Suspension cells

Plant Physiol, 2005 Jan, 137(1), 328 - 40 Epub 2004 Dec 23.
B1-phytoprostanes trigger plant defense and detoxification responses; Loeffler C et al.; Phytoprostanes are prostaglandin/jasmonate-like products of nonenzymatic lipid peroxidation that not only occur ubiquitously in healthy plants but also increase in response to oxidative stress . In this work, we show that the two naturally occurring B(1)-phytoprostanes (PPB(1)) regioisomers I and II (each comprising two enantiomers) are short-lived stress metabolites that display a broad spectrum of biological activities . Gene expression analysis of Arabidopsis (Arabidopsis thaliana) cell cultures treated with PPB(1)-I or -II revealed that both regioisomers triggered a massive detoxification and defense response . Interestingly, expression of several glutathione S-transferases, glycosyl transferases, and putative ATP-binding cassette transporters was found to be increased by one or both PPB(1) regioisomers, and hence, may enhance the plant's capacity to inactivate and sequester reactive products of lipid peroxidation . Moreover, pretreatment of tobacco (Nicotiana tabacum) suspension cells with PPB(1) considerably prevented cell death caused by severe CuSO(4) poisoning . Several Arabidopsis genes induced by PPB(1), such as those coding for adenylylsulfate reductase, tryptophan synthase beta-chain, and PAD3 pointed to an activation of the camalexin biosynthesis pathway that indeed led to the accumulation of camalexin in PPB(1) treated leaves of Arabidopsis . Stimulation of secondary metabolism appears to be a common plant reaction in response to PPB(1) . In three different plant species, PPB(1)-II induced a concentration dependent accumulation of phytoalexins that was comparable to that induced by methyl jasmonate . PPB(1)-I was much weaker active or almost inactive . No differences were found between the enantiomers of each regioisomer . Thus, results suggest that PPB(1) represent stress signals that improve plants capacity to cope better with a variety of stresses.

Biotechnol Bioeng . 2004 Dec 22; {Epub ahead of print}
Effect of nitric oxide on catharanthine production and growth of Catharanthus roseus suspension cells; Xu M et al.; Sodium nitroprusside (SNP) was used as the donor of nitric oxide (NO) to investigate its effect on catharanthine synthesis and the growth of Catharanthus roseus suspension cells . The results showed that SNP at high concentrations (10.0 and 20.0 mmol/L) stimulated catharanthine formation of C . roseus cells, but inhibited growth of the cells . Low concentrations of SNP (0.1 and 0.5 mmol/L) enhanced the growth of C . roseus cells, but had no effect on catharanthine synthesis . The maximum total catharanthine production was achieved by the addition of 0.5 and 10.0 mmol/L SNP to the cultures at day 0 and day 10, respectively, being about threefold of the control . NO-induced catharanthine production of C . roseus cells was strongly suppressed by jasmonic acid (JA) biosynthesis inhibitor ibuprofen (IBU) and nordihydroguaiaretic (NDGA) . The result suggests that the stimulatory role of NO on catharanthine production is partially JA-dependent . (c) 2004 Wiley Periodicals, Inc.

Plant Cell Physiol, 2004 Nov, 45(11), 1715 - 9
Ectopic expression of the NtSET1 histone methyltransferase inhibits cell expansion, and affects cell division and differentiation in tobacco plants; Shen WH et al.; The tobacco NtSET1 gene encodes a member of the SUV39H family of histone methyltransferases . Ectopic expression of NtSET1 causes an increase in methylated histone H3 lysine 9 and abnormal chromosome segregation in tobacco suspension cells, and inhibits tobacco plant growth . Here we show that the inhibition of plant growth was caused by reduced cell expansion as well as by abnormal cell division and differentiation . We found that deletion of the C-terminally located catalytic domain of the protein abolished the ectopic effects of NtSET1 on plant growth . Our results indicate that histone H3 lysine 9 methylation is a critical mark of epigenetic control for plant development.

Nucleosides Nucleotides Nucleic Acids, 2004 Oct, 23(8-9), 1451 - 4
Online fluorescent method to assess BCRP/ABCG2 activity in suspension cells; Hooijberg JH et al.; An online method was developed to monitor BCRP mediated efflux of fluorescent substrates in suspension cells . To this end, a 2-compartment system consisting of a transwell cup and a cuvette was used . In this system we were able to observe differences in efflux kinetics between BCRP overexpressing RPMI 8226/MR cells and parental myeloid RPMI 8226(s) cells using only 50,000 cells per experiment . 8226/MR cells displayed a larger cellular efflux rate of the BCRP substrate Hoechst 33342, as compared to the wildtype cells . This difference in efflux rate was completely decreased in the presence of the BCRP inhibitor Ko143.

Plant Cell Rep, 2004 Nov, 23(6), 371 - 6 Epub 2004 Nov.
Efficient plant regeneration from suspension cells of Allium cepa L; Zhang W et al.; Plant regeneration from calli of three cultivars of Allium cepa (Senshuki, O.Pki and Shojovaka) was investigated . Callus was induced on four variations of BDS medium containing different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BAP) . The regeneration frequency of calli of cvs . Senshuki and O.Pki subcultured on solid MS medium supplemented with BAP ranged from 50% to 80%; this frequency decreased to less than 30% after subculture in the dark in liquid BDS medium . By repeating the dark/light transitions of the culture protocol and by selecting for green cell clusters, we were able to increase the regeneration frequency to more than 80% in all three cultivars . These cell clusters maintained a high regeneration capacity in subsequent subcultures in the absence of light for 2 months . Most (97%) of the regenerated plantlets had a normal diploid karyotype (2 n=16) that was identical to that of the mother plants, although 3% of the regenerated plants of cv . Shojovaka had a tetraploid karyotype.

J Biomed Mater Res A, 2005 Jan 1, 72A(1), 1 - 9
Adhesion of human U937 macrophages to phosphorylcholine-coated surfaces; Gong YK et al.; A new type of amphiphilic phosphorylcholine (PC) polymer was used in this work to develop a cell culture surface that allows the attachment of U937 macrophages . The PC polymer was a random copolymer of N-isopropylacrylamide (45%), N-(phosphorylcholine)-N'-(ethylenedioxy-bis(ethyl)) acrylamide (41%), and the hydrophobic monomer N-(n-octadecyl) acrylamide (14%) . Polypropylene (PP) films (1 cm2) were coated with the polymer solution by immersion . U937 macrophage suspensions were applied on PC polymer-coated surfaces and incubated for up to 72 h at 37 degrees C . While U937 cells did not adhere to PP, ammonia, nitrogen, or oxygen plasma-treated surfaces, they attached rapidly on PC-coated surfaces (< 1 h), proliferated, and stayed attached to the modified surface for at least 72 h, suggesting that unique features of the PC polymer, and the U937 macrophages, are responsible for the attachment of these cells . We compared the effect of Co2+ and Cr3+ ions on the expression of bone-resorbing cytokines (TNF-alpha, IL-6, IL-1beta) in U937 macrophages cultured on PC-coated surfaces to the response of U937 macrophages in suspension . Cytokine gene expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR) . Addition of Co2+ and Cr3+ ions led to a significant increased expression of TNF-alpha in both cultured and suspension cells . On the other hand, Co2+ and Cr3+ ions had a weak stimulatory effect or no effect on IL-1beta and IL-6, respectively, in both cultured and suspension cells . In conclusion, the use of PC polymer-modified surfaces might offer promising new opportunities for the culture of human U937 cells and may also point to the mechanism by which macrophages interact with lipid bilayers of biological membranes.

Apoptosis, 2004 Nov, 9(6), 833 - 41
Acidic-rich region of amyloid precursor protein induces glial cell apoptosis; Sun KH et al.; Amyloid precursor protein (APP) has several caspase cleavage sites in its C-terminal cytoplasmic domain and N-terminal extracellular domain . Caspase cleavages of APP at its cytosolic tail may result in releasing the domain and inducing cell death . During apoptosis, the N-terminal domain may also be processed at amino acids 197 and 219 by caspases leading to unmasking of an acidic-rich region (AR) . In this study, AR-exposing APP was shown to inhibit cell growth after transfection into RBA-1 astrocytes and BV-2 microglial cells . The recombinant AR from residue 220 to 288 of APP (APP220-288) was produced and its biological activities were analyzed . APP220-288 induced morphological changes, cell death, and DNA fragmentation in BV-2 and RBA-1 cells . However, AR was determined to have no apparent effects in suspension cells, erythroleukemia K562 cells, and Jurkat T cells . The cytotoxicity was depending on negative charge cluster and the apoptotic activity of AR was attributed to the inhibition of cell adhesion . In BV-2 microglial cells, AR significantly stimulated Fas expression, although expressions of the pro-inflammatory cytokine genes were not detected . APP220-288 also induced nitric oxide synthase (iNOS) expression and nitric oxide (NO) production . These findings indicate that the acidic-rich domain of APP may have apoptotic activity due to inhibition of cell adhesion and induction of iNOS and Fas expressions . Moreover, unmasking the apoptosis-induced AR may activate and exacerbate glial cells which in turn lead to further progression of the death program.

Planta . 2004 Oct 6; {Epub ahead of print}
Catalase and alternative oxidase cooperatively regulate programmed cell death induced by beta-glucan elicitor in potato suspension cultures; Mizuno M et al.; In potato ( Solanum tuberosum L.) suspension cells, the expression of the gene encoding alternative oxidase (AOX) and H(2)O(2) accumulation were induced by treatment with beta-glucan elicitor . The inhibition of catalase activity enhanced both AOX mRNA expression and the production of H(2)O(2), whereas the ascorbate peroxidase inhibitor did not have any effect on these responses . Simultaneous inhibition of catalase and AOX activities in elicited cells dramatically increased H(2)O(2) accumulation, leading to the disruption of mitochondrial membrane potential (DeltaPsi(m)) and programmed cell death (PCD) . The results demonstrate, for the first time, that not only AOX but also catalase plays a central role in the suppression of mitochondrial DeltaPsi(m) breakdown and PCD induced by beta-glucan elicitor.

Appl Microbiol Biotechnol . 2004 Oct 5; {Epub ahead of print}
Elicitor-induced nitric oxide burst is essential for triggering catharanthine synthesis in Catharanthus roseus suspension cells; Xu M et al.; Elicitor prepared from the cell walls of Penicillium citrinum induced multiple responses in Catharanthus roseus suspension cells, including rapid generation of nitric oxide (NO), sequentially followed by enhancement of catharanthine production by C . roseus cells . Elicitor-induced catharanthine biosynthesis was blocked by NO-specific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and nitric oxide synthase (NOS) inhibitor S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea (PBITU) . PBITU also strongly inhibited elicitor-induced NO generation by C . roseus suspension cells . The inhibiting effect of PBITU on elicitor-induced catharanthine production was reversed by external application of NO via the NO-donor sodium nitroprusside . The results strongly suggested that NO, generated by NOS or NOS-like enzymes in C . roseus suspension cells when treated with the fungal elicitor, was essential for triggering catharanthine synthesis.

Plant Physiol Biochem, 2004 Jul-Aug, 42(7-8), 617 - 22
Characterization of DNA end-binding activities in higher plants; Yan KH et al.; DNA double-strand-breaks (DSB) are the most severe lesion in cells exposing to ionizing radiation and many other stress environments . Repair of DNA DSB is therefore critical to cellular survival . In this work, we observed the double-stranded DNA end-binding (DEB) like activities in rice (Oryza sativa L . cv . TN5) suspension cells and hypocotyls from etiolated mung bean (Vigna radiata L . TN5) seedlings . Higher plant DEB-like protein binds primarily to linearized double-stranded DNA ends . Competition of unlabeled probe was examined in double-stranded DEB assay of cell extracts from rice and mung bean . DEB-like activities of higher plants did not depend on sequence and types of double-stranded DNA ends . Distinct electrophoretic mobility shift patterns and binding features further indicate that DEB-like factors from various sources might not share identical structure and function, and probably belong to different types of DEB proteins from higher plants . Our evidence suggests that DEB proteins are certainly ubiquitous in all organisms probably for repairing and processing double-stranded DNA breaks from formidable lethal lesion .

FEBS Lett, 2004 Aug 27, 573(1-3), 51 - 4
Phosphate or phosphite addition promotes the proteolytic turnover of phosphate-starvation inducible tomato purple acid phosphatase isozymes; Bozzo GG et al.; Within 48 h of the addition of 2.5 mM phosphate (HPO42-, Pi) or phosphite (H2PO3-, Phi) to 8-day-old Pi-starved (-Pi) tomato suspension cells: (i) secreted and intracellular purple acid phosphatase (PAP) activities decreased by about 12- and 6-fold, respectively and (ii) immunoreactive PAP polypeptides either disappeared (secreted PAPs) or were substantially reduced (intracellular PAP) . The degradation of both secreted PAP isozymes was correlated with the de novo synthesis of two extracellular serine proteases having M(r)s of 137 and 121 kDa . In vitro proteolysis of purified secreted tomato PAP isozymes occurred following their 24 h incubation with culture filtrate from Pi-resupplied cells . The results indicate that Pi or Phi addition to -Pi tomato cells induces serine proteases that degrade Pi-starvation inducible extracellular proteins.

Plant Physiol, 2004 Aug, 135(4), 2330 - 47 Epub 2004 Aug 13.
Transcriptome profiling of the response of Arabidopsis suspension culture cells to Suc starvation; Contento AL et al.; Upon encountering nutrient stress conditions, plant cells undergo extensive metabolic changes and induce nutrient recycling pathways for their continued survival . The role of nutrient mobilization in the response of Arabidopsis suspension cells to Suc starvation was examined . Vacuolar autophagy was induced within 24 h of starvation, with increased expression of vacuolar proteases that are likely to be required for degradation of cytoplasmic components delivered to the vacuole, and thus for nutrient recycling . After 48 h of starvation, culture viability began to decrease, and substantial cell death was evident by 72 h . To provide further insight into the pathways required for survival during Suc deficit, transcriptional profiling during Suc starvation was performed using the ATH1 GeneChip array containing 22,810 probe sets . A significant increase in transcript levels was observed for 343 genes within 48 h of starvation, indicating a response to nutrient stress that utilizes the recycling of cellular components and nutrient scavenging for maintaining cell function, the protection of the cell from death through activation of various defense and stress response pathways, and regulation of these processes by specific protein kinases and transcription factors . These physiological and molecular data support a model in which plant cells initiate a coordinated response of nutrient mobilization at the onset of Suc depletion that is able to maintain cell viability for up to 48 h . After this point, genes potentially involved in cell death increase in expression, whereas those functioning in translation and replication decrease, leading to a decrease in culture viability and activation of cell death programs.

Plant Cell Rep . 2004 Jul 28; {Epub ahead of print}
Efficient plant regeneration from suspension cells of Allium cepa L; Zhang W et al.; Plant regeneration from calli of three cultivars of Allium cepa (Senshuki, O.Pki and Shojovaka) was investigated . Callus was induced on four variations of BDS medium containing different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BAP) . The regeneration frequency of calli of cvs . Senshuki and O.Pki subcultured on solid MS medium supplemented with BAP ranged from 50% to 80%; this frequency decreased to less than 30% after subculture in the dark in liquid BDS medium . By repeating the dark/light transitions of the culture protocol and by selecting for green cell clusters, we were able to increase the regeneration frequency to more than 80% in all three cultivars . These cell clusters maintained a high regeneration capacity in subsequent subcultures in the absence of light for 2 months . Most (97%) of the regenerated plantlets had a normal diploid karyotype (2 n=16) that was identical to that of the mother plants, although 3% of the regenerated plants of cv . Shojovaka had a tetraploid karyotype.

Plant Mol Biol, 2004 Feb, 54(3), 311 - 23
Purification, cloning and functional expression of hydroxyphenylpyruvate reductase involved in rosmarinic acid biosynthesis in cell cultures of Coleus blumei; Kim KH et al.; Hydroxyphenylpyruvate reductase (HPPR) is an enzyme involved in the biosynthesis of rosmarinic acid in Lamiaceae reducing hydroxyphenylpyruvates in dependence of NAD(P)H to the corresponding hydroxyphenyllactates . The HPPR protein was purified from suspension cells of Coleus blumei accumulating high levels of rosmarinic acid by ammonium sulfate precipitation, anion exchange chromatography, hydroxylapatite chromatography, chromatography on 2',5'-ADP-Sepharose 4B and SDS-polyacrylamide gel electrophoresis . The protein was tryptically digested and the peptides sequenced . Sequence information was used to isolate a full-length cDNA-clone for HPPR (EMBL accession number AJ507733) by RT-PCR, screening of a C . blumei cDNA-library and 5'-RACE-PCR . The open reading frame of the HPPR-cDNA consists of 939 nucleotides encoding a protein of 313 amino acid residues . The sequence showed that HPPR belongs to the family of D-isomer-specific 2-hydroxyacid dehydrogenases . The HPPR-cDNA was heterologously expressed in Escherichia coli and the protein was shown to catalyse the NAD(P)H-dependent reduction of 4-hydroxyphenylpyruvate to 4-hydroxyphenyllactate and 3,4-dihydroxyphenylpyruvate to 3,4-dihydroxyphenyllactate.

Plant Cell Rep, 2004 Oct, 23(4), 256 - 62 Epub 2004 Jul 24.
Expression of a salt-induced protein (SALT) in suspension-cultured cells and leaves of rice following exposure to fungal elicitor and phytohormones; Kim ST et al.; Phytohormones are essential signal compounds in the regulation of stress-related and defense-related genes . However, there is no clear evidence for any effect of these signal molecules and biotic elicitors on the regulation of the SALT gene in suspension-cultured rice cells . We characterized the expression of a SALT gene following treatment with fungal elicitor, phytohormones, cycloheximide, and inhibitors of protein kinase/phosphatases . SALT expression was up-regulated following treatment with a fungal elicitor, jasmonic acid (JA), abscisic acid (ABA), and NaCl . However, salicylic acid (SA) alone or in combination with one of the other elicitors not only strongly inhibited SALT gene expression but also exhibited an antagonistic effect in suspension cells and leaves . Cycloheximide inhibited SALT accumulation in suspension cells and in leaves, but the inhibitors of protein kinase/phosphatase did not . Immunolocalization revealed that SALT protein was present in xylem parenchyma cells of vascular bundles in the major and minor leaf veins.

