Nuclear/cytoplasmic localization of Akt activity in the cell cycle

Summary. The serine/threonine protein kinase Akt (also known as PKB) is a proto-oncogene and one of the most frequently hyperactivated kinases in human cancer. Its activation downstream of growth-factor-stimulated phosphatidylinositide-3′-OH kinase activity plays a role in the control of cell cycle, cell growth, apoptosis and cell energy metabolism. Akt phosphorylates some thousand downstream substrates, including typical cytoplasmic as well as nuclear proteins. Accordingly, it is not surprising that Akt activity can be found in both, the cytoplasm and the nucleus. Here we report the cell cycle regulation of nuclear and cytoplasmic Akt activity in mammalian cells. These data provide new insights into the regulation of Akt activity and have implications for future studies on the regulation of the wide variety of different nuclear and cytoplasmic Akt substrates.     

Isolation and characterization of two acyl-CoA-binding proteins from proembryogenic masses of Digitalis lanata Ehrh.

 Two acyl-CoA-binding-protein (ACBP) isoforms were isolated from proembryogenic masses of Digitalis lanata Ehrh. by column chromatography and preparative HPLC. The ACBPs had molecular masses of 9926 and 9997 Da, respectively. Partial sequence data indicated high similarity to each other and to ACBPs of other plant species such as Ricinus communis, Brassica napus and Arabidopsis thaliana. The isolated ACBPs bound palmitoyl-CoA with high affinity as determined by isoelectric-point shift. Received: 29 May 1999 / Accepted: 28 August 1999     

Ectopic expression of maize knotted1 results in the cytokinin-autotrophic growth of cultured tobacco tissues

The ectopic expression of knotted homologues has cytokinin-like effects on plant morphology. The functional relationship between knotted and cytokinins was investigated in cultures of leaf tissue established from tobacco (Nicotiana tabacum L. cv. Havana 425) plants transformed with the maize knotted1 (kn1) gene regulated by cauliflower mosaic virus 35S RNA expression signals. In contrast to leaf tissues of untransformed plants, leaf tissues of kn1 transformants were capable of sustained, cytokinin-autotrophic growth on auxin-containing medium and resembled the tobacco cytokinin-autotrophic mutants Hl-1 and Hl-2. The concentration of 18 cytokinins was measured in cultures initiated from leaves of three independent kn1 transformants and the Hl-1 and Hl-2 mutants. Although cytokinin contents were variable, the content of several cytokinins in Kn1, Hl-1 and Hl-2 tissue lines was at least 10-fold higher than that of wild-type tobacco tissues and in the range reported for other cytokinin-autotrophic tobacco tissues. These results suggest that the cytokinin-autotrophic growth of Kn1 lines could result from elevated steady-state levels of cytokinins. Received: 7 July 1999 / Accepted: 10 November 1999     

Eating the evidence? Manduca sexta larvae can not disrupt specific jasmonate induction in Nicotiana attenuata by rapid consumption

As feeding by the tobacco specialist Manduca sexta L. is known to result in significantly higher jasmonate (JA) concentrations in Nicotiana plants than do mechanical simulations of the larval damage, we investigated whether M. sexta, which is known to rapidly consume large quantities of leaf material, can impair this “recognition” response by consuming the leaf tissue before it can respond with amplified JA levels. We report that oral secretions (OS) from M. sexta, but not from the cabbage specialist Pieris rapae, amplified the wound-induced JA response of Nicotiana attenuata Torr. Ex Wats., regardless of larval diet, instar and molting stage, and were still active after boiling and when diluted to 1/1000. The largest JA response occurred within 40 min in tissues adjacent to the OS application site. When 3 mm of leaf tissue immediately adjacent to the OS application site was excised within 40 s, the signal that elicits JA amplification was found to travel rapidly into the leaf, beyond the mandibular reach of the larvae. We conclude that M. sexta is not able to consume the evidence of feeding activity. Received: 16 July 1999 / Accepted: 12 August 1999     

