare an integral over a few days (shellfish toxin content) and water samples. Furthermore, it seems that cells may have a variable specific toxicity. This work focuses on the variability in cell toxicity taking into account recent findings and using liquid chromatography coupled to mass spectrometry with an ion trap and electrospray interface. Esterified analogues of okadaic acid (DTX-4 and diol-esters) have been identified in cultures of Prorocentrum lima, another okadaic acid producer. These analogues are inactive on some protein phosphatases, contrary to okadaic acid, and seem to protect the cell from harmful effects by the toxin and to be enzymatically hydrolyzed during cell lysis. In order to document specific toxicity and to validate the presence of these analogues, D. acuminata concentrates were subjected to two separate heating and freeze/thaw procedures, respectively inhibiting or promoting hydrolysis. This paper reports on the high variability of D. acuminata specific toxicity and the presence of esters found in half of the samples only.     

Cytotoxin production by a merineLyngbya strain (cyanobacterium) in a large-scale laboratory bioreactor

A cytotoxic compound was produced by the marine cyanobacteriumLyngbya sp. Pearl strain in large laboratory-scale batch cultures. Adsorption and fractionation of methanol extracts with reverse phase (C-18) cartridges provided a rapid method for removal of bioassay interference from salts, biopolymers and pigments and concentration of the cytotoxic principles. Cytotoxicity to the murine leukemia cell line P-388 was produced in two cycles coinciding with the initiation of exponential growth and again during the late exponential growth phase. Antiviral activity against influenza virus PR8 was found in extracts prepared from early exponential growth phase cells but antiviral activity was not detected in extracts of mid-log or late-log growth phase cells. These differences in bioactivity suggests that the cytotoxic principles produced during early and late exponential growth may be different compounds. Cytotoxicity assays using murine P-388 leukemia indicates that the semi-pure compound has an IC₅₀ of < 0.25 μg ml⁻¹ to this cell line. P-388 cytotoxicity in cell extracts increased during the late exponential growth phase and the specific yield was estimated at approximately 0.14 mg g⁻¹ (dry cells).     

Maximizing okadaic acid content fromProrocentrum hoffmannianum Faust

Clonal cultures ofProrocentrum hoffmannianum Faust (clone 882a) were grown under optimal environmental conditions for maximal okadaic acid production. The environmental conditions of 25 °C and 86 µmol photon m^-2 s^-1 were used to cultivateP. hoffmannianum in a semi-continuous 36-L culture vessel with continuous cell suspension and pH control. Using these conditions, a 3-fold increase in harvestable biomass and okadaic acid content was observed when compared to batch culture techniques.Author for correspondence     

Plant cells at different stages in their growth curve differentially activate promutagens

Mutagenic activity of the promutagens 2-aminofluorene (2AF) and a contaminant of 4-nitro-o-phenylenediamine (NOP-X) was followed in Ames Salmonella strain TA98 following metabolism by cotton and carrot cell suspension cultures using the plant cell/microbe coincubation assay. Both cell lines were capable of activating each chemical. However, activation capacities of the cell lines differed relative to their respective stage of growth when used. For 2AF activation early-log phase cotton cells and mid-log phase carrot cells proved superior while mid-log phase cotton cells and stationary phase carrot cells proved superior for NOP-X activation. These data indicate that the phase of the growth cycle at which plant cells are harvested can significantly affect their promutagen activation potential.     

Hydration of cyanopyridine to nicotinamide by whole cell nitrile hydratase

Summary 3-cyanopyridine was hydrated to nicotinamide by whole cells ofBrevibacterium R-312 containing nitrile hydratase. Cells used for kinetic studies had an initial activity of 0.30 mg nicotinamide/mg cells(dry)-min and storage half-lives (pH 8) of approximately 100 days, 10 days, 5 days and less than 1 day at 4°C, 10°C, 25°C, and 30°C respectively. Temperature and pH maxima were 35°C and 8.0, respectively. Fermentations gave a maximum total hydratase activity of 1.25 mg nicotinamide/min, but at this maximum the amidase activity was unacceptably high (25% of the hydratase activity): nicotinamide was converted too rapidly to nicotinic acid. But systematic fermentation studies (7 1) showed that harvesting at mid-log phase (18–20 h) prior to the attainment of maximum total activity gave reasonably high levels of hydratase (0.3 mg nicotinamide/mg cells-min) and acceptable levels of amidase (0.03 mg nicotinic acid/mg cells-min).     