J Gen Virol, 2004 Aug, 85(Pt 8), 2459 - 69
Nucleo-cytoplasmic shuttling of the beet necrotic yellow vein virus RNA-3-encoded p25 protein; Vetter G et al.; The protein p25 encoded by beet necrotic yellow vein virus (BNYVV) RNA-3 is involved in symptom expression of infected plants . Confocal microscopy analysis of wild-type and mutated p25 fused to GFP and transiently expressed in BY-2 tobacco suspension cells identified a nuclear localization signal (NLS) in the N-terminal part of the protein . Functionality of the NLS was confirmed by pull-down assays using rice and pepper importin-alpha . Furthermore, it was demonstrated that p25 contains a nuclear export sequence sensitive to leptomycin B . The nuclear export signal (NES) was characterized by mutagenesis . A GFP-p25 fusion protein expressed during a BNYVV infection of Chenopodium quinoa leaves had the same subcellular localization as observed during transient expression in BY-2 cells . The symptom phenotype induced by expression of GFP-p25 during infection was similar to that induced by wild-type virus . Studies with mutated derivatives of GFP-p25 revealed that symptom phenotype was altered when the subcellular localization of GFP-p25 was modified.

Cells Tissues Organs, 2004, 177(1), 37 - 46
Establishment and characterization of a human gastric carcinoma cell line TMC-1; Shyu RY et al.; Established cancer cell lines are useful in the study of various cancers . We established a human gastric carcinoma cell line TMC-1 derived from the lymph node of a moderately differentiated adenocarcinoma of the stomach . TMC-1 cells grew in vitro as a mixture of attached and suspension cells, and exhibited spindle or ovoid morphology . They had a population doubling time of 15 h, a plating efficiency of 61%, formed colonies in semisolid agar, secreted the tumor marker CA 19-9, and were tumorigenic in athymic nude mice . The cells expressed E-cadherin and beta-catenin . The karyotypic analysis demonstrated hyperdiploid features with a modal chromosome of 53 . The cell had the deletion at chromosome 18q and gains at chromosome 2p13-25, 5p15, 5q21-35, 7, 8q24, 9q, 11, 12p, 14q24-32 and 20 . Analysis by fluorescence in situ hybridization showed the deletion at 7qtel and duplication at 7q11.2 at the rearranged chromosome 7 . Growth of TMC-1 cells was inhibited by 27-32% by interferon-alpha (2,000 U/ml) and by interferon-gamma with an IC50 of 125 U/ml . The cell line is tumorigenic in vivo, and its growth is moderately inhibited by interferon-alpha and interferon-gamma . It can be used to develop new modalities of human gastric cancer treatment .

Plant Physiol, 2004 Jul, 135(3), 1367 - 77 Epub 2004 Jul 02.
Signal peptide-dependent targeting of a rice alpha-amylase and cargo proteins to plastids and extracellular compartments of plant cells; Chen MH et al.; alpha-Amylases are important enzymes for starch degradation in plants . However, it has been a long-running debate as to whether alpha-amylases are localized in plastids where starch is stored . To study the subcellular localization of alpha-amylases in plant cells, a rice (Oryza sativa) alpha-amylase, alphaAmy3, with or without its own signal peptide (SP) was expressed in transgenic tobacco (Nicotiana tabacum) and analyzed . Loss-of-function analyses revealed that SP was required for targeting of alphaAmy3 to chloroplasts and/or amyloplasts and cell walls and/or extracellular compartments of leaves and suspension cells . SP was also required for in vitro transcribed and/or translated alphaAmy3 to be cotranslationally imported and processed in canine microsomes . alphaAmy3, present in chloroplasts of transgenic tobacco leaves, was processed to a product with Mr similar to alphaAmy3 minus its SP . Amino acid sequence analysis revealed that the SP of chloroplast localized alphaAmy3 was cleaved at a site only one amino acid preceding the predicted cleavage site . Function of the alphaAmy3 SP was further studied by gain-of-function analyses . beta-Glucuronidase (GUS) and green fluorescence protein fused with or without the alphaAmy3 SP was expressed in transgenic tobacco or rice . The alphaAmy3 SP directed translocation of GUS and green fluorescence protein to chloroplasts and/or amyloplasts and cell walls in tobacco leaves and rice suspension cells . The SP of another rice alpha-amylase, alphaAmy8, similarly directed the dual localizations of GUS in transgenic tobacco leaves . This study is the first evidence of SP-dependent dual translocations of proteins to plastids and extracellular compartments, which provides new insights into the role of SP in protein targeting and the pathways of SP-dependent protein translocation in plants.

Protoplasma, 2004 Jun, 223(2-4), 93 - 102 Epub 2004 Jun 22.
Plasmodesmata in Arabidopsis thaliana suspension cells; Bayer E et al.; A current challenge in plant biology is to identify the structural and functional components of plasmodesmata (PDs) . The use of plant tissue as a source material for plasmodesmal characterisation has had limited success, so we have explored the frequency and features of PDs occurring in suspension cell cultures of Arabidopsis thaliana . This material has the advantages of homogeneity, quantity, and ease of disruption . Using light and electron microscopy and immunostaining for callose and calreticulin, we showed that suspension cells laid down abundant PDs in division walls, and that vestiges of these structures were retained as half PDs even when the cell-to-cell contacts were disrupted during culture growth . Although callose was a reliable marker for PD distribution, which was deposited in an organised collar around the neck of PDs, it was not abundant in unstressed cells . Calreticulin and the chemical stain 3,3'-dihexyloxacarbocyanine iodide also provided useful markers when monitoring PDs in cell wall preparations by light microscopy . Purified cell walls were shown to be virtually free of contamination from cytoplasmic components, except for the presence of small amounts of cortical endoplasmic reticulum attached to PDs . Hence, clean cell walls from A . thaliana suspension cells provide a valuable resource for a proteomic approach to the analysis of plasmodesmal components.

Plant Physiol Biochem, 2004 May, 42(5), 429 - 35
Ergosterol elicits oxidative burst in tobacco cells via phospholipase A2 and protein kinase C signal pathway; Kasparovsky T et al.; Ergosterol, a typical fungal sterol, induced in tobacco (Nicotiana tabacum L . cv . Xanthi) suspension cells the synthesis of reactive oxygen species and alkalization of the external medium that are dependent on the mobilization of calcium from internal stores . We used specific inhibitors to elucidate the signal pathway triggered by ergosterol compared with cryptogein, a proteinaceous elicitor of Phytophthora cryptogea . Herbimycin A and genistein, inhibitors of tyrosine protein kinases, had no effect on the oxidative burst and pH changes induced by both elicitors . Similarly, H-89, an inhibitor of protein kinase A, had no effect on the induction of these defense reactions . However, the response to both elicitors was completely blocked by NPC-15437, a specific inhibitor of animal protein kinase C (PKC) . The responses induced by cryptogein but not those induced by ergosterol were inhibited by U73122 and neomycin, inhibitors of phospholipase C (PLC) . On the other hand, the activity of phospholipase A2 (PLA2) measured using a fluorogenic substrate was stimulated by ergosterol and not by cholesterol and cryptogein . A specific inhibitor of PLA2, arachidonic acid trifluoromethyl ketone (AACOCF3), inhibited the pathway stimulated by ergosterol but not that induced by cryptogein . These results suggest that the cryptogein-induced signal pathway leading to the oxidative burst and DeltapH changes includes PLC and PKC, whereas this response induced by ergosterol includes PLA2 and PKC .

Plant Cell Rep, 2004 Aug, 23(1-2), 1 - 8 Epub 2004 May 25.
Cryopreservation of embryogenic suspension cultures of Cyclamen persicum mill; Winkelmann T et al.; We have developed a simple protocol for the cryopreservation of embryogenic suspension cultures of Cyclamen persicum . Embryogenic suspension cultures in the linear growth phase 7-10 days after subculture were used for cryopreservation . Of the different cryoprotectants tested during a 2-day pre-culture, 0.6 M sucrose resulted in the highest re-growth rates of 75% . An additional pre-treatment with 0.6 M sucrose and 10% DMSO (dimethylsulfoxide) for 1 h also positively affected re-growth . Microscopic studies on viability revealed that only few small embryogenic cells survived cryopreservation, while vacuolated single cells died . Experiments in which the duration of the pre-culture period--i.e . the length of time the embryogenic suspension cells were exposed to 0.6 M sucrose--was varied showed that 2-4 days was the most optimal exposure time to 0.6 M sucrose . Callus re-grown after cryopreservation showed growth rates similar to that of unfrozen callus and regenerated even higher numbers of somatic embryos than unfrozen callus.

FEBS Lett, 2004 May 21, 566(1-3), 307 - 10
Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells; Unnamalai N et al.; We have used cationic oligopeptide polyarginine-12mer (POA) to deliver double-stranded RNA (dsRNA), prepared in vitro, to tobacco (Nicotiana tabacum) suspension cells . POA interacts electrostatically with dsRNA to form a complex . When dsRNA for the GUS or NPTII gene was delivered into cells carrying the same genes, the corresponding mRNA was degraded . Using RNase protection assay we were able to detect 21-bp small interfering RNA in dsRNA/POA-treated cells . These results demonstrate that POA can be used to deliver dsRNA to induce post-transcriptional gene silencing in plant cells.

Plant Physiol, 2004 May, 135(1), 231 - 43
Plasma membrane depolarization induced by abscisic acid in Arabidopsis suspension cells involves reduction of proton pumping in addition to anion channel activation, which are both Ca2+ dependent; Brault M et al.; In Arabidopsis suspension cells a rapid plasma membrane depolarization is triggered by abscisic acid (ABA) . Activation of anion channels was shown to be a component leading to this ABA-induced plasma membrane depolarization . Using experiments employing combined voltage clamping, continuous measurement of extracellular pH, we examined whether plasma membrane H(+)-ATPases could also be involved in the depolarization . We found that ABA causes simultaneously cell depolarization and medium alkalinization, the second effect being abolished when ABA is added in the presence of H+ pump inhibitors . Inhibition of the proton pump by ABA is thus a second component leading to the plasma membrane depolarization . The ABA-induced depolarization is therefore the result of two different processes: activation of anion channels and inhibition of H(+)-ATPases . These two processes are independent because impairing one did not suppress the depolarization . Both processes are however dependent on the {Ca2+}cyt increase induced by ABA since increase in {Ca(2+)}(cyt) enhanced anion channels and impaired H(+)-ATPases.

Plant Physiol, 2004 May, 135(1), 507 - 15 Epub 2004 May 07.
Characterization of GaWRKY1, a cotton transcription factor that regulates the sesquiterpene synthase gene (+)-delta-cadinene synthase-A; Xu YH et al.; The cotton (+)-delta-cadinene synthase (CAD1), a sesquiterpene cyclase, catalyzes a branch-point step leading to biosynthesis of sesquiterpene phytoalexins, including gossypol . CAD1-A is a member of CAD1 gene family, and its promoter contains a W-box palindrome with two reversely oriented TGAC repeats, which are the proposed binding sites of WRKY transcription factors . We isolated several WRKY cDNAs from Gossypium arboreum . One of them, GaWRKY1, encodes a protein containing a single WRKY domain and a putative N-terminal Leu zipper . Similar to genes encoding enzymes of cotton sesquiterpene pathway, GaWRKY1 was down-regulated in a glandless cotton cultivar that contained much less gossypol . GaWRKY1 showed a temporal and spatial pattern of expression comparable to that of CAD1-A in various aerial organs examined, including sepal, stigma, anther, and developing seeds . In suspension cells, expression of both GaWRKY1 and CAD1-A genes and biosynthesis of sesquiterpene aldehydes were strongly induced by a fungal elicitor preparation and methyl jasmonate . GaWRKY1 interacted with the 3x W-box derived from CAD1-A promoter in yeast (Saccharomyces cerevisiae) one-hybrid system and in vitro . Furthermore, in transgenic Arabidopsis plants, overexpression of GaWRKY1 highly activated the CAD1-A promoter, and transient assay in tobacco (Nicotiana tabacum) leaves demonstrated that W-box was required for this activation . These results suggest that GaWRKY1 participates in regulation of sesquiterpene biosynthesis in cotton, and CAD1-A is a target gene of this transcription factor.

Plant Physiol . 2004 Apr 30; {Epub ahead of print}
Plasma Membrane Depolarization Induced by Abscisic Acid in Arabidopsis Suspension Cells Involves Reduction of Proton Pumping in Addition to Anion Channel Activation, Which Are Both Ca2+ Dependent; Brault M et al.; In Arabidopsis suspension cells a rapid plasma membrane depolarization is triggered by abscisic acid (ABA) . Activation of anion channels was shown to be a component leading to this ABA-induced plasma membrane depolarization . Using experiments employing combined voltage clamping, continuous measurement of extracellular pH, we examined whether plasma membrane H(+)-ATPases could also be involved in the depolarization . We found that ABA causes simultaneously cell depolarization and medium alkalinization, the second effect being abolished when ABA is added in the presence of H(+) pump inhibitors . Inhibition of the proton pump by ABA is thus a second component leading to the plasma membrane depolarization . The ABA-induced depolarization is therefore the result of two different processes: activation of anion channels and inhibition of H(+)-ATPases . These two processes are independent because impairing one did not suppress the depolarization . Both processes are however dependent on the {Ca(2+)}cyt increase induced by ABA since increase in {Ca(2+)}cyt enhanced anion channels and impaired H(+)-ATPases.

In Vitro Cell Dev Biol Anim, 2003 Nov-Dec, 39(10), 420 - 3
Differential expression of mammalian or viral promoter-driven gene in adherent versus suspension cells; Feng G et al.; Although expression vectors using viral and mammalian promoters constitutively express genes of interest in adherent cells, few studies have examined whether the function of these vectors in suspended cells, such as in over-agar or soft agar assay (an in vitro cell transformation assay), is as robust as when they are in adherent cells . The selection of appropriate expression vector to optimally express genes in suspended cells would be useful in determining whether these genes play a critical role in maintaining colony formation or cell transformation . To compare promoter-driven expression vector function in adherent versus suspension cells, we performed transient transfection assays using viral (simian virus 40 {SV40} and cytomegalovirus {CMV}) and mammalian (beta-actin) promoters fused to luciferase or beta-galactosidase reporter gene . Over-agar assay was used to suspend cells on top of agar, which allowed cell retrieval and analysis . We found that beta-actin and SV40 promoters exhibited suppressed gene expression of 70 and 56%, respectively, in cells suspended on agar compared with those attached on plates . The suppressed response by the exogenous beta-actin promoter in suspension was consistent with the response of the endogenous beta-actin promoter activity because the steady-state level of beta-actin messenger ribonucleic acid in suspended cells was significantly reduced by 50% relative to that expressed in attached cells . In contrast to SV40 promoter, CMV promoter activity was not decreased in cells suspended in over-agar when compared with adherent cells . These studies show that regardless of mammalian or viral vectors, one cannot assume that all expression vectors behave similarly in both suspension and adherent state.

Plant Cell Rep, 2004 Jul, 22(12), 903 - 9 Epub 2004 Apr 07.
Transgenic Russian wildrye (Psathyrostachys juncea) plants obtained by biolistic transformation of embryogenic suspension cells; Wang ZY et al.; Russian wildrye (Psathyrostachys juncea (Fisch.) Nevski) is a cool-season forage species well adapted to semi-arid climates . We are interested in developing biotechnological methods to improve this monocot forage species . Single genotype-derived embryogenic suspension cultures were established from the Russian wildrye cultivar Bozoisky-Select, and were used as target cells for biolistic transformation . A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker, and a chimeric beta-glucuronidase (gusA) gene was co-transformed with hph . Resistant calli were obtained from 29% of the bombarded dishes after selection with 200 mg/l hygromycin . Plants were regenerated from 45% of the hygromycin resistant calli . Thirty-six transgenic Russian wildrye plants were recovered after microprojectile bombardment of suspension cells and subsequent hygromycin selection . The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples . When a second gene (gusA) was co-transformed with hph, a reasonably high co-transformation frequency of 78% was observed . Transgenic expression of gusA was confirmed by GUS staining of shoot and leaf tissues . Fertile transgenic plants were obtained after two winters of vernalization under field conditions . This is the first report on the generation of transgenic plants in Russian wildrye.

Biosci Biotechnol Biochem, 2004 Mar, 68(3), 705 - 13
Sequential Development of Cysteine Proteinase Activities and Gene Expression during Somatic Embryogenesis in Carrot; Mitsuhashi W et al.; Three bands of proteinase activity (Rf values of 0.5, 0.6, and 0.7) were detected on activity-stained gels after native gel electrophoresis of carrot (Daucus carota L . cv US-Harumakigosun) suspension cells . After the induction of somatic embryogenesis, one activity band (0.7 band) rapidly disappeared; the 0.6 band was absent at the heart-shaped embryo stage . However, the intensity of the 0.5 band increased during embryogenesis . An additional band (0.25 band) appeared after the torpedo-shaped stage . Three bands (0.25, 0.5, and 0.6) were also detected in zygotic seeds . Two activity bands (0.5 and 0.6) were classified as cysteine proteinases based on sensitivities to N-Ethylmaleimide (NEM) or L-3-trans-Carboxyoxirane-2-Carbonyl-L-Leucyl-Agmatine (E-64) . To find candidate genes for the cysteine proteinases, we cloned seven cDNAs encoding putative cysteine proteinases from suspension cells and developing somatic embryos . The expression patterns of the seven genes were categorized into three types (Type A, mRNAs increase concomitantly with somatic embryogenesis; Type B, mRNAs decrease quickly in organized cells; Type C, no significant change in transcript level during somatic embryogenesis).

Plant Cell Rep, 2004 Jun, 22(11), 848 - 58 Epub 2004 Mar 26.
Methylmalonate-semialdehyde dehydrogenase is induced in auxin-stimulated and zinc-stimulated root formation in rice; Oguchi K et al.; Proteins induced in rice by auxin and zinc were determined by proteome analysis . Cultured suspension cells of rice were treated with 2,4-dichlorophenoxyacetic acid and ZnSO4 and then proteins were separated by two-dimensional polyacrylamide gel electrophoresis; seven proteins were found to be induced by auxin and zinc . Of these seven, methylmalonate-semialdehyde dehydrogenase (MMSDH) was elevated by treatment with auxin alone . MMSDH was detected in cultured suspension cells, root and leaf sheath, but not in leaf blades . MMSDH responded to auxin and gibberellin, but did not respond to brassinolide and cytokinin . Furthermore, the amount of MMSDH in slr1, a constitutive gibberellin response mutant, was 2-fold that of wild type . MMSDH mRNA and protein were stimulated in root formation induced by auxin and/or zinc over a 4-week period . These results suggest that MMSDH may be necessary for root formation in rice induced by auxin and/or zinc .