The interaction of thin-film flow, bacterial swarming and cell differentiation in colonies of Serratia liquefaciens

 The rate of expansion of bacterial colonies of S. liquefaciens is investigated in terms of a mathematical model that combines biological as well as hydrodynamic processes. The relative importance of cell differentiation and production of an extracellular wetting agent to bacterial swarming is explored using a continuum representation. The model incorporates aspects of thin film flow with variable suspension viscosity, wetting, and cell differentiation. Experimental evidence suggests that the bacterial colony is highly sensitive to its environment and that a variety of mechanisms are exploited in order to proliferate on a variety of surfaces. It is found that a combination of effects are required to reproduce the variation of bacterial colony motility over a large range of nutrient availability and medium hardness. Received: 29 April 1999     

Biotechnology applications of amino acids in protein purification and formulations

Summary. Amino acids are widely used in biotechnology applications. Since amino acids are natural compounds, they can be safely used in pharmaceutical applications, e.g., as a solvent additive for protein purification and as an excipient for protein formulations. At high concentrations, certain amino acids are found to raise intra-cellular osmotic pressure and adjust to the high salt concentrations of the surrounding medium. They are called “compatible solutes”, since they do not affect macromolecular function. Not only are they needed to increase the osmotic pressure, they are known to increase the stability of the proteins. Sucrose, glycerol and certain amino acids were used to enhance the stability of unstable proteins after isolation from natural environments. The mechanism of the action of these protein-stabilizing amino acids is relatively well understood. On the contrary, arginine was accidentally discovered as a useful reagent for assisting in the refolding of recombinant proteins. This effect of arginine was ascribed to its ability to suppress aggregation of the proteins during refolding, thereby increasing refolding efficiency. By the same mechanism, arginine now finds much wider applications than previously anticipated in the research and development of proteins, in particular in pharmaceutical applications. For example, arginine solubilizes proteins from loose inclusion bodies, resulting in efficient production of active proteins. Arginine suppresses protein–protein interactions in solution and also non-specific adsorption to gel permeation chromatography columns. Arginine facilitates elution of bound proteins from various column resins, including Protein-A or dye affinity columns and hydrophobic interaction columns. This review covers various biotechnology applications of amino acids, in particular arginine.     

Programmed cell death: similarities and differences in animals and plants. A flower paradigm

Summary. After an overview of the criteria for the definition of cell death in the animal cell and of its different types of death, a comparative analysis of PCD in the plant cell is reported. The cytological characteristics of the plant cell undergoing PCD are described. The role of plant hormones and growth factors in the regulation of this event is discussed with particular emphasis on PCD activation or prevention by polyamine treatment (doses, timing and developmental stage of the organism) in a Developmental cell death plant model: the Nicotiana tabacum (tobacco) flower corolla. Some of the effects of polyamines might be mediated by transglutaminase catalysis. The activity of this enzyme was examined in different parts of the corolla during its life span showing an acropetal trend parallel to the cell death wave. The location of transglutaminase in some sub-cellular compartments suggests that it exerts different functions in the corolla DCD.     

Inactivation of DNA replication origins by the cell cycle regulator, trigonelline, in root meristems of Lactuca sativa

 The effects of trigonelline (TRG) on the cell cycle in root meristems of Lactuca sativa L. were examined in the knowledge that TRG is a cell cycle regulator that causes cell arrest in G2, and prevents ligation of replicons in S-phase. The hypothesis was tested that continuous exposure to TRG would perturb DNA replication which, in turn, would lengthen the cell cycle and impair root elongation. Using DNA fibre autoradiography, mean replicon size was 31 and 13 μm in the TRG (3 mM) and control treatments, respectively. Trigonelline also resulted in a lengthening of both S-phase and the cell cycle and a decrease in primary root elongation. Hence, replicon inactivation was responsible for the protracted S-phase. Trigonelline treatment also resulted in a 1.6-fold increase in fork rate (13.8 μm h⁻¹) compared with the control (8.4 m h⁻¹). The faster fork rate in the larger replicons is in accord with the highly significant positive relationship already established between fork rate and replicon size for various unrelated higher plants. Received: 11 October 1999 / Accepted: 23 December 1999     