Isolation of protoplasts from different Eucalyptus species and preliminary studies on regeneration

Protoplasts were isolated from different Eucalyptus clones and hybrids using mesophyll tissue, calli and cell suspension cultures. The protoplast yields differed greatly according to the starting material and adaptations of the basic procedure had to be designed in specific cases. Eucalyptus protoplasts are representative of recalcitrant woody plant systems since their proliferation is limited in culture. The best results were obtained with protoplasts from cell suspension cultures. A screening of factors increasing proliferation was performed. When some of these factors were combined the cell division frequency was enhanced and microcalli were obtained.Abbreviations B.A.P. Benzylaminopurine - 2-4D 2-4 dichlorophenoxy-acetic acid - F.D.A. Fluorescein diacetate - F.W. Fresh weight - M.E.S. Morpholino ethane sulfonic acid - M.S. Murashige & Skoog (1962) - N.A.A. Naphtalene acetic acid - TRIS Tri (hydroxymethyl amino methane) - V.KM. Medium-Kao and Michayluk medium modified by Vasil (Vasil & Vasil 1980)     

Metabolic discrimination of sucrose starvation from<Emphasis Type="Italic">Arabidopsis</Emphasis> cell suspension by<Superscript>1</Superscript>H NMR based metabolomics

Whole cell extracts ofArabidopsis cell cultures maintained on various sucrose concentrations (0,3, and 6%) were analyzed by¹H NMR spectroscopy to determine the comprehensive metabolic change in these cultures during sucrose starvation. The amount of sucrose, glucose, and fructose in the cells decreased to almost nothing after 12 h of culture in medium without sucrose. In contrast, the total free amino acid content of the cells increased as the culture proceeded. Among the free amino acids, phenylalanine and malic acid increased the most, followed by asparagine and alanine, whereas glutamic acid did not change significantly. These results are in agreement with previous studies using HPLC.¹H NMR spectroscopy enabled measurement of changes in the sugar and free amino acid content of whole cell extracts without fractionation and complicated sample preparation. These results indicate that comprehensive metabolic changes in the cells can be determined by a simple, rapid method using whole cell extracts and¹H NMR spectroscopy.     

Immunocytochemical study of cell cycle control by cytokinin in cultured soybean cells

An immunocytochemical method was used to determine the proportion of cells in the DNA synthesis (S phase) of the mitotic cycle in suspension cultures of soybean (Glycine max (L.) Merr. cv. Acme) callus of cotyledonary origin, the stably cytokinin-dependent tissue used in the cytokinin bioassay devised by Carlos O. Miller. A standard cell synchronization protocol involving hydroxyurea was used to demonstrate the applicability of the immunocytochemical method to this cell culture. Cells were brought to mitotic arrest by cytokinin withdrawal, and the cell division cycle was restarted by the addition of cytokinin. We have followed the pattern of resumption of S phase after the readdition of cytokinin. This pattern reveals the existence of three subpopulations of cells in cytokinin-starved cultures, consistent with the occurrence of three cytokinin-requiring events in the cell cycle: one in mitosis, one in S phase, and one in the G₁ phase.Abbreviations BrdU 5-bromo-2-deoxyuridine - DI deionized water - FITC fluorescein isothiocyanate - HU hydroxyurea - l-AOPP l--aminooxy--phenylpropionic acid - LI labeling index - PA polyamine - PI propidium iodide     

Effect of OA-inhibitor of protein phosphatases PP1 and PP2A — on initiation of DNA replication and mitosis in <Emphasis Type="Italic">Vicia faba</Emphasis> root meristems