Cell Res, 2004 Feb, 14(1), 27 - 33
Alpha-picolinic acid, a fungal toxin and mammal apoptosis-inducing agent, elicits hypersensitive-like response and enhances disease resistance in rice; Zhang HK et al.; Alpha-picolinic acid (PA), a metabolite of tryptophan and an inducer of apoptosis in the animal cell, has been reported to be a toxin produced by some of plant fungal pathogens and used in screening for disease resistant mutants . Here, we report that PA is an efficient apoptosis agent triggering cell death of hypersensitive-like response in planta . Confirmed by Fluorescence Activated Cell Sorter (FACS), rice suspension cells and leaves exhibited programmed cell death induced by PA . The PA-induced cell death was associated with the accumulation of reactive oxygen species that could be blocked by diphenylene iodonium chloride, indicating that the generation of reactive oxygen species was NADPH-oxidase dependent . We also demonstrated the induction of rice defense-related genes and subsequent resistant enhancement by PA against the rice blast fungus Magnaporthe grisea . Hence, it was concluded that the PA-stimulated defense response likely involves the onset of the hypersensitive response in rice, which also provides a simple eliciting tool for studying apoptosis in the plant cell.

Cell Res, 2004 Feb, 14(1), 8 - 15
Arabidopsis RAV1 is down-regulated by brassinosteroid and may act as a negative regulator during plant development; Hu YX et al.; RAV1 is a novel DNA-binding protein with two distinct DNA-binding domains unique in higher plants, but its role in plant growth and development remains unknown . Using cDNA array, we found that transcription of RAV1 is down-regulated by epibrassinolide (epiBL) in Arabidopsis suspension cells . RNA gel blot analysis revealed that epiBL-regulated RAV1 transcription involves neither protein phosphorylation/dephosphorylation nor newly synthesized protein, and does not require the functional BRI1, suggesting that this regulation might be through a new BR signaling pathway . Overexpressing RAV1 in Arabidopsis results in a retardation of lateral root and rosette leaf development, and the underexpression causes an earlier flowering phenotype, implying that RAV1 may function as a negative regulatory component of growth and development.

Int J Biochem Cell Biol, 2004 May, 36(5), 814 - 25
Methotrexate differentially affects growth of suspension and adherent cells; Kimura E et al.; The effects of low concentrations of methotrexate (MTX) on the growth of suspension (FM3A, 2B4 and THP-1) and adherent (NIH3T3 and V79) cells were compared . The concentration of methotrexate to cause the inhibition of cell growth was lower in suspension cells than in adherent cells . The IC(50) for FM3A, 2B4, THP-1, NIH3T3 and V79 cells were 3.5, 5, 9, 30 and 50 nM, respectively . The inhibition of cell growth was reversed completely by tetrahydrofolate and was fully or significantly reversed by adenosine and thymidine, suggesting that the effects of low concentrations of methotrexate result from the inhibition of biosynthesis of purines and pyrimidines . In suspension cells but not in adherent cells there was a decrease in the levels of S-adenosylmethionine and polyamines after methotrexate treatment . Growth of suspension but not adherent cells was significantly recovered by treatment with S-adenosylmethionine . However, treatment with spermidine did not reverse the effects of methotrexate in any of the cell lines . The preferential inhibitory effect of methotrexate in suspension cells versus adherent cells was due mainly to a more rapid uptake of methotrexate . This may be relevant to the in vivo effects of low doses of methotrexate, which have immunosuppressive and anti-inflammatory effects, because lymphocytes are suspension cells.

J Pineal Res, 2004 Mar, 36(2), 126 - 31
Attenuation of cold-induced apoptosis by exogenous melatonin in carrot suspension cells: the possible involvement of polyamines; Lei XY et al.; Pretreatment with 43 nM (10 ng/mL) to 86 nM melatonin for 5 days significantly attenuated cold-induced apoptosis in carrot suspension cells (Daucus carota L.) as evidenced by the TUNEL procedure, DNA fragmentation and the morphological changes revealed by electronic microscopy observations . The antiapoptotic effect of melatonin was initially thought to be a result of its antioxidant actions . In our study, however, reactive oxygen species (ROS) generation remained unaffected by melatonin treatment, suggesting that melatonin plays its protective role not related to its direct ROS scavenger . At the same time, notable increases in putrescine and spermidine levels were observed in melatonin-treated cells, which may be responsible for the alleviation of the cold-induced apoptosis . The possible involvement of polyamines in the antiapoptotic effect of melatonin was further confirmed by the inhibitory effect of exogenous polyamines on apoptosis as displayed by the DNA laddering assay.

Plant J, 2004 Mar, 37(5), 654 - 67
Phosphorylation and 14-3-3 binding of Arabidopsis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Kulma A et al.; Fructose 2,6-bisphosphate (fru-2,6-P2) is a signalling metabolite that regulates photosynthetic carbon partitioning in plants . The content of fru-2,6-P2 in Arabidopsis leaves varied in response to photosynthetic activity with an abrupt decrease at the start of the photoperiod, gradual increase through the day, and modest decrease at the start of the dark period . In Arabidopsis suspension cells, fru-2,6-P2 content increased in response to an unknown signal upon transfer to fresh culture medium . This increase was blocked by either 2-deoxyglucose or the protein phosphatase inhibitor, calyculin A, and the effects of calyculin A were counteracted by the general protein kinase inhibitor K252a . The changes in fru-2,6-P2 at the start of dark period in leaves and in the cell experiments generally paralleled changes in nitrate reductase (NR) activity . NR is inhibited by protein phosphorylation and binding to 14-3-3 proteins, raising the question of whether fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase protein from Arabidopsis thaliana (AtF2KP), which both generates and hydrolyses fru-2,6-P2, is also regulated by phosphorylation and 14-3-3s . Consistent with this hypothesis, AtF2KP and NR from Arabidopsis cell extracts bound to a 14-3-3 column, and were eluted specifically by a synthetic 14-3-3-binding phosphopeptide (ARAApSAPA) . 14-3-3s co-precipitated with recombinant glutathione S-transferase (GST)-AtF2KP that had been incubated with Arabidopsis cell extracts in the presence of Mg-ATP . 14-3-3s bound directly to GST-AtF2KP that had been phosphorylated on Ser220 (SLSASGpSFR) and Ser303 (RLVKSLpSASSF) by recombinant Arabidopsis calcium-dependent protein kinase isoform 3 (CPK3), or on Ser303 by rat liver mammalian AMP-activated protein kinase (AMPK; homologue of plant SNF-1 related protein kinases (SnRKs)) or an Arabidopsis cell extract . We have failed to find any direct effect of 14-3-3s on the F2KP activity in vitro to date . Nevertheless, our findings indicate the possibility that 14-3-3 binding to SnRK1-phosphorylated sites on NR and F2KP may regulate both nitrate assimilation and sucrose/starch partitioning in leaves.

FEBS Lett, 2004 Jan 30, 558(1-3), 85 - 8
Histidine-containing phosphotransfer domain extinction by RNA interference turns off a cytokinin signalling circuitry in Catharanthus roseus suspension cells; Papon N et al.; We previously reported that cytokinins (CK) induce the fast and specific transcription of CrRR1, a gene encoding a type A response regulator in Catharanthus roseus cell cultures . Here, we characterized the CrHPt1 gene that encodes a histidine-containing phosphotransfer domain . CrHPt1 was silenced through RNA interference (RNAi) to test its possible implication in the CK signalling pathway . In transgenic lines stably transformed with an intron-spliced construct, the degradation of CrHPt1 transcripts abolishes the CK inductive effect on CrRR1 transcription . These result give a new in vivo functional argument for the crucial role of HPt proteins in the CK signalling pathway leading to the expression of the genes encoding type A response regulators . They also show that RNAi is a powerful strategy to turn off the CK signalling circuitry.

Cell Motil Cytoskeleton, 2004 Apr, 57(4), 246 - 58
Microtubules become more dynamic but not shorter during preprophase band formation: a possible "search-and-capture" mechanism for microtubule translocation; Vos JW et al.; The dynamic behavior of the microtubule cytoskeleton plays a crucial role in cellular organization, but the physical mechanisms underlying microtubule (re)organization in plant cells are poorly understood . We investigated microtubule dynamics in tobacco BY-2 suspension cells during interphase and during the formation of the preprophase band (PPB), the cytoskeletal structure that defines the site of cytokinesis . Here we show that after 2 h of microtubule accumulation in the PPB and concurrent disappearance elsewhere in the cortex, the PPB is completed and starts to breakdown exponentially already 20 min before the onset of prometaphase . During formation of the PPB, the dynamic instability, i.e., the stochastic alternating between growing and shrinking phases, of the cortical microtubules outside the PPB increases significantly, but the microtubules do not become shorter . Based on this, as well as on the cross-linking of microtubules in the PPB and the lack of evidence for motor involvement, we propose a "search-and-capture" mechanism for PPB formation, in which the regulation of dynamic instability causes the cortical microtubules to become more dynamic and possibly longer, while the microtubule cross-linking activity of the developing PPB preferentially stabilizes these "searching" microtubules . Thus, microtubules gradually disappear from the cortex outside the PPB and aggregate to the forming PPB .

Cytometry A, 2004 Feb, 57(2), 100 - 7
Comparison of flow cytometry and laser scanning cytometry for the analysis of CD34+ hematopoietic stem cells; Oswald J et al.; BACKGROUND: Characterization of hematopoietic stem cells (HSCs) by laser scanning cytometry (LSC) was compared with conventional flow cytometry (FCM) . The method was evaluated for application in the development of advanced cell culture substrates that were supposed to support the ex vivo expansion of HSC . For this purpose, adherent HSCs were grown in culture on thin polymer films coated with reconstituted collagen I fibrils and subsequently analyzed by LSC . METHODS: CD34+ HSCs were isolated from cord blood by immunomagnetic separation and cultivated on polymer films coated with reconstituted collagen I fibrils . Cell surface antigens (CD34, CD29) were stained with antibodies, and nuclei were labeled with a DNA stain (TO-PRO-3 iodide) that does not interfere with the fluorochromes of the antibodies . Fluorescence intensity of the adherent cells was measured by means of LSC . Before and after in vitro expansion for time periods of up to 7 days, suspension cells were analyzed with LSC and FCM . RESULTS: LSC-based analysis enabled reliable quantification of CD34+ cells with bright antigen expression before cell culture . At this stage, LSC and FCM data for CD34 expression at given HSC samples largely coincided . After in vitro expansion, LSC data deviated from FCM data for cells with dim CD34 antigen expression, whereas the fluorescence intensity of the CD29 antigen remained comparable . The deviation between LSC and FCM data for CD34dim was attributed to the better resolution of weak fluorescence by FCM . Based on the preceding evaluation of the method, LSC analysis could be applied to characterize HSCs cultivated on collagen I-coated polymer films without detachment of the cells from the substrate . CONCLUSIONS: LSC-based analysis allows for the automated evaluation of adherent HSCs . Although resolution of weakly expressed antigens can be achieved more precisely with FCM, the method provides a valuable tool to study interactions of HSCs with bioartificial substrates .

J Zhejiang Univ Sci, 2004 Feb, 5(2), 137 - 43
Programmed cell death features in apple suspension cells under low oxygen culture; Xu CJ et al.; Suspension-cultured apple fruit cells (Malus pumila Mill . cv . Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions . Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342) . DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding . About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability . Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen . The results suggest that apple cell death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

Biosci Biotechnol Biochem, 2003 Nov, 67(11), 2438 - 47
Gibberellin is essentially required for carrot (Daucus carota L.) somatic embryogenesis: dynamic regulation of gibberellin 3-oxidase gene expressions; Mitsuhashi W et al.; A GA biosynthesis inhibitor, uniconazole, caused many shrunken embryos when it was supplied to cultured carrot (Daucus carota L.) cells at the induction of somatic embryos . The abnormality was prevented by exogenous GA(1) or GA(4) . To analyze the status of GA biosynthesis during somatic embryogenesis, expression patterns of newly isolated genes encoding GA biosynthetic enzymes, two GA 20-oxidases, three GA 3-oxidases, and two GA 2-oxidases were observed by using a semi-quantitative reverse-transcription-polymerase chain reaction with gene-specific primers . Transcript levels of GA 20-oxidases and GA 2-oxidases did not change greatly during development of the somatic embryo . On the other hand, drastic changes were found in three GA 3-oxidase genes . Strikingly, expression of a GA 3-oxidase gene, DcGA3ox2, was elevated once in somatic embryogenesis, but not in the non-induced suspension cells . The enzymatic functions of these gene products were also confirmed using recombinant proteins expressed in Escherichia coli . Our results indicate that GA biosynthesis is required for carrot somatic embryogenesis.

Mol Genet Genomics, 2004 Jan, 270(6), 485 - 96 Epub 2003 Nov 21.
Proteome analysis of rice tissues by two-dimensional electrophoresis: an approach to the investigation of gibberellin regulated proteins; Tanaka N et al.; Protein databases constructed using high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) were used to explore the proteome expressed in various rice tissues . Proteins from leaf sheath, root, and cultured suspension cells were systematically analyzed using 2D-PAGE, mass spectrometry and Edman sequencing, followed by database searching . In all, 79 of the 431 spots detected by 2D-PAGE in the leaf sheath, 73 of the 508 spots in the root and 140 of the 962 spots in the cultured suspension cells could be identified . Protein lists were constructed for each tissue and used to investigate the effects of gibberellin (GA) treatment . In the leaf sheath, root and cultured suspension cells, 8, 21, and 14 of the identified proteins, respectively, were regulated by GA . These proteins included polypeptides involved in general metabolism, energy production, transcriptional regulation and signal transduction in the leaf sheath; in metabolism and defense in the root; and in metabolism, energy production, cell growth, defense and signal transduction in the cultured suspension cells . These results indicate that the proteome databases assembled in these studies will be useful for the rapid assessment of changes in protein content in specific tissues, and that proteins regulated by GA may play a significant role in tissue growth.

J Biomol Screen, 2002 Dec, 7(6), 515 - 25
A homogeneous enzyme fragment complementation cyclic AMP screen for GPCR agonists; Golla R et al.; In the new high-throughput screening (HTS) campaign, receptor functional assays, 3',5'-cyclic adenosine monophosphate (cAMP), intracellular {Ca(2)+}(i), phosphatidylinositol turnover, and reporter-based assays are being used as primary screens as they are now developed as homogeneous and automation-friendly assays . FlashPlate assay and scintillation proximity assay using radiolabeled cAMP have been used for measuring cAMP . A nonradioactive homogeneous HTS assay using HitHunter trade mark enzyme fragment complementation (EFC) technology was evaluated for measuring cAMP in adherent and suspension cells overexpressing a Galpha(s)-coupled receptor . In the EFC-cAMP assay, the beta-galactosidase (beta-gal) donor fragment-cAMP (ED-cAMP) conjugate complements with the beta-gal enzyme acceptor (EA) fragment to form an active beta-gal enzyme . Binding of ED-cAMP conjugate to the anti-cAMP antibody prevents its complementation with the EA fragment to form an active enzyme . Cyclic AMP in the samples compete with ED-cAMP to bind to the anti-cAMP antibody, thus increasing the free ED-cAMP that can complement with the EA fragment to form an active enzyme that is assayed with a luminescent substrate . Thus, this assay results in a positive signal unlike other technologies, wherein the signal is completed by cAMP in the sample . Glucagon-like peptide (GLP)-1 binds to GLP-1 receptor (with a Kd of 0.2 nM) signals through Galpha(s) to activate adenylate cyclase, which results in an increase of intracellular cAMP (EC(50) of 0.3 nM) . GLP-1 stimulation of cAMP levels measured by the EFC method was similar in both adherent and suspension cell formats (EC(50)~0.3 nM) at different cell numbers . The assay was further validated with forskolin, exendin, and several active GLP-1 peptide analogues . The stimulation of cAMP by GLP-1 and forskolin was effectively inhibited by the adenylate cyclase inhibitors MDL-12330A and SQ-22536, confirming that the increased cAMP is through the AC pathway . The assay tolerates dimethyl sulfoxide (DMSO) up to 10%, and tartrazine does not interfere with the assay with the adherent cells up to 1 mM and affects minimally up to 10 microM in suspension cells . The assay is very robust, with a Z' value of 0.7 to 0.8 . The assay was validated with several plates of low molecular weight nonpeptide compounds and peptide agonists with different potencies . The suspension cell protocol is a robust homogeneous assay that involves fewer steps than the adherent cell protocol and is suitable for HTS . The cAMP assay using EFC technology is advantageous in that it has a greater dynamic range of detection; is nonradioactive, very sensitive, robust; has minimal interference from DMSO and colored compounds; and is amenable for automation . An added advantage of this assay is that the cAMP is measured as a positive signal, thereby reducing the incidence of false positives.

Biochem J, 2004 Jan 15, 377(Pt 2), 419 - 28
Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures; Bozzo GG et al.; An intracellular acid phosphatase (IAP) from P(i)-starved (-P(i)) tomato ( Lycopersicon esculentum ) suspension cells has been purified to homogeneity . IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (lambda(max)=546 nm) and was insensitive to L-tartrate . PAGE, periodic acid-Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (alpha-subunit) and 57 kDa (beta-subunit) polypeptides . However, the nine N-terminal amino acids of the alpha- and beta-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs . Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to P(i)-deprivation was correlated with similar increases in the amount of antigenic IAP alpha- and beta-subunits . IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg(2+), but was potently inhibited by Zn(2+), Cu(2+), Fe(3+), molybdate, vanadate, fluoride and P(i) . Although IAP demonstrated broad substrate selectivity, its specificity constant ( V (max)/ K (m)) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate . IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg(2+) or molybdate . This IAP is proposed to scavenge P(i) from intracellular phosphate esters in -P(i) tomato . A possible secondary IAP role in the metabolism of reactive oxygen species is discussed . IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of -P(i) tomato cells {Bozzo, Raghothama and Plaxton (2002) Eur . J . Biochem . 269, 6278-6286}.

J Exp Bot, 2003 Nov, 54(392), 2587 - 8 Epub 2003 Sep 09.
CrMYC1, a Catharanthus roseus elicitor- and jasmonate-responsive bHLH transcription factor that binds the G-box element of the strictosidine synthase gene promoter; Chatel G et al.; A cDNA encoding a bHLH transcription factor was isolated by the yeast one-hybrid system from a Catharanthus roseus cDNA library using the G-box element of the Strictosidine synthase gene promoter as bait . The corresponding protein (named CrMYC1) was shown to bind specifically to the G-box in yeast . In C . roseus suspension cells CrMYC1 mRNA levels are induced by fungal elicitor and jasmonate suggesting that CrMYC1 may be involved in the regulation of gene expression in response to these signals.