Amino acid analysis by hydrophilic interaction chromatography coupled on-line to electrospray ionization mass spectrometry

Summary. Collagens form a common family of triple-helical proteins classified in 21 types. This unique structure is further stabilized by specific hydroxylation of distinct lysyl and prolyl residues forming 5-hydroxylysine and hydroxyproline (Hyp) isomers, mostly 4-trans and 3-trans-Hyp. The molecular distribution of the Hyp-isomers among the different collagen types is still not well investigated, even though disturbances in the hydroxylation of collagens are likely to be involved in several diseases such as osteoporosis and autoimmune diseases. Here, a new approach to analyze underivatized amino acids by hydrophilic interaction chromatography (HILIC) coupled on-line to electrospray ionization mass spectrometry (ESI-MS) is reported. This method can separate all three studied Hyp-isomers, Ile, and Leu, which are all isobaric, allowing a direct qualitative and quantitative analysis of collagen hydrolysates. The sensitivity and specificity was increased by a neutral loss scan based on the loss of formic acid (46 u).     

Taurine trophic modulation of goldfish retinal outgrowth and its interaction with the optic tectum

Summary. Goldfish retinal explant outgrowth in the presence of fetal calf serum is stimulated by taurine. In the absence of it, but with glucose in the medium, length of neurites is still elevated by the amino acid. Using the medium in the presence of glucose, but in the absence of fetal calf serum, we explored the effect of optic tectum medium from cultures of them coming from goldfish without crush of the optic nerve or 3, 5, 10, 14 and 20 days after crush. Retinal explants, intact or from goldfish with crush of the optic nerve 10 days prior to starting the culture, were employed in order to measure the possible effect of optic tectum media and the inter action with taurine. In other type of experiments the optic nerve was crushed 1, 2, 4, 7 and 10 days before dissection of the optic tectum, and then co-cultured with intact or 10 days post-crush retinal explants. Optic tectum media produced a time-dependent effect on outgrowth in lesioned retinas with a maximum effect around 5 days after the lesion for the corresponding optic tectum. Taurine, 4 mM, did not further affect the outgrowth in the presence of optic tectum media, but did significantly increase length of neurites either in intact or in post-lesion retinas. Co-culture of optic tectum at different days post-lesion and retinas at 10 days post-lesion increased the outgrowth around 4 days post-lesion, in a preparation resulting in mutual effects of both types of tissues. The addition of taurine in these conditions did not further increase outgrowth, rather inhibited it according to the time after lesion of optic nerve corresponding to the co-cultured optic tectum. The effect of taurine was concentration-dependent, since 0.2 mM was more effective than 2 or 4 mM in the presence of optic tectum with lesion of 2 days. These results demonstrate the time-course of the regeneration processes in the visual system of goldfish, indicating the crucial periods after crush in which the tectum could produce stimulation and later decrease or no effect on outgrowth from the retina. In addition, they are evidences of the interaction between taurine and optic tectum production of time-produced specific agents. The mechanisms underlying these effects are closely related to calcium, as it was demonstrated by the addition of extracellular or intracellular chelators to the medium, which inhibited the effects of the optic tectum and the trophic properties of taurine in this system. The inhibitor of taurine transport, guanidoethylsulfonate, also decreased the stimulatory effects of the optic tectum and of taurine, indicating an interaction of substances produced by the tectum with taurine, and an effect of taurine mediated through its entrance to the cells. Overall, retinal explants outgrowth in the absence of fetal calf serum, the interaction of agents of the optic tectum and taurine modulates outgrowth from the retina, and these effects are mediated by calcium levels and by the levels of intracellular taurine.     