According to the principal control point (PCP) hypothesis, experiments with excised, carbohydrate-starved stationary root meristems of Vicia faba var. minor have demonstrated that cells which previously divided asynchronously were preferentially blocked in G₁ (PCP1) and G₂ (PCP2) phases. When stationary phase meristems are supplied with exogenous carbohydrate (2 % sucrose), the G1- and G2-arrested cells start out DNA replication and mitotic divisions, respectively. The resumption of DNA synthesis and mitosis is not immediate and the delays of G₁- and G₂-arrested cells are found different. Using this model, we examined the effects of 4 pulse incubations with okadaic acid (OA), a specific inhibitor of PP1 and PP2A, on the duration of intervals elapsing between the provision of sucrose and the first appearance of S- and M-phase cells. We have found that depending on the period during which OA had been applied, the release from G1 and G2 phase arrest-points becomes prolonged, showing different time-course modifications. The obtained data provide evidence that activation of PP1 and PP2A is required to allow the cells for both PCP1→S and PCP2→M transitions in root meristems of V. faba.     

Okadaic acid co-induces vimentin expression and cell cycle arrest in MPC-11 mouse plasmacytoma cells

The effect of the tumor promoter okadaic acid on cell cycle progression and on vimentin expression in MPC-11 mouse plasmacytoma cells was compared with that of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Cell cycle progression of asynchronously grown MPC-11 cells was inhibited by both agents, but, in contrast to the G1 phase arrest caused by TPA, okadaic acid gave rise to G2/M phase and S phase arrest. This effect of okadaic acid was delayed significantly compared to the TPA-caused arrest. Furthermore, okadaic acid was able to induce vimentin expression to an extent comparable to the TPA response. However, vimentin expression was markedly delayed in okadaic acid-treated relative to TPA-treated cells. Another protein phosphatase inhibitor, calyculin A, also induced cell cycle changes and vimentin expression at concentrations at or above 1 × 10^?9M. Based on these observations, we suggest an involvement of protein phosphatase 1 (possibly also phosphatase 2A and/or other phosphatases) in both the G2/M cell cycle block and the induction of vimentin expression in MPC-11 cells by okadaic acid. © 1995 Wiley-Liss, Inc.     

Phosphoinositol 3 kinase inhibitor, LY294002 increases bcl-2 protein and inhibits okadaic acid-induced apoptosis in Bcl-2 expressing renal epithelial cells

Protein phosphorylation plays an indispensable role in cellular regulation of mitosis, metabolism, differentiation, and death. We previously reported that the protein phosphatase inhibitor okadaic acid (OKA) induces apoptosis in renal epithelial cells in culture. In the present study, we examined the role of phosphotidylinositol 3 (PI3) kinase signaling in okadaic acid-induced apoptosis by pre-treating normal rat kidney renal epithelial cells expressing human bcl-2 with the PI3 kinase inhibitors, LY294002 and wortmannin, followed by apoptosis-inducing concentrations of okadaic acid. Given the reported cell survival activity of PI3 kinase signaling mostly attributed to Akt kinase activation, we hypothesized that inhibition of PI3 kinase would enhance okadaic-induced apoptosis. Surprisingly, our data show that pretreatment with LY294002, but not wortmannin, attenuated okadaic acid-induced apoptosis. In contrast, to LY294002, wortmannin enhanced apoptosis. Interestingly, we also found that LY294002 treatment increased bcl-2 protein levels in normal rat kidney epithelial cells expressing bcl-2 (NRK-bcl-2). In untreated cells, bcl-2 appeared to be mainly perinuclear, coincident with the nuclear membrane, or in the cytosol. In OKA treated cells that were pre-treated with Ly294002, bcl-2 was highly co-localized with mitochondria, but in cells treated with okadaic acid alone, bcl-2 was associated with fragmented chromatin. In this model, it appears that LY294002 may exert anti-apoptotic effects by a previously unreported treatment related increase in bcl-2. Although it is widely accepted that bcl-2 protein can inhibit apoptosis, we propose that the subcellular location of bcl-2 is an important determinant in whether bcl-2 effectively inhibits apoptosis.     