Plant Cell, 2003 Sep, 15(9), 2058 - 75
Involvement of the secretory pathway and the cytoskeleton in intracellular targeting and tubule assembly of Grapevine fanleaf virus movement protein in tobacco BY-2 cells; Laporte C et al.; Grapevine fanleaf virus (GFLV) is one of a large class of plant viruses whose cell-to-cell transport involves the passage of virions through tubules composed of virus-encoded movement protein (MP) . The tubules are embedded within modified plasmodesmata, but the mechanism of targeting of MP to these sites is unknown . To study intracellular GFLV MP trafficking, a green fluorescent protein-MP fusion (GFP:MP) was expressed in transgenic tobacco BY-2 suspension cells under the control of an inducible promoter . We show that GFP:MP is targeted preferentially to calreticulin-labeled foci within the youngest cross walls, where it assembles into tubules . During cell division, GFP:MP colocalizes in the cell plate with KNOLLE, a cytokinesis-specific syntaxin, and both proteins are linked physically, as shown by coimmunoprecipitation of the two proteins from the same microsomal fraction . In addition, treatment with various drugs has revealed that a functional secretory pathway, but not the cytoskeleton, is required for tubule formation . However, correct GFP:MP targeting to calreticulin-labeled foci seems to be cytoskeleton dependent . Finally, biochemical analyses have revealed that at least a fraction of the MP behaves as an intrinsic membrane protein . These findings support a model in which GFP:MP would be transported to specific sites via Golgi-derived vesicles along two different pathways: a microtubule-dependent pathway in normal cells and a microfilament-dependent default pathway when microtubules are depolymerized.

Planta, 2003 Dec, 218(2), 204 - 16 Epub 2003 Aug 21.
The association of peroxisomes with the developing cell plate in dividing onion root cells depends on actin microfilaments and myosin; Collings DA et al.; We have investigated changes in the distribution of peroxisomes through the cell cycle in onion ( Allium cepa L.) root meristem cells with immunofluorescence and electron microscopy, and in leek ( Allium porrum L.) epidermal cells with immunofluorescence and peroxisomal-targeted green fluorescent protein . During interphase and mitosis, peroxisomes distribute randomly throughout the cytoplasm, but beginning late in anaphase, they accumulate at the division plane . Initially, peroxisomes occur within the microtubule phragmoplast in two zones on either side of the developing cell plate . However, as the phragmoplast expands outwards to form an annulus, peroxisomes redistribute into a ring immediately inside the location of the microtubules . Peroxisome aggregation depends on actin microfilaments and myosin . Peroxisomes first accumulate in the division plane prior to the formation of the microtubule phragmoplast, and throughout cytokinesis, always co-localise with microfilaments . Microfilament-disrupting drugs (cytochalasin and latrunculin), and a putative inhibitor of myosin (2,3-butanedione monoxime), inhibit aggregation . We propose that aggregated peroxisomes function in the formation of the cell plate, either by regulating hydrogen peroxide production within the developing cell plate, or by their involvement in recycling of excess membranes from secretory vesicles via the beta-oxidation pathway . Differences in aggregation, a phenomenon which occurs in onion, some other monocots and to a lesser extent in tobacco BY-2 suspension cells, but which is not obvious in the roots of Arabidopsis thaliana (L.) Heynh., may reflect differences within the primary cell walls of these plants.

Planta, 2003 Dec, 218(2), 233 - 9 Epub 2003 Aug 14.
Phosphite accelerates programmed cell death in phosphate-starved oilseed rape (Brassica napus) suspension cell cultures; Singh VK et al.; Phosphite (H(2)PO(3)(-), Phi) prevents the acclimation of plants and yeast to orthophosphate (Pi, HPO(4)(2-)) deprivation by specifically obstructing the derepression of genes encoding proteins characteristic of their Pi-starvation response . In this study, we report that prolonged (i.e., 3-4 weeks) culture of Brassica napus L . suspension cells in Pi-deficient (-Pi) media leads to programmed cell death (PCD) . However, when the B . napus cells were subcultured into -Pi media containing 2 mM Phi, they initiated PCD within 5 days, with 95% cell death observed by day 9 . Dying cells exhibited several morphological and biochemical features characteristic of PCD, including protoplast shrinkage, chromatin condensation, and fragmentation of nuclear DNA . Immunoblotting indicated that B . napus cells undergoing PCD upregulated a 30-kDa cysteine endoprotease that is induced during PCD in the inner integument cells of developing B . napus seeds . It is concluded that PCD in B . napus suspension cells is triggered by extended Pi starvation, and that Phi treatment greatly accelerates this process . Our results also infer that the adaptive value of acclimating at the molecular level to Pi-stress is to extend the viability of -Pi B . napus cell cultures by about 3 weeks.

Plant Cell Rep, 2003 Aug, 21(12), 1199 - 206 Epub 2003 May 15.
A promoter derived from taro bacilliform badnavirus drives strong expression in transgenic banana and tobacco plants; Yang IC et al.; Taro bacilliform virus (TaBV) is a pararetrovirus of the genus Badnavirus which infects the monocotyledonous plant, taro ( Colocasia esculenta) . A region of the TaBV genome spanning nucleotides 6,281 to 12 (T1200), including the 3' end of open reading frame 3 (ORF 3) and the intergenic region to the end of the tRNA(met)-binding site, was tested for promoter activity along with four different 5' deletion fragments (T600, T500, T250 and T100) . In transient assays, only the T1200, T600, T500 fragments were shown to have promoter activity in taro leaf, banana suspension cells and tobacco callus . When these three promoters were evaluated in stably transformed, in vitro-grown transgenic banana and tobacco plants, all were found to drive near-constitutive expression of either the green fluorescent protein or beta-glucuronidase (GUS) reporter gene in the stem (or pseudostem), leaves and roots, with strongest expression observed in the vascular tissue . In transgenic banana leaves, the T600 promoter directed four-fold greater GUS activity than that of the T1200, T500 and the maize polyubiquitin-1 promoters . In transgenic tobacco leaves, the levels of GUS expression directed by the three promoters was between four- and ten-fold lower than that of the double Cauliflower mosaic virus 35S promoter . These results indicate that the TaBV-derived promoters may be useful for the high-level constitutive expression of transgenes in either monocotyledonous or dicotyledonous species.

World J Gastroenterol, 2003 Jul, 9(7), 1594 - 7
Effects of carbon dioxide and nitrogen on adhesive growth and expressions of E-cadherin and VEGF of human colon cancer cell CCL-228; Cai KL et al.; AIM: To study the effects of carbon dioxide on the metastatic capability of cancer cells, and to compare them with that of nitrogen . METHODS: The colon cancer cell CCL-228 was treated with 100 % carbon dioxide or nitrogen at different time points and then cultured under normal condition . Twelve hours after the treatment, the survival rates of suspension cells and the expressions of e-cadherin and VEGF were examined . RESULTS: After 60 min of carbon dioxide and longer time of nitrogen treatment, the suspended cells increased and the expression of e-cadherin decreased while the expression of VEGF was enhanced significantly . And the effects of nitrogen were similar to, but weaker than, those of carbon dioxide . CONCLUSION: Carbon dioxide may improve the metastatic capability of cancer cells and its effects are significantly stronger than that of nitrogen . A sequential use of carbon dioxide and nitrogen in pneumoperitoneum may take the advantage of both gases.

Biocell, 2003 Apr, 27(1), 47 - 55
Inhibition of focal adhesion kinase by antisense oligonucleotides enhances the sensitivity of breast cancer cells to camptothecins; Satoh TH et al.; This study shows a strong association between cell attachment to substratum and activation of beta 1-integrin-signaling with resistance to the camptothecin derivative topotecan (TPT) in breast cancer cells . We propose a mechanistic-driven approach to sensitize the cells to camptothecins . ZR-75-1 anchorage-dependent breast cancer cell line, its derivative 9D3S suspension cells (9D3S-S), and 9D3S cells attached to fibronectin-coated plates (9D3S-A) were treated with TPT (1 microM) or CPT-11 (40 microM) for 48 h . Programmed cell death (PCD), as shown by poly(ADP-ribose) polymerase (PARP), pro-caspase-3 and pro-caspase-9 cleavage, was observed in 9D3S-S cells but not in ZR-75-1 or 9D3S-A cells . Because p125 focal adhesion kinase (FAK) is a transducer in the beta 1-integrin signaling pathway, it is essential to cell adhesion and it is overexpressed in metastatic breast cancer, we hypothesized that attenuation of FAK might enhance the sensitivity of breast cancer cells to camptothecins . Moreover, inhibition of FAK gene expression by a phosphorothioated antisense oligodeoxynucleotide targeting the portion of the gene encoding amino acids 262-268, increased the sensitivity of ZR-75-1, MDA-MB-231 and MCF7 breast cancer cells to treatment with TPT or CPT-11.

J Exp Bot, 2003 Jul, 54(388), 1793 - 5
Differential expression of two type-A response regulators in plants and cell cultures of Catharanthus roseus (L.) G . Don; Papon N et al.; Two full-length cDNAs named CrRR2 and CrRR3 have been isolated by PCR from a C . roseus cDNA library . The first one encodes a 154 amino acid putative protein with a high percentage of identity with the Arabidopsis thaliana response regulators ARR16 and ARR17, and is not expressed in C . roseus organs and cell cultures . The second one encodes a 188 amino acid ORF sharing the highest homologies with the A . thaliana ARR8 and ARR9 response regulators . Its expression is root-specific and the transcripts are transiently up-regulated after trans-zeatin treatment in C . roseus suspension cells . CrRR3 protein might be involved in the cytokinin-enhanced alkaloid production in C . roseus cell cultures.

J Biol Chem, 2003 Sep 12, 278(37), 35732 - 42 Epub 2003 Jun 16.
Equilibrative nucleoside transporters of Arabidopsis thaliana . cDNA cloning, expression pattern, and analysis of transport activities; Li G et al.; Equilibrative nucleoside transporters (ENTs) occur in diverse organisms . In the model plant Arabidopsis thaliana, eight potential ENTs (AtENTs) have been predicted by genome sequencing . We here report the cloning of the cDNAs for AtENTs 2, 3, 4, 6, 7, and 8 . Conceptual translation of the cDNAs of AtENTs 2, 3, 4, 6, 7, and 8 yielded polypeptides possessing strong similarities to ENTs characterized previously . Eleven putative transmembrane domains were identified in each of the six AtENTs . In suspension cells, the transcription of AtENTs 1, 3, 4, 6, and 8 was increased by two treatments (nitrogen deprivation, application of 5-fluorouracil and methotrexate) that inhibited the de novo pathway of nucleotide synthesis, indicating that multiple members of the Arabidopsis ENT family may function in the salvage pathway of nucleotide synthesis . Except for AtENT1, the transcription of the remaining six AtENTs showed varying degrees of organ specificity . However, all seven AtENTs were expressed in the leaf and flower . In plant, insect, and yeast cells, ectopically expressed AtENT3 was targeted to the plasma membrane . AtENT3 expressed in yeast cells transported adenosine and uridine with high affinity . Furthermore, the activities of AtENT3 appear not to require a transmembrane proton gradient because protonophores did not abolish adenosine or uridine transport . In competition experiments, the transport of {3H}adenosine by AtENT3 was most significantly inhibited by a number of different purine and pyrimidine nucleosides and 2'-deoxynucleosides, although certain nucleobases and nucleotides were also found to have some inhibitory effect . This indicates that AtENT3 may possess broad substrate specificity . Adenosine and uridine transport by AtENT3, although partly sensitive to the vasodilator drugs dilazep and dipyridamole, was resistant to the nucleoside analogue nitrobenzylmercaptopurine ribonucleoside . We conclude that AtENT3 represents the first ei type ENT characterized from higher plants . The potential functions of ENTs in the biology of A . thaliana are discussed.

Protoplasma, 2003 Mar, 220(3-4), 111 - 8
Development and disintegration of phragmoplasts in living cultured cells of a GFP::TUA6 transgenic Arabidopsis thaliana plant; Ueda K et al.; Cultured suspension cells of Arabidopsis thaliana that stably express a green-fluorescent protein-alpha-tubulin 6 fusion protein were used to follow the development and disintegration of phragmoplasts . The development and disintegration of phragmoplasts in the living cultured cells could be successively observed by detecting the green-fluorescent protein fluorescence of the microtubules . In the early telophase spindle, where two kinetochore groups and two daughter chromosome groups had completely separated from one another, fluorescence appeared in the interzone between the two chromosome groups . The fluorescent region was gradually condensed at the previous equator and increased in fluorescence intensity, and finally it formed the initial phragmoplast . The initial phragmoplast moved from the cell center towards the cell periphery, and it lost fluorescence at its center and became double rings in shape . The expansion orientation of the phragmoplast was not always the same as that of the future new cell wall before it came in contact with the cell wall . The phragmoplast did not usually come in contact with the cell wall simultaneously with its entire length . A portion of the phragmoplast which was earlier in contact with the cell wall disappeared earlier than other portions of the phragmoplast . The duration of contact between any portions of the phragmoplast and the plasma membrane of the cell wall was 15-30 min . The fluorescence intensity of the cytoplasm did not seem to be elevated by the disintegration of the strongly fluorescent phragmoplast.

J Biol Chem, 2003 Jun 13, 278(24), 21395 - 407 Epub 2003 Mar 19.
Expression of the telomeric repeat binding factor gene NgTRF1 is closely coordinated with the cell division program in tobacco BY-2 suspension culture cells; Yang SW et al.; Telomeres are vital for preserving chromosome integrity during cell division . Several genes encoding potential telomere-binding proteins have recently been identified in higher plants, but nothing is known about their function or regulation during cell division . In this study, we have isolated and characterized a cDNA clone, pNgTRF1, encoding a putative double-stranded telomeric repeat binding factor of Nicotiana glutinosa, a diploid tobacco plant . The predicted protein sequence of NgTRF1 (Mr = 75,000) contains a single Myb-like domain with significant homology to a corresponding motif in human TRF1/Pin2 and TRF2 . Gel retardation assays revealed that bacterially expressed full-length NgTRF1 was able to form a specific complex only with probes containing three or more contiguous telomeric TTTAGGG repeats . The Myb-like domain of NgTRF1 is essential, but not sufficient, to bind the telomeric repeat sequence . The glutamine-rich extreme C-terminal region, which does not exist in animal proteins, was additionally required to form a specific telomere-protein complex . The dissociation constant (Kd) of the Myb motif plus the glutamine-rich domain of NgTRF1 to the two-telomeric repeat sequence was evaluated to be 4.5 +/- 0.2 x 10-9 m, which is comparable to that of the Myb domain of human TRF1 . Expression analysis showed that NgTRF1 gene activity was inversely correlated with the cell division capacity of tobacco root cells and during the 9-day culture period of BY-2 suspension cells, while telomerase activity was positively correlated with cell division . In synchronized BY-2 cells, NgTRF1 was selectively expressed in G1 phase, whereas telomerase activity peaked in S phase . These findings suggest that telomerase activity and NgTRF1 expression are differentially regulated in an opposing fashion during growth and cell division in tobacco plants . The possible physiological functions of NgTRF1 in tobacco cells are also discussed.

J Cell Physiol, 2003 Apr, 195(1), 108 - 18
Lipid factor (bVLF) from bovine vitreous body evokes in EGFR-T17 cells a Ca2+-dependent K+ current associated with inositol 1,4,5-trisphosphate-independent Ca2+ mobilization; Camina JP et al.; Bovine vitreous lipid factor (bVLF) is a complex phospholipid isolated from bovine vitreous body with strong Ca(2+)-mobilizing activity . In this study, the effects of bVLF on membrane potential were investigated in EGFR-T17 fibroblasts with the whole-cell patch clamp technique on monolayer cells, as well as with the fluorescent dye bis-oxonol as membrane potential-sensitive probe on monolayer and suspension cells . bVLF induced a transient hyperpolarization characterized by an initial peak and subsequent return to resting membrane potential levels within 1-2 min . The increase of {Ca(2+)}(i) was concomitant with an outward current responsible for the hyperpolarizing response . Results with: (a) high {K(+)}(o) media; (b) the monovalent cation ionophore gramicidin; and (c) substitution of K(+) with Cs(+) in the intracellular solution were consistent with the involvement of K(+) channels . The bVLF-induced hyperpolarization was blocked by the K(+) channel blockers, quinine and tetraethylamonium chloride, and partially affected by 4-aminopyridine . The calcium ionophore ionomycin caused a similar hyperpolarization as bVLF . When intracellular calcium was buffered by adding BAPTA to the pipette solution, bVLF-activated outward current was prevented . Moreover, the hyperpolarization response was strongly reduced at low doses (3 nM) of specific Ca(2+)-activated K(+) channel blockers, charybdotoxin and iberiotoxin . Based on these observations we conclude that bVLF hyperpolarizes the cells via the activation of a Ca(2+)-dependent K(+) current . In addition, it was observed that bVLF did not have a significant effect on intercellular communication measured by a single patch-electrode technique . Thus, membrane potential changes appeared to belong to the earliest cellular responses triggered by bVLF, and are closely associated with phosphatidic acid-dependent {Ca(2+)}(i) mobilization .

Biochim Biophys Acta, 2003 Feb 20, 1625(3), 261 - 8
Identification and characterization of two new members of the GRAS gene family in rice responsive to N-acetylchitooligosaccharide elicitor; Day RB et al.; We identified two new members of the GRAS gene family from rice, CIGR1 and CIGR2, which are rapidly induced upon N-acetylchitooligosaccharide elicitor perception . The predicated proteins encoded by CIGR1 and CIGR2 possess significant sequence similarity with previously identified members of the GRAS family, such as Arabidopsis SCARECROW, GAI, RGA, tomato Lateral suppressor, and rice SLR1, all of which have VHIID regions, likely to play a role in cellular signaling . Fusions of CIGR1 and CIGR2 with Green Fluorescent Protein were detected exclusively in the nuclei of onion epidermal cells . The expression of CIGR1 and CIGR2 was dependent on the structure of N-acetylchitooligosaccharides, which parallels the structural specificity for chitin binding to the plasma membrane-localized chitin-binding protein, and independent of de novo protein synthesis . Co-cultivation of rice cells with rice blast fungus strongly induced the expression of CIGR1 and CIGR2, whereas inoculation of suspension cells with phytopathogenic bacteria did not . We hypothesize that CIGR1 and CIGR2 act as transcriptional regulators in the early events of the elicitor-induced defense response in rice.