Chemical and toxicological studies of products resulting from sorbic acid and methylamine interaction in food conditions

Summary. Sorbic acid has a system of conjugated double bonds which makes it able to undergo nucleophilic addition reactions with certain functions. The interactions between sorbic acid and an amine present in the endogenous constituents of food were quantified. We demonstrated the formation of new products and studied the underlying mechanisms using ethyl sorbate and various amines. HPLC, GC, GC-SM and NMR analyses of the reaction mixes enabled the isolation and identification of the products. The addition reactions led, at 20°C, to linear monoadducts and, at 50°C, to cyclic derivatives resulting from double addition. Mutagenesis studies, involving Ames test and genotoxicity studies with HeLa cells and on plasmid DNA, in cyclic interaction products, showed that none of the products studied presented neither mutagenic nor genotoxic activities. Received September 28, 1999     

Decrease of phosphoribulokinase activity by antisense RNA in transgenic tobacco: definition of the light environment under which phosphoribulokinase is not in large excess

 To test the hypothesis that the contribution of phosphoribulokinase (PRK) to the control of photosynthesis changes depending on the light environment of the plant, the response of transgenic tobacco (Nicotiana tabacum L.) transformed with antisense PRK constructs to irradiance was determined. In plants grown under low irradiance (330 μmol m⁻² s⁻¹) steady-state photosynthesis was limited in plants with decreased PRK activity upon exposure to higher irradiance, with a control coefficient of PRK for CO₂ assimilation of 0.25 at and above 800 μmol m⁻² s⁻¹. The flux control coefficient of PRK for steady-state CO₂ assimilation was zero, however, at all irradiances in plant material grown at 800 μmol m⁻² s⁻¹ and in plants grown in a glasshouse during mid-summer (alternating shade and sun 300–1600 μmol m⁻² s⁻¹). To explain these differences between plants grown under low and high irradiances, Calvin cycle enzyme activities and metabolite content were determined. Activities of PRK and other non-equilibrium Calvin cycle enzymes fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase and ribulose-1,5-bisphosphate carboxylase-oxygenase were twofold higher in plants grown at 800 μmol m⁻² s⁻¹ or in the glasshouse than in plants grown at 330 μmol m⁻² s⁻¹. Activities of equilibrium enzymes transketolase, aldolase, ribulose-5-phosphate epimerase and isomerase were very similar under all growth irradiances. The flux control coefficient of 0.25 in plants grown at 330 μmol m⁻² s⁻¹ can be explained because low ribulose-5-phosphate content in combination with low PRK activity limits the synthesis of ribulose-1,5-bisphosphate. This limitation is overcome in high-light-grown plants because of the large relative increase in activities of sedoheptulose-1,7-bisphosphatase and fructose-1,6-bisphosphatase under these conditions, which facilitates the synthesis of larger amounts of ribulose-5-phosphate. This potential limitation will have maintained evolutionary selection pressure for high concentrations of PRK within the chloroplast. Received: 15 November 1999 / Accepted: 27 January 2000     

Vacuolar granules in Chlamydomonas reinhardtii: polyphosphate and a 70-kDa polypeptide as major components

The alga Chlamydomonas reinhardtii contains cytoplasmic vacuoles that are often filled with a dense granule that is released from the cell by exocytosis. Purified granules contained polyphosphate, complexed with calcium and magnesium, as the predominant inorganic components. Antiserum was raised against the major 70-kDa protein in granules purified from wall-deficient (cw15) mutants, which reacted on immunoblots with larger glycoprotein complexes in purified cell wall fractions from wild-type cells. Confocal fluorescence microscopy detected binding of these antibodies predominantly at the periphery of wall-containing C. reinhardtiiy1 cells but primarily to loci in the interior of cells of the cw15 strain. Immunoelectron microscopy demonstrated that the 70-kDa protein was localized in vacuolar granules and the trans-Golgi network in sections of cw15 cells but not in the cytosol or chloroplast. Treatment of cells with a dye, fluorescent in its protonated form, indicated that the pH within vacuoles was lower than that in the cytosol, which suggested that the vacuoles are similar to lysosomes. Thus, the vacuoles may serve a dual function to provide an environment for degradation within the cell and also serve as a vehicle for secretion of specific proteins. Received: 29 September 1999 / Accepted: 20 November 1999     