Protein kinase activities in the growth cycle of suspension cultured plant cells

In batch suspension cultures of Nicotiana tabacum and Datura innoxia protein kinase activity extracted from the whole cells and assayed with casein as substrate was followed over the growth cycle. In one case (N. tabacum) the activity was also determined in the nuclei preparation obtained from the suspension cultured cells. Immediately at the onset of the growth curve the protein kinase level increases strongly and reaches a maximum value at the early phase of proliferation; the enzyme level from the nuclei is slightly delayed. A comparison with protein synthesis shows that protein kinases are among the first proteins synthesized in the growth cycle. Chromatographic separation of the enzymes contributing to the total activity revealed that both in the extract of whole cells and in the nuclei two enzyme species are present. Their time course is similar to that of the total protein kinase level, although the activity corresponding to the enzyme with the higher molecular weight in the case of the whole cell extract is slightly delayed. The possible significance of protein phosphorylation in the growth cycle is discussed.     

Okadaic acid accumulation in macrofilter feeders subjected to natural blooms of Dinophysis acuminata

Thermaikos Gulf is a eutrophic area located in the Northwestern part of the Aegean Sea in the Eastern Mediterranean. Interspecific differences among various filter feeders in their ability to accumulate okadaic acid, were observed during natural blooms of Dinophysis acuminata in the gulf. Okadaic acid analyses by high performance liquid chromatography (HPLC) were performed on benthic specimens and D. acuminata cell densities and cell toxin content were estimated in water samples. Seven filter feeding species were collected in the gulf during two DSP outbreaks in May 2003 and March 2004. The various species showed a different potential to accumulate okadaic acid in their tissues. The highest concentrations were found in the mussel populations (Mytilus galloprovincialis and Modiolus barbatus), while among the non-bivalve filter feeders, ascidians were the main accumulators of okadaic acid. The rest of shellfish populations (Flexopecten proteus, Chlamys varia and Venus verrucosa) were found to contain toxins only during 2004, when D. acuminata densities were found above 10000 cells l⁻¹. M. galloprovincialis was proved to be the most appropriate indicator for a safe warning of okadaic acid contamination in Thermaikos Gulf.     

Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells.

Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration-dependent manner with a half-maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40-60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2-M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S-phase. The absence of a change in the proportion of S-phase cells indicates that G1-specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti-tubulin demonstrated that 10 nM okadaic acid-treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid-treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression.     

Influence of the carbon source on growth and rosmarinic acid production in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei were characterized with respect to growth and rosmarinic acid formation in media with different sugars and various sugar concentrations. Sucrose is the sugar with the highest stimulating effect on growth and rosmarinic acid accumulation, followed by glucose and fructose. The sugar alcohol mannitol cannot be metabolized by the plant cells. Sucrose is cleaved into glucose and fructose by the Coleus cells. Sucrose concentrations from 1 to 5% have an increasing positive effect on growth and rosmarinic acid synthesis in the cell cultures with a maximum rosmarinic acid content of 12% of the dry weight in medium with 5% sucrose; in medium with 6% sucrose rosmarinic acid accumulation obviously did not reach its highest level in the culture period of 14 days. A very high yield of rosmarinic acid (2 mg ml^-1 suspension) could also be achieved by maintaining a sucrose concentration of 2% during the whole culture period. The start of rosmarinic acid synthesis by the cell cultures seems to be regulated by the growth limitation when a nutrient, e.g. phosphate is depleted from the medium. The rate of rosmarinic acid accumulation is related to the amount of carbon left in the medium when growth ceases.Abbreviations RA rosmarinic acid     

Glyceraldehyde-3-phosphate dehydrogenase,a glycolytic enzyme present in the periplasm of Aeromonas hydrophila