Zhong Yao Cai, 2000 Jan, 23(1), 1 - 4
{Effects of phytohormones on growth and content of depsides in Salvia miltiorrhiza suspension cells}; Huang L et al.; This paper deals with the effects of 2,4-D, BA and GA3 on the growth and content of two depsides (rosmarinic acid and lithospermic acid B) in suspension cells of Salvia miltiorrhiza . The results showed cell growth and rosmarinic acid content reached the maximum on the 12th day and lithospermic acid B content on the 16th day after incubating cells in the subculture medium MS + 2, 4-D 1 mg/L + KT 0.1 mg/L . This cell line was a growth-product-associated . With the same concentration (1 mg/L), 2, 4-D stimulated the cell growth but prohibited the formation of lithospermic acid B; GA3 inhabited the cell growth but stimulated the formation of two depsides; The effects of BA is between 2, 4-D and GA3 . The concentration optimum of phytohormones tested displayed 3 mg/L for BA and 1 mg/L for GA3 . For the optimum time of adding BA and GA3 was in med-term (the 8th day) and early term (at the beginning) of culture period respectively . The synergiatic function of BA and GA3 on the depside formation was also showed that adding GA3 (1 mg/L) was favour for the formation of lithospermic acid B of the suspension cell cultured in the medium containing BA 3 mg/L . The suitable time of adding GA3 was the 6the day after incubating cells in the medium of MS + BA 3 mg/L.

Plant Cell Physiol, 2003 Jan, 44(1), 93 - 5
The plastid clpP gene may not be essential for plant cell viability; Cahoon AB et al.; The plastid gene clpP is widely regarded as essential for chloroplast function and general plant cell survival . In this note we provide evidence that certain lines of non-photosynthetic maize (Zea mays) Black Mexican Sweet (BMS) suspension cells do not carry clpP in their plastid genomes . We also discuss several incidences in the literature where clpP is either missing or not expressed in other non-green cell lines and plants . We conclude that clpP is not required for general plant cell survival but instead may only be essential for the development and/or function of plastids with active gene expression.

Shi Yan Sheng Wu Xue Bao, 2000 Mar, 33(1), 85 - 8
{Factors influence on transformation by particle bombardment in Indica rice}; Tao LZ et al.; Four factors influence on transformation of indica rice, which were high osmotic treatment; different explant as the target tissue; pressure of rupture disk and quantity of plasmid DNA, were investigated in this experiment . High osmotic treatment of target tissue prior to and after bombardment increased 3.2-fold for Gus transient expression than control . The best treatment of high osmotic was that the target tissues were kept in the target-bed medium which contained 0.4-0.6 mol/L sorbitol and manitol each for 4 h prior to bombardment and for 16 h after bombardment . Four explants: scutellum from mature seed, young panicle, embryogenic callus and suspension cells of indica rice were tested as target explant by particle bombardment . The results of Gus transient showed that the highest expression was scutellum and for other three explants, the order from high to low was young panicle, embryogenic callus and suspension cell . Transgenic plants were obtained from all of the explants except young panicle . For the pressure of rupture disk on transformation, 1100 psi or 1300 psi of the pressure of rupture disk were best one for the transformation and higher than 1300 psi could damage the target tissue which become black and died in the following culture duration . For the quantity of plasmid DNA, the results showed that 0.83 microgram of plasmid DNA per bombardment was preferred for the transformation of indica rice.

Shi Yan Sheng Wu Xue Bao, 1999 Sep, 32(3), 271 - 6
{Studies on the application of antifreeze proteins in cryopreservation of rice suspension cells}; Wang JH et al.; AFP from winter flounder was utilized in cryopreservation of plant cells . During cryopreservation of rice suspension cells by two-step method, AFP at 0.01 mg/ml damaged the cells extremely . The data obtained at relatively high concentration, however, decreased the variability of survival rate . During vitrification of rice cells, AFP at 0.2 mg/ml enhanced the viability . However, high concentration AFP (> 5 mg/ml) decreased the recovery rate . Studies indicated that the results of application of AFP in cryopreservation were closely related to the concentration of cryoprotectant . The amount of ice crystal in environment, the concentration of AFP and cryoprotectant, and the composition of plasma membrane were several key factors affecting the results of AFP application . In mechanism analysis, the authors suggested that on one hand AFP can interact with ice crystal, which inhibits ice recrystallization and prevent the cells from devitrification . On the other hand, AFP also can interact with cell membrane, resulting in the ice growth around the plasma membrane.

Shi Yan Sheng Wu Xue Bao, 1999 Mar, 32(1), 88 - 92
{Involvement of anion channel in signal transduction of early defense responses elicited by harpinPss in tobacco}; Qiu JL et al.; No matter when anion channel inhibitors, DIDS (4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid) and A9C (anthracene-9-carboxylic acid) added (before, at the same time of or after harpinPss treatment), they can inhibit harpinPss-induced hypersensitive response in tobacco seedlings and release of active oxygen and extracellular alkalinization in tobacco suspension cells . DIDS and A9C also inhibit harpinPss-induced Ca2+ influx . In all these cases, DIDS is more efficient than A9C . It is postulated that anion channel positively regulates calcium channel in plasma membrane, and harpinPss may function through signal transduction mediated by anion channel and calcium channel to regulate cellular Ca2+ concentration and defense responses.

Eur J Biochem, 2002 Dec, 269(24), 6278 - 86
Purification and characterization of two secreted purple acid phosphatase isozymes from phosphate-starved tomato (Lycopersicon esculentum) cell cultures; Bozzo GG et al.; Two secreted acid phosphatases (SAP1 and SAP2) were markedly up-regulated during Pi-starvation of tomato suspension cells . SAP1 and SAP2 were resolved during cation-exchange FPLC of culture media proteins from 8-day-old Pi-starved cells, and purified to homogeneity and final p-nitrophenylphosphate hydrolyzing specific activities of 246 and 940 micro mol Pi produced.min-1 mg.protein-1, respectively . SDS/PAGE, periodic acid-Schiff staining and analytical gel filtration demonstrated that SAP1 and SAP2, respectively, exist as 84 and 57 kDa glycosylated monomers . SAP1 and SAP2 are purple acid phosphatases (PAPs) as they displayed an absorption maximum at 518 and 538 nm, respectively, and were not inhibited by l-tartrate . The respective sequence of a SAP1 and SAP2 tryptic peptide was very similar to a portion of the deduced sequence of several putative Arabidopsis thaliana PAPs . CNBr peptide mapping indicated that SAP1 and SAP2 are structurally distinct . Both isozymes displayed a pH optimum of approximately pH 5.3 and were heat stable . Although they exhibited wide substrate specificities, the Vmax of SAP2 with various phosphate-esters was significantly greater than that of SAP1 . SAP1 and SAP2 were activated by up to 80% by 5 mm Mg2+, and demonstrated potent competitive inhibition by molybdate, but mixed and competitive inhibition by Pi, respectively . Interestingly, both SAPs exhibited significant peroxidase activity, which was optimal at approximately pH 8.4 and insensitive to Mg2+ or molybdate . This suggests that SAP1 and SAP2 may be multifunctional proteins that operate: (a) PAPs that scavenge Pi from extracellular phosphate-esters during Pi deprivation, or (b) alkaline peroxidases that participate in the production of extracellular reactive oxygen species during the oxidative burst associated with the defense response of plants to pathogen infection.

Plant Physiol, 2002 Oct, 130(2), 999 - 1007
Activation of phospholipases C and D is an early response to a cold exposure in Arabidopsis suspension cells; Ruelland E et al.; The signaling events generated by a cold exposure are poorly known in plants . We were interested in checking the possible activation of enzymes of the phosphoinositide signaling pathway in response to a temperature drop . In Arabidopsis suspension cells labeled with (33)PO(4)(3-), a cold treatment induces a rapid increase of phosphatidic acid (PtdOH) content . This production was due to the simultaneous activation of phospholipase C (through diacylglycerol kinase activity) and phospholipase D, as monitored by the production of inositol triphosphate and of transphosphatidylation product, respectively . Moreover, inhibitors of the phosphoinositide pathway and of diacylglycerol kinase reduced PtdOH production . Enzyme activation occurred immediately after cells were transferred to low temperature . The respective contribution of both kind of phospholipases in cold-induced production of PtdOH could be estimated . We created conditions where phospholipids were labeled with (33)PO(4)(3-), but with ATP being nonradioactive . In such conditions, the apparition of radioactive PtdOH reflected PLD activity . Thus, we demonstrated that during a cold stress, phospholipase D activity accounted for 20% of PtdOH production . The analysis of composition in fatty acids of cold-produced PtdOH compared with that of different phospholipids confirmed that cold-induced PtdOH more likely derived mainly from phosphoinositides . The addition of chemical reagents modifying calcium availability inhibited the formation of PtdOH, showing that the cold-induced activation of phospholipase pathways is dependent on a calcium entry.

Plant Mol Biol, 2002 Nov, 50(4-5), 735 - 42
Regulation of alternative oxidase gene expression in soybean; Djajanegara I et al.; Soybean (Glycine max cv . Stevens) suspension cells were used to investigate the expression of the alternative oxidase (Aox) multigene family . Suspension cells displayed very high rates of cyanide-insensitive respiration, but Aox3 was the only isoform detected in untreated cells . Incubation with antimycin A, citrate, salicylic acid or at low temperature (10 degrees C) specifically induced the accumulation of the Aox1 isoform . Aox2 was not observed under any conditions in the cells . Increases in Aox1 protein correlated with increases in Aox1 mRNA . Treatment of soybean cotyledons with norflurazon also induced expression of Aox1 . Reactive oxygen species (ROS) were detected upon incubation of cells with antimycin, salicylic acid or at low temperature, but not during incubation with citrate . Aox1 induction by citrate, but not by antimycin, was prevented by including the protein kinase inhibitor staurosporine in the medium . The results suggest that multiple pathways exist in soybean to regulate expression of Aox genes and that Aox1 specifically is induced by a variety of stress and metabolic conditions via at least two independent signal transduction pathways.

Planta, 2002 Oct, 215(6), 914 - 23 Epub 2002 Jul 25.
Nitric oxide induces transcriptional activation of the nitric oxide-tolerant alternative oxidase in Arabidopsis suspension cells; Huang X et al.; Nitric oxide (NO) is a double-edged sword - it can be either beneficial and activate defence responses in plants and animals or, together with reactive oxygen species, it can kill not only the pathogen but also the host . A prime target of NO is the cytochrome c-dependent respiration . Only plants possess alternative-pathway respiration with alternative oxidase (AOX) as a terminal electron acceptor . AOX has been suggested to be barely affected by NO . Here we show that NO affects cytochrome-dependent respiration in Arabidopsis thaliana (L.) Heynh . At the same time, treatment of Arabidopsis cell cultures with NO actually strongly induced AOX1a transcription, as determined by using a cDNA microarray and by Northern analysis . In accordance with transcript accumulation, NO treatment of suspension cells resulted in increased respiration through the alternative pathway . Addition of an AOX inhibitor to Arabidopsis cell cultures resulted in dramatically increased NO-sensitivity and cell death . In all, our data suggest that NO induces the AOX1a gene and that AOX may participate to counteract the toxicity of NO . Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00425-002-0828-z.

Planta, 2002 Sep, 215(5), 880 - 4 Epub 2002 Jul 26.
Regeneration of flowering plants from difficile lily protoplasts by means of a nurse culture; Horita M et al.; The regeneration of difficile lily protoplasts isolated from suspension cells of the Oriental hybrid lily ( Lilium L.) cultivars Casablanca, Siberia and Acapulco was achieved by using the nurse-culture method . The divided protoplasts grew into colonies with nurse cells that have no regeneration ability, and developed to visible calli on a medium containing picloram . Many plantlets were formed on the calli after transfer of the proliferated calli to hormone-free medium . We were able to transplant the plantlets to soil in pots without acclimatization, and the plantlets grew in a greenhouse until flowering 2 years later.

Mol Plant Microbe Interact, 2002 Sep, 15(9), 932 - 8
The indolic compound hypaphorine produced by ectomycorrhizal fungus interferes with auxin action and evokes early responses in nonhost Arabidopsis thaliana; Reboutier D et al.; Signals leading to mycorrhizal differentiation are largely unknown . We have studied the sensitivity of the root system from plant model Arabidopsis thaliana to hypaphorine, the major indolic compound isolated from the basidiomycetous fungus Pisolithus tinctorius . This fungi establishes ectomycorrhizas with Eucalyptus globulus . Hypaphorine controls root hair elongation and counteracts the activity of indole-3-acetic acid on root elongation on A . thaliana, as previously reported for the host plant . In addition, we show that hypaphorine counteracts the rapid upregulation by indole-3-acetic acid and 1-naphthalenic-acetic acid of the primary auxin-responsive gene IAA1 and induces a rapid, transient membrane depolarization in root hairs and suspension cells, due to the modulation of anion and K+ currents . These early responses indicate that components necessary for symbiosis-related differentiation events are present in the nonhost plant A . thaliana and provide tools for the dissection of the hypaphorine-auxin interaction.

Plant Physiol, 1994 Dec, 106(4), 1503 - 1510
Molecular Genetic Alteration of Plant Respiration (Silencing and Overexpression of Alternative Oxidase in Transgenic Tobacco); Vanlerberghe GC et al.; The alternative oxidase (AOX) of plant mitochondria is encoded by the nuclear gene Aox1 . Sense and antisense DNA constructs of Nicotiana tabacum Aox1 were introduced into tobacco, and transgenic plants with both increased and decreased levels of mitochondrial AOX protein were identified . Suspension cells derived from wild-type and transgenic plants were grown in heterotrophic batch culture . Transgenic cells with increased AOX protein had an increased capacity for cyanide-resistant, salicylhydroxamic acid-sensitive respiration compared to wild-type cells, whereas transgenic cells with decreased AOX protein had a decreased capacity for such respiration . Thus, genetic alteration of the level of AOX protein was sufficient to alter the capacity for electron transport through the alternative pathway . Under our standard growth conditions, "antisense" cells with dramatically reduced levels of AOX protein had growth and respiration rates similar to the wild type . However, whereas wild-type cells were able to grow under conditions that severely suppressed cytochrome pathway activity, antisense cells could not survive this treatment . This suggests that a critical function of AOX may be to support respiration when the cytochrome pathway is impaired . The much higher level of AOX protein in "sense" cells compared to the wild type did not appreciably alter the steady-state partitioning of electrons between the cytochrome path and the alternative pathway in vivo, suggesting that this partitioning may be subject to additional regulatory factors.

Plant Physiol, 1994 Nov, 106(3), 1213 - 1216
Comparison of Dehydrin Gene Expression and Freezing Tolerance in Bromus inermis and Secale cereale Grown in Controlled Environments, Hydroponics, and the Field; Robertson AJ et al.; There have been very few reports on the expression of stress-responsive genes in field-grown material . A barley dehydrin cDNA was used to investigate the expression of dehydrin-like transcripts after low-temperature and abscisic acid-induced acclimation of bromegrass (Bromus inermis Leyss) suspension cells and of bromegrass and rye (Secale cereale) plants grown in the field and under controlled environmental conditions . Field-acclimated plants accumulated high levels of dehydrin transcripts and were very freezing tolerant . Plants grown in pots and hydroponics under controlled environments also accumulated dehydrin transcripts and showed increased freezing tolerance . Simulation of a combined drought and freezing stress in pots resulted in expression of dehydrin-like transcripts comparable to those observed in field-acclimated material.

Plant Physiol, 1994 Oct, 106(2), 459 - 467
Rice Triosephosphate Isomerase Gene 5{prime} Sequence Directs {beta}-Glucuronidase Activity in Transgenic Tobacco but Requires an Intron for Expression in Rice; Xu Y et al.; In rice (Oryza sativa L.), cytosolic triosephosphate isomerase (TPI) is encoded by a single gene . TPI catalyzes a vital step in glycolysis, and RNA blots showed that the tpi gene is expressed in all vegetative tissues (root, culm, and leaves) and in rice suspension cells . No effect of light on expression was detected, but submergence of rice seedlings resulted in elevated levels of TPI mRNA in roots and culms . The 2767-bp 5{prime} upstream sequence of the tpi gene was fused translationally with the {beta}-glucuronidase (gusA) gene, and the resulting construct, TPI-GUS, was found to express constitutive, high levels of GUS activity in transgenic tobacco (Nicotiana tabacum) plants . However, the same construct yielded no GUS activity in stably transformed rice plants, and RNA blots showed that no GUS mRNA could be detected even though stable integration of functional copies of the construct was confirmed by Southern blot and genomic polymerase chain reaction analyses . Transient assays using particle bombardment yielded high levels of GUS expression from the TPI-GUS construct in tobacco leaves, but essentially no expression in rice, barley, or maize leaves . When the first intron of the tpi gene was included in the construct (TPI-int1-GUS), transient GUS activity was routinely obtained in rice leaves, revealing that the first intron of the rice tpi gene is crucial for its expression in rice . TPI-int1-GUS also directed transient GUS expression in maize and barley leaves, but little or no activity was obtained from this construct in tobacco, tomato, or soybean leaves . These results with the rice tpi promoter are in accordance with mounting evidence that differences in gene expression exist between monocots and dicots.

Plant Physiol, 1994 May, 105(1), 89 - 94
Pretreatment of Parsley (Petroselinum crispum L.) Suspension Cultures with Methyl Jasmonate Enhances Elicitation of Activated Oxygen Species; Kauss H et al.; Suspension-cultured cells of parsley (Petroselinum crispum L.) were used to demonstrate an influence of jasmonic acid methyl ester (JAME) on the elicitation of activated oxygen species . Preincubation of the cell cultures for 1 d with JAME greatly enhanced the subsequent induction by an elicitor preparation from cell walls of Phytophtora megasperma f . sp . glycinea (Pmg elicitor) and by the polycation chitosan . Shorter preincubation times with JAME were less efficient, and the effect was saturated at about 5 {mu}M JAME . Treatment of the crude Pmg elicitor with trypsin abolished induction of activated oxygen species, an effect similar to that seen with elicitation of coumarin secretion . These results suggest that JAME conditioned the parsley suspension cells in a time-dependent manner to become more responsive to elicitation, reminiscent of developmental effects caused by JAME in whole plants . It is interesting that pretreatment of the parsley cultures with 2,6-dichloroisonicotinic and 5-chlorosalicylic acid only slightly enhanced the elicitation of activated oxygen species, whereas these substances greatly enhanced the elicitation of coumarin secretion . Therefore, these presumed inducers of systemic acquired resistance exhibit a specificity different from JAME.