Non-sufficient cell cycle control as possible clue for the resistance of human malignant glioma cells to clinically relevant treatment conditions

Summary. Objectives. Human gliomas have a catastrophic prognosis with a median survival in the range of one year even after therapeutic treatment. Relatively high resistance towards apoptotic stimuli is the characteristic feature of malignant gliomas. Since cell cycle control has been shown to be the key mechanism controlling both apoptosis and proliferation, this study focuses on DNA damage analysis and protein expression patterns of essential cell cycle regulators P53 and P21^waf1/cip1 in glioma under clinically relevant therapeutic conditions. Material and methods. U87MG cell line, characterised by wild p53-phenotype relevant for the majority of primary malignant glioblastomas, was used. Glioma cells underwent either irradiation or temozolomide treatment alone, or combined radio/chemo treatment. DNA damage was analysed by the “Comet Assay”. Expression rates of target proteins were analysed using “Western-Blot” technique. Results and conclusions. “Comet Assay” demonstrated extensive DNA damage caused by temozolomide treatment alone and in combination with irradiation, correlating well with the low survival rate observed under these treatment conditions. In contrast, irradiation alone resulted in a relatively low DNA damage, correlating well with a high survival rate and indicating a poor therapeutic efficiency of irradiation alone. Unusually low up-regulation of P53 and P21^waf1/cip1 expression patterns was produced by the hereby tested stressful conditions. A deficit in cell cycle control might be the clue to the high resistance of malignant glioma cells to established therapeutic approaches.     

Chemical-induced apoptotic cell death in tomato cells: involvement of caspase-like proteases

 A new system to study programmed cell death in plants is described. Tomato (Lycopersicon esculentum Mill.) suspension cells were induced to undergo programmed cell death by treatment with known inducers of apoptosis in mammalian cells. This chemical-induced cell death was accompanied by the characteristic features of apoptosis in animal cells, such as typical changes in nuclear morphology, the fragmentation of the nucleus and DNA fragmentation. In search of processes involved in plant apoptotic cell death, specific enzyme inhibitors were tested for cell-death-inhibiting activity. Our results showed that proteolysis plays a crucial role in apoptosis in plants. Furthermore, caspase-specific peptide inhibitors were found to be potent inhibitors of the chemical-induced cell death in tomato cells, indicating that, as in animal systems, caspase-like proteases are involved in the apoptotic cell death pathway in plants. Received: 5 August 1999 / Accepted: 14 March 2000     

Growth, ageing and death of a photoautotrophic plant cell culture

 Batch cultures of photoautotrophic cell suspensions of Chenopodiumrubrum L., growing in an inorganic medium on CO₂ under a daily balanced light–dark regime of 16 : 8 h could be maintained for approximately 100 d without subcultivation. The long-lived cultures showed an initial cell division phase of 4 weeks, followed by a stationary phase of another 4 weeks, after which ageing and progressive cell death reduced the number of living cells and the cultures usually expired after another 3–4 weeks. These developmental phases of the cell culture were characterised with respect to photosynthetic performance, dark respiration, content of phytohormones and capacity of cell division. Cell division of the majority of the cells finished in the G1- or G0-phase of the cell cycle, caused by a pronounced decline in the endogenous levels of auxin and cytokinins. Supply of these growth factors to resting cells resulted in resumption of cytokinesis, at least by some of the cells. However, responsiveness to the phytohomones declined during the stationary phase, and subcultivation was no longer possible beyond day 60 when the phases of ageing and death commenced. Ageing was characterised by a further decline in the photosynthetic capacity of the cells, by a climacteric enhancement of dark respiration, but also by a slight increase in the level of IAA and cytokinins concomitant with a decrease in ethylene. Similarities and differences between the development of batch-cultured photoautotrophic cells of C. rubrum and that of a leaf are discussed with respect to using the cell culture as a model for a leaf. Received: 30 April 1999 / Accepted: 21 August 1999