This is the first report describing the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a protein associated with the cell envelope of a gram-negative bacterium (Aeromonas hydrophila). Dose-dependent GAPDH activity was detected in whole bacterial cells from exponentially growing cultures, indicating that an active form of GAPDH is located outside the plasma membrane. This activity represents roughly 10–20% of total cell activity, and it is not reduced by pretreatment of the cells with trypsin. Assays with soluble GAPDH indicate that the activity measured in intact cells does not originate by rebinding to intact cells of cytosolic enzyme released following cell lysis. GAPDH activity levels detected in intact cells varied during the growth phase. The relationship between GAPDH activity and cell culture density was not linear, showing this activity as a major peak in the late-logarithmic phase (A₆₀₀ = 1.1–1.3), and a decrease when cells entered the stationary phase. The late exponential growing cells showed a GAPDH activity 3 to 4-fold higher than early growing or stationary cells. No activity was detected in culture supernatants. Enzymatic and Western-immunoblotting analysis of subcellular fractions (cytosol, whole and outer membranes, and periplasm) showed that GAPDH is located in the cytosol, as expected, and also in the periplasm. These results place the periplasmic GAPDH of A. hydrophila into the family of multifunctional microbial cell wall-associated GAPDHs which retain their catalytic activity. This revised version was published online in June 2006 with corrections to the Cover Date.     

Factors influencing protoplast isolation from Coffea arabica cells

Cultured plant cells such as Coffea arabica L. cells, accumulate low concentration of secondary metabolites. One way to obtain high-producing plant cell cultures is to prepare single cell clones by using protoplast systems. Identification of limiting factors should facilitate the development of an isolation procedure that can generate adequate yields of intact and viable protoplasts Coffea arabica L. suspension cells. The most suitable conditions for protoplasting were as follows: 6 g of fresh tissue were plasmolysed in 100 ml of K ₃ salts (Nagy & Maliga 1976) containing 0.5 M sucrose for 1 h at 24°C. Then, 1 g of preplasmolysed cells were incubated in 10 ml of cellulase R10 (1%), macerozyme R10 (0.8%) and driselase (0.5%) in preplasmolysis medium. The protoplasts were collected and purified after 15 h of lytic reaction in the dark, at 28°C. More than 75% and 95% of the cells were converted into protoplasts when 5 and 8 day-old suspensions respectively were used for the release step. A number of viable protoplasts ranging from 3.5×10⁶ to 4.6×10⁶ P g^-1 fresh weight was obtained corresponding to an increase by a factor 10 to 15 of the protoplast yield obtained by Acuna & De Pena (1991).Abbreviations BAP 6-benzylamino purine - BSA Bovine Serum Albumin - 2,4-d 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - MES 2-(N-morpholino)ethanesulfonic acid - NAA naphthalene acetic acid - PI propidium iodide - PCV Packed Cell Volume - fw fresh weight     

Okadaic acid activates atypical protein kinase C (zeta/lambda) in rat and 3T3/L1 adipocytes. An apparent requirement for activation of Glut4 translocation and glucose transport.

Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, is known to provoke insulin-like effects on GLUT4 translocation and glucose transport, but the underlying mechanism is obscure. Presently, we found in both rat adipocytes and 3T3/L1 adipocytes that okadaic acid provoked partial insulin-like increases in glucose transport, which were inhibited by phosphatidylinositol (PI) 3-kinase inhibitors, wortmannin and LY294002, and inhibitors of atypical protein kinase C (PKC) isoforms, zeta and lambda. Moreover, in both cell types, okadaic acid provoked increases in the activity of immunoprecipitable PKC-zeta/lambda by a PI 3-kinase-dependent mechanism. In keeping with apparent PI 3-kinase dependence of stimulatory effects of okadaic acid on glucose transport and PKC-zeta/lambda activity, okadaic acid provoked insulin-like increases in membrane PI 3-kinase activity in rat adipocytes; the mechanism for PI 3-kinase activation was uncertain, however, because it was not apparent in phosphotyrosine immunoprecipitates. Of further note, okadaic acid provoked partial insulin-like increases in the translocation of hemagglutinin antigen-tagged GLUT4 to the plasma membrane in transiently transfected rat adipocytes, and these stimulatory effects on hemagglutinin antigen-tagged GLUT4 translocation were inhibited by co-expression of kinase-inactive forms of PKC-zeta and PKC-lambda but not by a double mutant (T308A, S473A), activation-resistant form of protein kinase B. Our findings suggest that, as with insulin, PI 3-kinase-dependent atypical PKCs, zeta and lambda, are required for okadaic acid-induced increases in GLUT4 translocation and glucose transport in rat adipocytes and 3T3/L1 adipocytes.