Plant Physiol, 1993 Nov, 103(3), 963 - 969
Characterization of Paraquat Transport in Protoplasts from Maize (Zea mays L.) Suspension Cells; Hart JJ et al.; Protoplasts isolated from maize (Zea mays L.) suspension cells were used to study transport of paraquat . {14C}Paraquat uptake was measured in 400-{mu}L centrifuge tubes using silicon oil centrifugation techniques . Approximately 50% of accumulation from a 100 {mu}M paraquat solution occurred in the first 10 s, and net accumulation reached a maximum after about 10 min . Membrane binding accounted for about 30% of apparent accumulation . Concentration-dependent uptake kinetics were characterized by a non-saturating curve, which was resolved into a linear and a saturable component . The Km of the saturable component was 132 {mu}M, and the Vmax was 0.512 nmol {mu}L of protoplasts-1 min-1 . In the absence of sucrose, the Vmax of the saturable component was reduced by 52%, suggesting that paraquat uptake across the plasmalemma is energy dependent . Measurement of concentration-dependent binding of paraquat to burst protoplasts showed a linear response . This suggests that the linear component from intact protoplast concentration kinetics represented paraquat binding to the plasmalemma surface . Calcium inhibited the saturable component, and this inhibition was shown by Lineweaver-Burk analysis to be noncompetitive . Putrescine, a divalent cationic polyamine with a charge distribution similar to that of paraquat, competitively inhibited paraquat uptake . These results show that paraquat transport characteristics at the plasmalemma of maize protoplasts are similar to those reported earlier for paraquat transport in roots of intact maize seedlings.

Plant Physiol, 1993 Jun, 102(2), 459 - 466
Conditioning of Parsley (Petroselinum crispum L.) Suspension Cells Increases Elicitor-Induced Incorporation of Cell Wall Phenolics; Kauss H et al.; The elicitor-induced incorporation of phenylpropanoid derivatives into the cell wall and the secretion of soluble coumarin derivatives (phytoalexins) by parsley (Petroselinum crispum L.) suspension cultures can be potentiated by pretreatment of the cultures with 2,6-dichloroisonicotinic acid or derivatives of salicylic acid . To investigate this phenomenon further, the cell walls and an extracellular soluble polymer were isolated from control cells or cells treated with an elicitor from Phytophthora megasperma f . sp . glycinea . After alkaline hydrolysis, both fractions from elicited cells showed a greatly increased content of 4-coumaric, ferulic, and 4-hydroxybenzoic acid, as well as 4-hydroxybenzaldehyde and vanillin . Two minor peaks were identified as tyrosol and methoxytyrosol . The pretreatment effect is most pronounced at a low elicitor concentration . Its specificity was elaborated for coumarin secretion . When the parsley suspension cultures were preincubated for 1 d with 2,6-dichloroisonicotinic, 4- or 5-chlorosalicylic, or 3,5- dichlorosalicylic acid, the cells exhibited a greatly increased elicitor response . Pretreatment with isonicotinic, salicylic, acetylsalicylic, or 2,6-dihydroxybenzoic acid was less efficient in enhancing the response, and some other isomers were inactive . This increase in elicitor response was also observed for the above-mentioned monomeric phenolics, which were liberated from cell walls upon alkaline hydrolysis and for "lignin-like" cell wall polymers determined by the thioglycolic acid method . It was shown for 5-chlorosalicylic acid that conditioning most likely improves the signal transduction leading to the activation of genes encoding phenylalanine ammonia lyase and 4-coumarate: coenzyme A ligase . The conditioning thus sensitizes the parsley suspension cells to respond to lower elicitor concentrations . If a similar mechanism were to apply to whole plants treated with 2,6-dichloroisonicotinic acid, a known inducer of systemic acquired resistance, one can hypothesize that fungal pathogens might be recognized more readily and effectively.

Plant Physiol, 1993 Feb, 101(2), 499 - 506
Characterization of Maize Acetyl-Coenzyme A Carboxylase; Egli MA et al.; Maize (Zea mays L.) leaf acetyl-CoA carboxylase (ACCase) was purified about 500-fold by ammonium sulfate fractionation and gel filtration and blue Sepharose affinity and anion-exchange chromatography . Most ACCase activity (85%) recovered from the anion-exchange column was found in a highly purified fraction (specific activity 5.5 {mu}mol acid-stable product min-1 mg-1) that consisted primarily of a single 227-kD biotinylated polypeptide . The fraction represented 29% of the original activity and was designated ACCase I . A second partially purified ACCase activity (ACCase II) eluted earlier during anion-exchange chromatography, contained a single biotinylated polypeptide of 219 kD, was poorly recognized by antiserum raised against the ACCase I polypeptide, and was less inhibited by the herbicides haloxyfop or sethoxydim than was ACCase I . ACCase I and II both utilized propionyl-CoA as substrate about 50% as effectively as acetyl-CoA, and neither utilized methylcrotonyl-CoA . Immunoprecipitation with antiserum and protein blotting of crude extracts of leaf, embryo, and endosperm tissue and suspension cells indicated that most ACCase activity in these tissues was immunologically similar and consisted of ACCase I . Only leaves contained significant amounts of the ACCase II polypeptide; however, no ACCase II polypeptide was found in isolated mesophyll chloroplasts . The ACCase I and II polypeptides appear to be subunits of distinct ACCase isoforms.

Plant Physiol, 2002 Sep, 130(1), 265 - 72
Plasmalemma abscisic acid perception leads to RAB18 expression via phospholipase D activation in Arabidopsis suspension cells; Hallouin M et al.; Abscisic acid (ABA) plays a key role in the control of stomatal aperture by regulating ion channel activities and water exchanges across the plasma membrane of guard cells . Changes in cytoplasmic calcium content and activation of anion and outward-rectifying K(+) channels are among the earliest cellular responses to ABA in guard cells . In Arabidopsis suspension cells, we have demonstrated that outer plasmalemma perception of ABA triggered similar early events . Furthermore, a Ca(2+) influx and the activation of anion channels are part of the ABA-signaling pathway leading to the specific expression of RAB18 . Here, we determine whether phospholipases are involved in ABA-induced RAB18 expression . Phospholipase C is not implicated in this ABA pathway . Using a transphosphatidylation reaction, we show that ABA plasmalemma perception results in a transient stimulation of phospholipase D (PLD) activity, which is necessary for RAB18 expression . Further experiments showed that PLD activation was unlikely to be regulated by heterotrimeric G proteins . We also observed that ABA-dependent stimulation of PLD was necessary for the activation of plasma anion current . However, when ABA activation of plasma anion channels was inhibited, the ABA-dependent activation of PLD was unchanged . Thus, we conclude that in Arabidopsis suspension cells, ABA stimulation of PLD acts upstream from anion channels in the transduction pathway leading to RAB18 expression.

Plant Physiol, 1996 Jul, 111(3), 721 - 724
Growth Inhibition in Suspension-Cultured Rice Cells under Phosphate Deprivation Is Mediated through Putrescine Accumulation; Shih CY et al.; The effects of phosphate deprivation on the growth and polyamine levels of suspension-cultured rice (Oryza sativa) cells were investigated . When rice suspension cells were deprived of phosphate, cell growth was markedly inhibited . Phosphate deprivation resulted in a higher putrescine level and lower spermidine and spermine levels in rice suspension cells . The growth of rice cells cultured in the absence of phosphate did not recover as a result of spermidine and spermine addition . D-Arginine and {alpha}-methylornithine, inhibitors of putrescine biosynthesis, caused a reduced level of putrescine in rice suspension cells cultured under phosphate deprivation . The growth of rice cells cultured in the absence of phosphate was completely recovered after the addition of D-arginine but not {alpha}-methylornithine . Our results indicate that putrescine accumulation is a factor causing growth inhibition of suspension-cultured rice cells under phosphate deprivation.

Plant Physiol, 1996 Jun, 111(2), 589 - 595
Signals Regulating the Expression of the Nuclear Gene Encoding Alternative Oxidase of Plant Mitochondria; Vanlerberghe GC et al.; Suspension cells of tobacco (Nicotiana tabacum L . cv Bright Yellow) were used to investigate signals regulating the expression of the nuclear gene Aox1 encoding the mitochondrial alternative oxidase (AOX) protein responsible for cyanide-resistant respiration in plants . We found that an increase in the tricarboxylic acid cycle intermediate citrate (either after its exogenous supply to cells or after inhibition of aconitase by monofluoroacetate) caused a rapid and dramatic increase in the steady-state level of Aox1 mRNA and AOX protein . This led to a large increase in the capacity for AOX respiration, defined as the amount of salicylhydroxamic acid-sensitive O2 uptake by cells in the presence of potassium cyanide . The results indicate that citrate may be an important signal metabolite regulating Aox1 gene expression . A number of other treatments were also identified that rapidly induced the level of Aox1 mRNA and AOX capacity . These included short-term incubation of cells with 10 mM acetate, 2 {mu}M antimycin A, 5 mM H2O2, or 1 mM cysteine . For some of these treatments, induction of AOX occurred without an increase in cellular citrate level, indicating that other signals (possibly related to oxidative stress conditions) are also important in regulating Aox1 gene expression . The signals influencing Aox1 gene expression are discussed with regard to the potential function(s) of AOX to modulate tricarboxylic acid cycle metabolism and/or to prevent the generation of active oxygen species by the mitochondrial electron transport chain.

Plant Physiol, 1996 Feb, 110(2), 465 - 470
Diauxic Growth in Rice Suspension Cells Grown on Mixed Carbon Sources of Acetate and Glucose; Lee TK et al.; Diauxic growth was observed in rice (Oryza sativa L.) suspension cells growing on acetate (10 mM) and glucose (10 mM) . Cells used acetate during the first growth phase and the acetate level in the medium was rapidly decreased, whereas the level of glucose remained essentially unchanged . After acetate was depleted from the medium, cells started to use glucose, forming the second growth phase . It appears that uptake of {14C}glucose was repressed during the first growth phase and became active during the second growth phase . In contrast, uptake of {14C}acetate occurred actively throughout the diauxic growth . By further demonstrating the specific induction of isocitrate lyase (EC 4.1.3.1), a glyoxylate cycle enzyme, and hexokinase (EC 2.7.1.1), a glycolysis enzyme, during the first and second growth phases, respectively, it was clearly shown that rice cells use acetate first and do not use both carbon sources simultaneously . This kind of diauxic growth pattern has been observed in bacteria . To our knowledege, this study is the first report demonstrating the presence of diauxic growth in plant cells.

Plant Physiol, 1996 Jan, 110(1), 249 - 257
Expression of a Late Embryogenesis Abundant Protein Gene, HVA1, from Barley Confers Tolerance to Water Deficit and Salt Stress in Transgenic Rice; Xu D et al.; A late embryogenesis abundant (LEA) protein gene, HVA1, from barley (Hordeum vulgare L.) was introduced into rice suspension cells using the Biolistic-mediated transformation method, and a large number of independent transgenic rice (Oryza sativa L.) plants were generated . Expression of the barley HVA1 gene regulated by the rice actin 1 gene promoter led to high-level, constitutive accumulation of the HVA1 protein in both leaves and roots of transgenic rice plants . Second-generation transgenic rice plants showed significantly increased tolerance to water deficit and salinity . Transgenic rice plants maintained higher growth rates than nontransformed control plants under stress conditions . The increased tolerance was also reflected by delayed development of damage symptoms caused by stress and by improved recovery upon the removal of stress conditions . We also found that the extent of increased stress tolerance correlated with the level of the HVA1 protein accumulated in the transgenic rice plants . Using a transgenic approach, this study provides direct evidence supporting the hypothesis that LEA proteins play an important role in the protection of plants under water-or salt-stress conditions . Thus, LEA genes hold considerable potential for use as molecular tools for genetic crop improvement toward stress tolerance.

Plant Physiol, 2002 Aug, 129(4), 1473 - 81
High-level and ubiquitous expression of the rice cytochrome c gene OsCc1 and its promoter activity in transgenic plants provides a useful promoter for transgenesis of monocots; Jang IC et al.; Expression patterns of a rice (Oryza sativa) cytochrome c gene OsCc1 and its promoter activity were characterized in transgenic rice plants . OsCc1 transcripts accumulate in most cell types, but to varying levels . Large amounts of OsCc1 transcripts are found in the roots, calli, and suspension cells, but relatively lower in mature leaves, demonstrating its higher levels of expression in non-photosynthetic tissues . Unlike the human cytochrome c gene, which is responsive to cAMP, OsCc1 expression is not enhanced in various rice tissues after dibutyryl cAMP treatments . OsCc1 promoter was linked to the sgfp gene and its activities in different tissues and cell types of transgenic rice plants were analyzed in comparison with the Act1 and RbcS promoters . OsCc1 promoter directs expression in virtually all organs of transgenic plants including roots, leaves, calli, embryos, and suspension cells, showing a particularly high activity in calli and roots . Activity of the OsCc1 promoter was 3-fold higher than Act1 in calli and roots and comparable with RbcS in leaves, representing a useful alternative to the maize (Zea mays) Ubi1 and the rice Act1 promoters for transgene expression in monocots.

Chemosphere, 2002 Jun, 47(9), 957 - 62
Transformation of {14C} trichloroethylene by poplar suspension cells; Shang TQ et al.; Trichloroethylene (TCE) is one of the most prevalent environmental contaminants, and it poses an expensive remediation problem . Phytoremediation has been investigated as a potential tool for the removal of TCE from ground water and soil, and has shown promise in preliminary trials . However, the fate of TCE in plants is largely unknown . Radiolabel studies showed that once taken up and transformed, most of the TCE is incorporated into plant tissue as a non-volatile, un-extractable residue . We describe here an assay for TCE transformation by poplar suspension cells . Using this assay, it was shown that two different activities contribute to the fixation of TCE by poplar cells, one associated with cell walls and insoluble residues, the other associated with a high molecular weight, heat labile fraction of the cell extract . It appears that plant enzymes catalyze some of the transformations.

Phytochemistry, 2002 Jul, 60(5), 467 - 74
Increase of free cysteine and citric acid in plant cells exposed to cobalt ions; Oven M et al.; Cobalt complexation was investigated in a suspension cell culture of the cobalt hyperaccumulator Crotalaria cobalticola . C . cobalticola cells were more tolerant towards cobalt ions than the suspension cells of the non-accumulators Rauvolfia serpentina and Silene cucubalus . While the concentration of various compounds increased in cells of C . cobalticola challenged with cobalt ions, phytochelatin biosynthesis was not induced . Instead, the exposure to cobalt ions resulted in the increase of citrate and cysteine in cells . Size exclusion chromatography demonstrated the co-elution of cobalt and cysteine in C . cobalticola cell extracts . A significant increase in cysteine was observed also in cells of R . serpentina and S . cucubalus when they were exposed to cobalt ions . These results suggest that free cysteine is involved in cobalt ion complexation in plant cells.

DNA Cell Biol, 2002 Mar, 21(3), 213 - 39
Cytoplasmic intermediate filaments are stably associated with nuclear matrices and potentially modulate their DNA-binding function; Tolstonog GV et al.; The tight association of cytoplasmic intermediate filaments (cIFs) with the nucleus and the isolation of crosslinkage products of vimentin with genomic DNA fragments, including nuclear matrix attachment regions (MARs) from proliferating fibroblasts, point to a participation of cIFs in nuclear activities . To test the possibility that cIFs are complementary nuclear matrix elements, the nuclei of a series of cultured cells were subjected to the Li-diiodosalicylate (LIS) extraction protocol developed for the preparation of nuclear matrices and analyzed by immunofluorescence microscopy and immunoblotting with antibodies directed against lamin B and cIF proteins . When nuclei released from hypotonically swollen L929 suspension cells in the presence of digitonin or Triton X-100 were exposed to such strong shearing forces that a considerable number were totally disrupted, a thin, discontinuous layer of vimentin IFs remained tenaciously adhering to still intact nuclei, in apparent coalignment with the nuclear lamina . Even in broken nuclei, the distribution of vimentin followed that of lamin B in areas where the lamina still appeared intact . The same retention of vimentin together with desmin and glial IFs was observed on the nuclei isolated from differentiating C2C12 myoblast and U333 glioma cells, respectively . Nuclei from epithelial cells shed their residual perinuclear IF layers as coherent cytoskeletal ghosts, except for small fractions of vimentin and cytokeratin IFs, which remained in a dot-to cap-like arrangement on the nuclear surface, in apparent codistribution with lamin B . LIS extraction did not bring about a reduction in the cIF protein contents of such nuclei upon their transformation into nuclear matrices . Moreover, in whole mount preparations of mouse embryo fibroblasts, DNA/chromatin emerging from nuclei during LIS extraction mechanically and chemically cleaned the nuclear surface and perinuclear area from loosely anchored cytoplasmic material with the production of broad, IF-free annular spaces, but left substantial fractions of the vimentin IFs in tight association with the nuclear surface . Accordingly, double-immunogold electron microscopy of fixed and permeabilized fibroblasts disclosed a close neighborhood of vimentin IFs and lamin B, with a minimal distance between the nanogold particles of ca . 30 nm . These data indicate an extremely solid interconnection of cIFs with structural elements of the nuclear matrix, and make them, together with their susceptibility to crosslinkage to MARs and other genomic DNA sequences under native conditions, complementary or even integral constituents of the karyoskeleton.

Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 7172 - 7 Epub 2002 May 07.
Carbocyclic fatty acids in plants: biochemical and molecular genetic characterization of cyclopropane fatty acid synthesis of Sterculiafoetida; Bao X et al.; Fatty acids containing three-member carbocyclic rings are found in bacteria and plants . Bacteria synthesize cyclopropane fatty acids (CPA-FAs) only by the addition of a methylene group from S-adenosylmethionine to the cis-double bond of monoenoic phospholipid-bound fatty acids . In plants CPA-FAs are usually minor components with cyclopropene fatty acids (CPE-FAs) more abundant . Sterculia foetida seed oil contains 65-78% CPE-FAs, principally sterculic acid . To address carbocyclic fatty acid synthesis in plants, a cDNA library was constructed from developing seeds during the period of maximum oil deposition . About 0.4% of 5,300 expressed sequence tags were derived from one gene, which shared similarities to the bacterial CPA-FA synthase . However, the predicted protein is twice as large as the bacterial homolog and represents a fusion of an FAD-containing oxidase at the N terminus and a methyltransferase at the C terminus . Functional analysis of the isolated full-length cDNA was conducted in tobacco suspension cells where its expression resulted in the accumulation of up to 6.2% dihydrosterculate of total fatty acids . In addition, the dihydrosterculate was specifically labeled by {methyl-(14)C}methionine and by {(14)C}oleic acid in the transgenic tobacco cells . In in vitro assay of S . foetida seed extracts, S-adenosylmethionine served as a methylene donor for the synthesis of dihydrosterculate from oleate . Dihydrosterculate accumulated largely in phosphatidylcholine in both systems . Together, a CPA-FA synthase was identified from S . foetida, and the pathway in higher plants that produce carbocyclic fatty acids was defined as by transfer of C(1) units, most likely from S-adenosylmethionine to oleate.

J Cell Sci, 2002 May 15, 115(Pt 10), 2233 - 9
PLC-gamma1 is required for IGF-I protection from cell death induced by loss of extracellular matrix adhesion; Chattopadhyay A et al.; Phospholipase C-gamma1, a tyrosine kinase substrate, hydrolyses phosphatidylinositol 4,5-bisphosphate to produce inositol 1,4,5-trisphosphate and diacylglycerol, which act as second messenger moleculesto mobilize intracellular calcium and activate protein kinase C, respectively . We have investigated the role of phospholipase C-gamma1 in anoikis, or cell death, induced by the loss of extracellular matrix adhesion . Spontaneously immortalized mouse embryonic fibroblasts nullizygous at the Plcg1 locus (Plcg1(-/-)), referred to as Null cells, were derived from targeted gene disruption experiments . Subsequently, phospholipase C-gamma1 was re-expressed in these cells to derive Null+ cells . The Null and Null+ cells were then placed in suspension to induce cell death, which was measured directly as well as by the induction of caspase 3, as an index of programmed cell death or apoptosis . The results demonstrate that insulin-like growth factor can rescue Null+ cells but not Null cells from suspension-induced cell death . This demonstrates that phospholipase C-gamma1 is required for insulin-like growth factor dependent cell survival under these conditions . Lastly, the data demonstrate that insulinlike growth factor stimulated tyrosine phosphorylation of phospholipase C-gamma1 in both adherent and suspension cells.

Plant J, 2002 Apr, 30(1), 71 - 81
The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH; Gerbeau P et al.; Mechanisms that regulate water channels in the plant plasma membrane (PM) were investigated in Arabidopsis suspension cells . Cell hydraulic conductivity was measured with a cell pressure probe and was reduced 4-fold as compared to control values when calcium was added in the pipette and in bathing solution . To assess the significance of these effects in vitro, PM vesicles were isolated by aqueous two-phase partitioning and their water transport properties were characterized by stopped-flow spectrophotometry . Membrane vesicles isolated in standard conditions exhibited reduced water permeability (P(f)) together with a lack of active water channels . In contrast, when prepared in the presence of chelators of divalent cations, PM vesicles showed a 2.3-fold higher P(f) and active water channels . Furthermore, equilibration of purified PM vesicles with divalent cations reduced their P(f ) and water channel activity down to the basal level of membranes isolated in standard conditions . Ca2+ was the most efficient with a half-inhibition of P(f) at 50-100 microM free Ca2+ . Water transport in purified PM vesicles was also reversibly blocked by H+, with a half-inhibition of P(f )at pH 7.2-7.5 . Thus, both Ca2+ and H+ contribute to a membrane-delimited switch from active to inactive water channels that may allow coupling of water transport to cell signalling and metabolism.

Yao Xue Xue Bao, 1998, 33(2), 132 - 7
{Effects of phenylalanine, sucrose and mannitol on the growth and production of taxol, baccatin III and 10-deacetylbaccatin III in suspension cells of Taxus media}; Chen Y et al.; The effects of phenylalanine, sucrose and mannitol on the cell growth and the production of taxol, baccatin III and 10-deacetylbaccatin III in the suspension cells of Taxus media were studied . The results showed that phenylalanine 1.0 mmol.L-1 or 2.0 mmol.L-1 initially added into the medium, and sucrose 73.0 mmol.L-1 and mannitol 173.3 mmol.L-1 added into the medium at the 28th d of culture strongly promoted the cell growth and the formation of the three taxanes in the suspension cells . Compared with those of the control, the cell biomass of the treatments supplemented with phenylalanine and added with sucrose and mannitol at the 28th d of culture increased by 0.6-0.8-fold, taxol yield by 9-10-fold, baccatin III yield by 2.5-3.0-fold, and 10-deacetylbaccatin III yield by 7-fold . Addition of sucrose 73.0 mmol.L-1 at the 28th d of culture significantly promoted the cell growth, but showed little effect on the contents of the three taxanes in the suspension cultures.

Planta, 2002 Feb, 214(4), 537 - 45
A critical role for ethylene in hydrogen peroxide release during programmed cell death in tomato suspension cells; de J et al.; Camptothecin, a topo isomerase-I inhibitor used in cancer therapy, induces apoptosis in animal cells . In tomato (Lycopersicon esculentum Mill.) suspension cells, camptothecin induces cell death that is accompanied by the characteristic nuclear morphological changes such as chromatin condensation and nuclear and DNA fragmentation that are commonly associated with apoptosis in animal systems . These effects of camptothecin can effectively be blocked by inhibitors of animal caspases, indicating that, in tomato suspension cells, camptothecin induces a form of programmed cell death (PCD) with similarities to animal apoptosis (A.J . De Jong et al . (2000) Planta 211:656-662) . Camptothecin induced cell death was employed to study processes involved in plant PCD . Camptothecin induced a transient increase in H2O2 production starting within 2 h of application . Both camptothecin-induced cell death and the release of H2O2 were effectively blocked by application of the calcium-channel blocker lanthanum chloride, the caspase-specific inhibitor Z-Asp-CH2-DCB, or the NADPH oxidase inhibitor diphenyl iodonium, indicating that camptothecin exerts its effect on cell death through a calcium- and caspase-dependent stimulation of NADPH oxidase activity . In addition, we show that ethylene is an essential factor in camptothecin-induced PCD . Inhibition of either ethylene synthesis or ethylene perception by L-alpha-(2-aminoethoxyvinyl)glycine or silver thiosulphate, respectively, blocked camptothecin-induced H2O2 production and PCD . Although, in itself, insufficient to trigger H2O2 production and cell death, exogenous ethylene greatly stimulated camptothecin-induced H2O2 production and cell death . These results show that ethylene is a potentiator of the camptothecin-induced oxidative burst and subsequent PCD in tomato cells . The possible mechanisms by which ethylene stimulates cell death are discussed.

Arch Biochem Biophys, 2002 Apr 1, 400(1), 54 - 62
Molecular and regulatory properties of leucoplast pyruvate kinase from Brassica napus (rapeseed) suspension cells; Plaxton WC et al.; Plastidic pyruvate kinase (PK(p)) from Brassica napus suspension cells was purified 431-fold to a final specific activity of 28 micromol phosphoenolpyruvate (PEP) utilized/min/mg protein . SDS-PAGE, immunoblot and gel filtration analyses indicated that this PK(p) exists as a 380-kDa heterohexamer composed of equal proportions of 64- (alpha-subunit) and 58-kDa (beta-subunit) polypeptides . The N-terminal sequence of the PK(p) alpha- and beta-subunits exhibited maximal identity with the corresponding regions deduced from putative PK genes of Arabidopsis thaliana and Methylobacterium extorquens, respectively . B . napus PK(p) displayed a sharp pH optimum of pH 8.0, and hyperbolic saturation kinetics with PEP and ADP (K(m) = 0.052 and 0.14 mM, respectively) . 6-Phosphogluconate functioned as an activator (K(a) = 0.12 mM) by increasing V(max) by approximately 35% while decreasing the K(m)(PEP) and K(m)(ADP) values by 40 and 50%, respectively . 2-Oxoglutarate and oxalate were the most effective inhibitors (I(50) = 8.3 and 0.23 mM, respectively) . A model is presented which highlights the role of 6-phosphogluconate in coordinating stromal NADPH and ATP production for anabolic processes of B . napus leucoplasts.

J Biol Chem, 2002 May 31, 277(22), 19304 - 14 Epub 2002 Mar 19.
Mlo, a modulator of plant defense and cell death, is a novel calmodulin-binding protein . Isolation and characterization of a rice Mlo homologue; Kim MC et al.; Transient influx of Ca(2+) constitutes an early event in the signaling cascades that trigger plant defense responses . However, the downstream components of defense-associated Ca(2+) signaling are largely unknown . Because Ca(2+) signals are mediated by Ca(2+)-binding proteins, including calmodulin (CaM), identification and characterization of CaM-binding proteins elicited by pathogens should provide insights into the mechanism by which Ca(2+) regulates defense responses . In this study, we isolated a gene encoding rice Mlo (Oryza sativa Mlo; OsMlo) using a protein-protein interaction-based screening of a cDNA expression library constructed from pathogen-elicited rice suspension cells . OsMlo has a molecular mass of 62 kDa and shares 65% sequence identity and scaffold topology with barley Mlo, a heptahelical transmembrane protein known to function as a negative regulator of broad spectrum disease resistance and leaf cell death . By using gel overlay assays, we showed that OsMlo produced in Escherichia coli binds to soybean CaM isoform-1 (SCaM-1) in a Ca(2+)-dependent manner . We located a 20-amino acid CaM-binding domain (CaMBD) in the OsMlo C-terminal cytoplasmic tail that is necessary and sufficient for Ca(2+)-dependent CaM complex formation . Specific binding of the conserved CaMBD to CaM was corroborated by site-directed mutagenesis, a gel mobility shift assay, and a competition assay with a Ca(2+)/CaM-dependent enzyme . Expression of OsMlo was strongly induced by a fungal pathogen and by plant defense signaling molecules . We propose that binding of Ca(2+)-loaded CaM to the C-terminal tail may be a common feature of Mlo proteins.

Plant Cell, 2002 Jan, 14(1), 197 - 210
The rice cyclin-dependent kinase-activating kinase R2 regulates S-phase progression; Fabian-Marwedel T et al.; Cyclin-dependent kinases (CDKs) are the central components of eukaryotic cell cycle regulation . Phosphorylation of CDKs at a conserved threonine residue is required for their full activity and is mediated by a CDK-activating kinase (CAK) . The CAK R2 from rice belongs to those CAKs that phosphorylate not only CDKs but also the C-terminal domain (CTD) of RNA polymerase II . We showed that R2 is a nuclear protein with increased expression and increased CTD kinase activity in S-phase . Increasing R2 abundance through a transgenic approach accelerated S-phase progression and overall growth rate in suspension cells . In planta, the CTD kinase activity of R2 was induced by a growth-promoting signal . R2 regulation, therefore, may constitute a plant-specific adaptive mechanism that is used to adjust the rate of cell proliferation in response to a changing environment.

Plant Cell, 2002 Jan, 14(1), 71 - 86
Uptake of a fluorescent marker in plant cells is sensitive to brefeldin A and wortmannin; Emans N et al.; We assessed FM1-43 {N-(3-triethylammoniumpropyl)-4-(4-{dibutylamino}styryl)pyridinium dibromide} as a fluorescent endocytosis marker in intact, walled plant cells . At 4 degrees C, FM1-43 stained the plasma membrane, and after 30 to 120 min of incubation at 26 degrees C, FM1-43 labeled cytoplasmic vesicles and then the vacuole . Fluorimetric quantitation demonstrated dye uptake temperature sensitivity (approximately 65% reduction at 16 degrees C, >90% at 4 degrees C) . FM1-43 uptake in suspension cells was stimulated more than twofold by brefeldin A and inhibited approximately 0.4-fold by wortmannin . FM1-43 delivery to the vacuole was largely inhibited by brefeldin A, although overall uptake was stimulated, and brefeldin A treatment caused the accumulation of large prevacuolar endosomal vesicles heavily labeled with FM1-43 . Three-dimensional time lapse imaging revealed that FM1-43-labeled vacuoles and vesicles are highly dynamic . Thus, FM1-43 serves as a fluorescent marker for imaging and quantifying membrane endocytosis in intact plant cells.

Cryo Letters, 2001 Jul-Aug, 22(4), 221 - 8
Pregrowth-desiccation: a simple and efficient procedure for the cryopreservation of rice (Oryza sativa L.) embryogenic suspension cells; Zhang YX et al.; Rice embryogenic suspension cells were successfully cryopreserved by a pregrowth-desiccation procedure . Cells were precultured in liquid AA medium containing 0.175 mol/L sucrose for 3 d and then in liquid AA medium containing 0.4 mol/L sorbitol for 1 d . After air-drying for about 20 h to a water content of 10%, the cells were placed into cryotubes and quenched into liquid nitrogen . Using this pregrowth-desiccation procedure, a survival rate of 96+/-6% (TTC reduction assay) or 100% (cell clump regrowth) was achieved . Cryostored cells revived very quickly during the recovery culture and they retained the ability to regenerate fertile plants . In conclusion, air-drying, a method usually employed in cryopreservation of seeds or shoot tips, can be used as a simple and efficient procedure for the cryopreservation of precultured rice suspension cells.

Cryo Letters, 2001 May-Jun, 22(3), 175 - 82
The dual effect of antifreeze protein on cryopreservation of rice (Oryza sativa l.) embryogenic suspension cells; Wang JH et al.; The effects of fish antifreeze protein AFP-I on cryopreservation of rice suspension cells by three different protocols were investigated . During the two-step method, AFP-I at 0.01 mg/ml significantly lowered the viability of both precultured and non-precultured cells . During the vitrification method, AFP-I at 0.2 mg/ml improved the viability of suboptimally thawed cells; however, much higher doses of this protein (10mg/ml) attenuated the cell viability . During rapid freezing of rice cells in the solutions with relatively high (but non-vitrifying) concentrations of cryoprotectant, AFP-I displayed protective action in the higher concentrated cryoprotectants and detrimental effect in more dilute ones . Taken together, it was concluded that, depending upon a number of factors discussed in the present paper, both positive effect and negative effect could be observed during application of AFP to cryopreservation of rice cells . The possible mechanism of this dual character was discussed.

Cryo Letters, 2001 Jan-Feb, 22(1), 43 - 50
Cryopreservation of Taxus chinensis suspension cell cultures; Kim SI et al.; A simple cryopreservation method for suspension cells of Taxus chinensis was established . In this procedure 7 days old suspension cells were used without any pre-culture treatment . At first, cells were incubated in cryoprotectant solution (0.5M DMSO and 0.5M glycerol) on ice for 30 min and then frozen at a cooling rate of 1 degree C/min to -40 degrees C prior to immersion in liquid nitrogen . The average viability of frozen-thawed cells was between 30 to 40% . The recovery of cryopreserved cells in liquid nitrogen for 1 month was accomplished . After rapid thawing, cells were transferred to solid medium and cultivated for 4-6 weeks . The treatment of trehalose as a cryoprotectant enhanced re-growth of frozen-thawed cells . The stable maintenance of paclitaxel biosynthetic ability in cryopreserved cells was confirmed by comparing with that of regularly sub-cultured suspension cells.

J Cell Biochem, 2002, 84(2), 301 - 8
Proline-rich transcript of the brain (prtb) is a serum-responsive gene in osteoblasts and upregulated during adhesion; Sommerfeldt DW et al.; To characterize the temporal expression of genes that play a functional role during the process of osteoblast adhesion, we used differential display (DD-PCR) on mRNA isolated from attached vs . suspended osteoblasts . A 200-bp fragment displaying upregulated expression after 30 and 60 min adhesion was isolated, sequenced, and showed 97% homology to prtb, previously showed to be expressed in mouse brain . Northern analysis confirmed a two-fold increase in prtb message during adhesion to tissue culture polystyrene, both in the presence or absence of surface-adsorbed serum proteins . Serum stimulation alone was also able to induce prtb expression, although to a lesser extent, in suspension cells . Strong prtb expression was also detected in both brain and bone of adult rats . Furthermore, prtb expression analysis during MC3T3-E1 cell differentiation revealed high expression levels independent of proliferation (day 0-7), matrix maturation (day 7-14), and mineralization (day 14-31) . Time course analysis of prtb expression during adhesion of sensitized osteoblasts to serum-protein coated surfaces showed robust mRNA expression at 5 min post-plating and a peak at 10 min . The two known serum-inducible immediate early genes c-fos and c-jun showed similar expression kinetics, with c-jun mRNA levels peaking at 15 min and c-fos at 20 min . Based on these data, we hypothesize that prtb may function as an immediate early, serum-responsive, and adhesion-inducible gene with possible involvement in processes such as cell cycle control, adhesion, and proliferation .

Biochim Biophys Acta, 2001 Nov 26, 1550(1), 52 - 63
Purification and properties of Arabidopsis thaliana type 1 protein phosphatase (PP1); Stubbs MD et al.; The Arabidopsis thaliana type 1 protein phosphatase (PP1) catalytic subunit was released from its endogenous regulatory subunits by ethanol precipitation and purified by anion exchange and microcystin affinity chromatography . The enzyme was identified by MALDI-TOF mass spectrometry from a tryptic digest of the purified protein as a mixture of PP1 isoforms (TOPP 1-6) indicating that at least 4-6 of the eight known PP1 proteins are expressed in sufficient quantities for purification from A . thaliana suspension cells . The enzyme had a final specific activity of 8950 mU/mg using glycogen phosphorylase a as substrate, had a subunit molecular mass of 35 kDa as determined by SDS-PAGE and behaved as a monomeric protein of approx . 39 kDa on Superose 12 gel filtration chromatography . Similar to the mammalian type 1 protein phosphatases, the A . thaliana enzyme was potently inhibited by Inhibitor-2 (IC(50)=0.65 nM), tautomycin (IC(50)=0.06 nM), microcystin-LR (IC(50)=0.01 nM), nodularin (IC(50)=0.035 nM), calyculin A (IC(50)=0.09 nM), okadaic acid (IC(50)=20 nM) and cantharidin (IC(50)=60 nM) . The enzyme was also inhibited by fostriecin (IC(50)=22 microM), NaF (IC(50)=2.1 mM), Pi (IC(50)=9.5 mM), and PPi (IC(50)=0.07 mM) . Purification of the free catalytic subunit allowed it to be used to probe protein phosphatase holoenzyme complexes that were enriched on Q-Sepharose and a microcystin-Sepharose affinity matrix and confirmed several proteins to be PP1 targeting subunits.

J Immunol, 2001 Dec 1, 167(11), 6403 - 11
Surrogate light chain-mediated interaction of a soluble pre-B cell receptor with adherent cell lines; Bradl H et al.; Signals initiated by the precursor B cell receptor (pre-BCR) are critical for B cell progenitors to mature into precursor B cells . The pre-BCR consists of a homodimer of microH chains, the covalently associated surrogate L (SL) chain composed of VpreB and lambda5, and the transmembrane signal molecules Ig(alpha) and Igbeta . One way to explain how maturation signals are initiated in late progenitor B cells is that the pre-BCR is transported to the cell surface and interacts from there with a ligand on stroma cells . To address this hypothesis, we first produced soluble Fab-like pre-BCR and BCR fragments, as well as SL chain, in baculovirus-infected insect cells . Flow cytometry revealed that, in contrast to Fab-like BCR fragments, the soluble pre-BCR binds to the surface of stroma and several other adherent cell lines, but not to B and T lymphoid suspension cells . The specific binding of the soluble pre-BCR to stroma cells is saturable, sensitive to trypsin digestion, and not dependent on bivalent cations . The binding of pre-BCR seems to be independent of the H chain of IgM (microH chain), because SL chain alone was able to interact with stroma cells . Finally, soluble pre-BCR specifically precipitated a 135-kDa protein from ST2 cells . These findings not only demonstrate for the first time the capacity of a pre-BCR to specifically bind to a structure on the surface of adherent cells, but also suggest that the pre-BCR interacts via its SL chain with a putative ligand on stroma cells.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9538 - 42
Phosphate-starvation response in plant cells: de novo synthesis and degradation of acid phosphatases; Duff SM et al.; Induction of phosphatase activity is an important component of the plant cell response to phosphate deficiency . Suspension cell cultures of Brassica nigra contain two major inducible acid phosphatase (APase) isozymes; vacuolar phosphoenolpyruvate (PEP) APase and cell wall nonspecific APase . Polyclonal antibodies raised against purified PEP-APase crossreacted specifically with both isozymes . Furthermore, anti-(PEP-APase) IgG detected proteins from a wide range of higher plants, suggesting that the major plant APase isozymes have diverged from a common ancestral form . Quantification on immunoblots indicated that in B . nigra suspension cells experiencing transition from Pi sufficiency to deficiency or vice versa, the amount of total antigenic APase protein correlated closely with total enzyme activity . This was also shown in intact plant roots . Therefore, the activity was governed by the synthesis and degradation of APases . Increases in the amounts of both major APase isozymes occurred simultaneously following Pi deprivation of B . nigra suspension cells, suggesting the involvement of a common regulatory mechanism.

Mol Plant Microbe Interact, 2001 Sep, 14(9), 1063 - 74
Intracellular localization and movement phenotypes of alfalfa mosaic virus movement protein mutants; Huang M et al.; Thirteen mutations were introduced in the movement protein (MP) gene of Alfalfa mosaic virus (AMV) fused to the green fluorescent protein (GFP) gene and the mutant MP-GFP fusions were expressed transiently in tobacco protoplasts, tobacco suspension cells, and epidermal cells of tobacco leaves . In addition, the mutations were introduced in the MP gene of AMV RNA 3 and the mutant RNAs were used to infect tobacco plants . Ten mutants were affected in one or more of the following functions of MP: the formation of tubular structures on the surface of protoplasts, association with the endoplasmic reticulum (ER) of suspension cells and epidermal cells, targeting to punctate structures in the cell wall of epidermis cells, movement from transfected cells to adjacent cells in epidermis tissue, cell-to-cell movement, or long-distance movement in plants . The mutations point to functional domains of the MP and support the proposed order of events in AMV transport . Studies with several inhibitors indicate that actin or microtubule components of the cytoskeleton are not involved in tubule formation by AMV MP . Evidence was obtained that tubular structures on the surface of transfected protoplasts contain ER- or plasmalemma-derived material.

Cell Transplant, 2001, 10(4-5), 459 - 64
Isolation, culture, and characterization of endocrine cells from 6-month-old porcine pancreas; Hori H et al.; Porcine endocrine cells were isolated from pancreas of 6-month-old pigs by two-step enzymatic digestion procedures . They were separated by the density gradient (isopycnic) centrifugation method using Histopaque-1077 . Isolated cells were cultured and divided into two groups: suspension cells and adhesion cells . Suspension cells maintained their cell numbers on and after 7 days in culture . Approximately 1 x 10(7) cells were obtained from single pancreas of a 6-month-old pig . The cultured suspension cells took up dithizone (DTZ) staining 14 days after isolation in culture and indicated the presence of beta-cells . In in vitro study, the suspension cells were capable of secreting insulin into the culture medium . The suspension cells were tested for insulin and glucagon staining by Western blot analysis . These results indicated the maintenance of endocrine cell function after isolation . However, cultured adhesion cells failed to maintain their function during culture . In in vivo study, the suspension cells were transplanted into diabetes-induced nude mice . Reduction in blood glucose level was obtained after transplantation . Intraperitoneal glucose tolerance test (IPGTT) results showed a normal pattern of blood glucose clearance . After 1 week, the transplanted endocrine cells were detected with anti-insulin antibody by immunostaining and it showed the presence of viable beta-cells under the renal capsule of nude mice . Collectively, our results suggest that isolated and cultured suspension porcine endocrine cells maintained their endocrine function . These endocrine cells can be used as isolated islets for further study, including transplantation experiments.

Physiol Plant, 1989 Jul, 76(3 Pt 1), 431 - 7
Preparatory studies for the use of plant protoplasts in space research; Rasmussen O et al.; An experiment using plant protoplasts has been accepted for the IML-1 mission to be flown on a space shuttle in 1991 . Preparatory experiments include studies of cell wall formation, cell division, the effect of simulated weightlessness using fast and slow rotating clinostats, and the development and testing of hardware for the IML-1 mission . After 24 h at 25 degrees C, protoplasts isolated from hypocotyls or leaves of rapeseed seedlings, or from carrot suspension cells, show 60, 20 and 15% cell wall formation, respectively . The time course of formation of the cell wall and cell division could be delayed by treatment at low temperatures or immobilization in alginate or agarose . This aspect is of importance in connection with problems of late access to the space shuttle before launch . At 4 degrees C only 18% of the rapeseed hypocotyl protoplasts had formed cell walls after 24 h . Protoplasts immobilised in agarose or alginate gradually regain their cell division capacity and after 72 h the frequencies are 51 and 26%, respectively, compared to non-immobilised control protoplasts . A significant decrease in cell division activity is observed after rotation for 6 h on the slow clinostat . A similar effect is not observed on the fast clinostat . Protoplasts, cultured in the specially designed plant chamber for up to 14 days established cell aggregates which have further developed into plants.

Plant Mol Biol, 2001 Jul, 46(4), 459 - 68
A tomato homologue of the human protein PIRIN is induced during programmed cell death; Orzaez D et al.; Programmed cell death (PCD), with similarities to animal apoptosis, was induced in tomato suspension cells by the topoisomerase I inhibitor camptothecin . Previously, a differential display screening was performed to isolate genes differentially expressed during camptothecin-induced cell death . As a result, the new tomato gene Le-pirin was isolated, whose mRNA levels dramatically increase during camptothecin-induced PCD . Le-pirin mRNA accumulation is also observed when cell death is triggered by the mycotoxin fumonisin-B1, but not when the suspension cells are treated with stress-related compounds such as ethylene, methyl jasmonate or salicylic acid . The caspase inhibitor Z-Asp-CH2-DCB and the calcium channel blocker LaCl3 effectively delayed whereas ethylene greatly stimulated camptothecin-induced PCD and the accumulation of Le-pirin mRNA . The Le-pirin encoded protein shows 56% identity with the human protein PIRIN, a nuclear factor reported to interact with the human oncogene Bcl-3 . Human PIRIN stabilizes the formation of quaternary complexes between Bcl-3, the anti-apoptotic transcription factor NF-kappaB and its DNA target sequences in vitro . The isolation of Le-pirin and its implication in plant PCD provides new clues on the role of putative NF-kappaB-associated pathways in plant defence mechanisms.

Physiol Plant, 2001 Jul, 112(3), 327 - 333
Mitochondrial alternative oxidase acts to dampen the generation of active oxygen species during a period of rapid respiration induced to support a high rate of nutrient uptake; Yip JY et al.; When wild type (wt) tobacco (Nicotiana tabacum L . cv . Petit Havana SR1) suspension cells were grown under phosphate (P) limitation, they contained large amounts of mitochondrial alternative oxidase (AOX) . When these cells were resupplied with P, there was a large, immediate and sustained stimulation of respiration to support a period of rapid P uptake . Two lines of evidence suggest that the abundant level of AOX present in wt cells contributed to this stimulated rate of respiration . First, when P-limited transgenic antisense tobacco cells (AS8) lacking AOX were resupplied with P, the stimulation of respiration was much less dramatic even though these cells displayed similar rates of P uptake . Second, while the stimulated rate of respiration in AS8 cells was insensitive (as expected) to the AOX inhibitor n-propyl gallate (nPG), much of the stimulated rate of respiration in wt cells could be inhibited by nPG . Given the non-phosphorylating nature of AOX respiration, wt cells required higher rates of electron transport to O2 than AS8 cells to support similar rates of P uptake . The utilization of AOX by wt cells during P uptake was apparently not occurring because the cytochrome (Cyt) pathway alone could not fully support the rate of P uptake, as the respiration of cells lacking AOX (either untreated AS8 cells or wt cells treated with nPG) supported similar rates of P uptake as wt cells with abundant AOX . Rather, we provide in vivo evidence that the utilization of AOX during the period of high respiration supporting P uptake was to dampen the mitochondrial generation of active oxygen species (AOS).

Plant Physiol, 2001 Jul, 126(3), 1092 - 104
The Ca(2+) status of the endoplasmic reticulum is altered by induction of calreticulin expression in transgenic plants; Persson S et al.; To investigate the endoplasmic reticulum (ER) Ca(2+) stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca(2+)-binding protein . NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter . ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca(2+) uptake and release . We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent (45)Ca(2+) accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls . Furthermore, after treatment with the Ca(2+) ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of (45)Ca(2+) released, and a 2- to 3-fold increase in the amount of (45)Ca(2+) retained compared with wild type . These data indicate that altering the production of CRT affects the ER Ca(2+) pool . In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects . We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca(2+)-containing medium to Ca(2+)-depleted medium . Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca(2+) stores and thereby enhances the survival of plants grown in low Ca(2+) medium.

Physiol Plant, 2001 Jun, 112(2), 280 - 284
4-Hydroxybenzyl alcohol accumulates in suspension-cell cultures and inhibits somatic embryogenesis in carrot; Kobayashi T et al.; Somatic embryogenesis in carrot (Daucus carota L.) is strongly inhibited by certain factors that accumulate in culture medium of high-density cultures of embryogenic cells . We previously identified 4-hydroxybenzyl alcohol (4HBA) as one of the inhibitory factors . In this study, we analyzed the accumulation pattern of 4HBA in the cultures of carrot suspension cells . When somatic embryogenesis was induced by culturing embryogenic cells in phytohormone-free Murashige and Skoog medium at various initial cell densities, 4HBA accumulated in the culture medium . The concentration of 4HBA in high cell density cultures was higher than in low cell density cultures . The accumulation of 4HBA in high cell density cultures was rapid during the early days of culture . This rapid accumulation of 4HBA in high cell density cultures might result in the strong inhibition of somatic embryogenesis . The production of 4HBA decreased as the somatic embryos developed . In addition, embryogenic cells released larger amount of 4HBA into the culture medium compared with non-embryogenic cells . These results suggest that the production of 4HBA is both related to embryogenic competence and developmentally regulated during somatic embryogenesis.

J Biol Chem, 2001 Sep 14, 276(37), 35103 - 10 Epub 2001 Jul 10.
Intracellular delivery of proteins with a new lipid-mediated delivery system; Zelphati O et al.; There are many very effective methods to introduce transcriptionally active DNA into viable cells but approaches to deliver functional proteins are limited . We have developed a lipid-mediated delivery system that can deliver functional proteins or other bioactive molecules into living cells . This delivery system is composed of a new trifluoroacetylated lipopolyamine (TFA-DODAPL) and dioleoyl phosphatidylethanolamine (DOPE) . This cationic formulation successfully delivered antibodies, dextran sulfates, phycobiliproteins, albumin, and enzymes (beta-galactosidase and proteases) into the cytoplasm of numerous adherent and suspension cells . Two systems were used to demonstrate that the proteins were delivered in a functionally active form . First, intracellular beta-galactosidase activity was clearly demonstrated within X-gal-stained cells after TFA-DODAPL:DOPE-mediated delivery of the enzyme . Second, the delivery system mediated delivery of several caspases (caspase 3, caspase 8, and granzyme B) into cultured cell lines and primary cells triggering apoptosis . Mechanistic studies showed that up to 100% of the protein mixed with the lipid formulation was captured into a lipid-protein complex, and up to 50% of the input protein associated with cells . This lipid-mediated transport system makes protein delivery into cultured cells as convenient, effective, and reliable as DNA transfection.

J Cell Biol, 2001 Jun 25, 153(7), 1381 - 90
Role of the F-box protein Skp2 in adhesion-dependent cell cycle progression; Carrano AC et al.; Cell adhesion to the extracellular matrix (ECM) is a requirement for proliferation that is typically lost in malignant cells . In the absence of adhesion, nontransformed cells arrest in G1 with increased levels of the cyclin-dependent kinase inhibitor p27 . We have reported previously that the degradation of p27 requires its phosphorylation on Thr-187 and is mediated by Skp2, an F-box protein that associates with Skp1, Cul1, and Roc1/Rbx1 to form the SCF(Skp2) ubiquitin ligase complex . Here, we show that the accumulation of Skp2 protein is dependent on both cell adhesion and growth factors but that the induction of Skp2 mRNA is exclusively dependent on cell adhesion to the ECM . Conversely, the expression of the other three subunits of the SCF(Skp2) complex is independent of cell anchorage . Phosphorylation of p27 on Thr-187 is also not affected significantly by the loss of cell adhesion, demonstrating that increased p27 stability is not dependent on p27 dephosphorylation . Significantly, ectopic expression of Skp2 in nonadherent G1 cells resulted in p27 downregulation, entry into S phase, and cell division . The ability to induce adhesion-independent cell cycle progression was potentiated by coexpressing Skp2 with cyclin D1 but not with cyclin E, indicating that Skp2 and cyclin D1 cooperate to rescue proliferation in suspension cells . Our study shows that Skp2 is a key target of ECM signaling that controls cell proliferation.

FEBS Lett, 2001 Jun 15, 499(1-2), 161 - 5
Leucine and its keto acid enhance the coordinated expression of genes for branched-chain amino acid catabolism in Arabidopsis under sugar starvation; Fujiki Y et al.; Branched-chain alpha-keto acid dehydrogenase (BCKDH), a multienzyme complex, plays a key role in branched-chain amino acid catabolism . However, it remains unclear whether expression of each subunit is coordinately regulated in plants, which should be important for the efficient assembly of subunits into a functional multienzyme complex . We show that the transcripts from the Arabidopsis E1alpha subunit gene accumulated in dark-adapted leaves and in sugar-starved suspension cells . These results are complementary to our previous report that the transcripts for the E1beta and E2 subunit genes accumulated in sugar-starved cells . Expression of the E1alpha gene is likely to be regulated by hexokinase-mediated sugar signaling, indicating that sugar plays a regulatory role in the coordinated expression of BCKDH subunit genes . Furthermore, Leu and its metabolite alpha-ketoisocaproate have synergistic effects on the enhanced expression of BCKDH subunit genes under sugar starvation . We hence suggest that branched-chain amino acids activate their own degradation pathway in sugar-starved cells through co-induction of each subunit gene of BCKDH.

Arch Biochem Biophys, 2001 Jun 15, 390(2), 265 - 78
Taxol biosynthesis: differential transformations of taxadien-5 alpha-ol and its acetate ester by cytochrome P450 hydroxylases from Taxus suspension cells; Wheeler AL et al.; The biosynthesis of the diterpenoid antineoplastic drug Taxol in Taxus species involves the cyclization of the ubiquitous isoprenoid intermediate geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P450-mediated hydroxylation (with allylic rearrangement) of this olefin precursor to taxa-4(20),11(12)-dien-5 alpha-ol, and further oxygenation and acylation reactions . Based on the abundances of naturally occurring taxoids, the subsequent order of oxygenation of the taxane core is considered to occur at C10, then C2 and C9, followed by C13, and finally C7 and C1 . Circumstantial evidence suggests that the acetylation of taxadien-5 alpha-ol may constitute the third specific step of Taxol biosynthesis . To determine whether taxadienol or the corresponding acetate ester serves as the direct precursor of subsequent oxygenation reactions, microsomal preparations isolated from induced Taxus cells and optimized for cytochrome P450 catalysis were incubated with each potential substrate . Both taxadienol and taxadienyl acetate were oxygenated to the level of a diol and to higher polyols at comparable rates by cytochrome P450 enzymes of the microsomal preparation . Preparative-scale incubation allowed the isolation of sufficient quantities of the diol derived from taxadienol to permit the NMR-based structural elucidation of this metabolite as taxa-4(20),11(12)-dien-5 alpha,13 alpha-diol, which may represent an alternate route of taxoid metabolism in induced cells . GC-MS-based structural definition of the diol monoacetate derived in microsomes from taxadienyl acetate confirmed this metabolite as taxa-4(20),11(12)-dien-5 alpha-acetoxy-10 beta-ol, thereby indicating that acetylation at C5 of taxadienol precedes the cytochrome P450-mediated insertion of the C10-beta-hydroxyl group of Taxol .

Novartis Found Symp, 2001, 236, 85 - 95; discussion 95-6
Regulation of gene expression by small molecules in rice; Zhang S et al.; A system for the regulation of gene expression by small molecules in transgenic rice was developed . This gene switch system consists of two components: (1) a hybrid chemically activated transcription factor, and (2) a synthetic target promoter . The two elements were transformed into rice suspension cells and transgenic plants were regenerated . A luciferase reporter under control of the gene switch system displayed as high as 10,000-fold inducibility following exposure to the small molecule ligand . The dose-response and induction time-course were determined . Regulated luciferase activity in activated plants decreased one day following removal of ligand and could be reactivated multiple times without apparent cosuppression . Analysis of luciferase activity following ligand application to media surrounding the roots suggests that ligand can be absorbed and transported systemically . In contrast, reporter activation was limited to a small area when ligand was applied directly to the leaf surface . The described gene switch system represents an important tool for situations requiring conditional gene expression in a monocot species.

FEBS Lett, 2001 Apr 6, 494(1-2), 44 - 7
Immunocytological localization of two plant fatty acid desaturases in the endoplasmic reticulum; Dyer JM et al.; The subcellular location of two integral membrane-bound fatty acid desaturases (Fads), Fad2 and Fad3, was elucidated by immunofluorescence microscopic analyses of tobacco suspension cells transiently transformed with different epitope-tagged versions of the enzymes . Both myc- or hemagglutinin-tagged Fad2 and Fad3 localized to the same region of the endoplasmic reticulum (ER), as evidenced by their co-localization with the ER lumenal protein calreticulin . Results from differential permeabilization experiments revealed that the N-termini of both epitope-tagged Fad2 and Fad3 were exposed on the cytosolic side of ER membranes . These data define the subcellular location and topological orientation of plant desaturases in ER membranes.

J Biotechnol, 2001 Apr 27, 87(1), 1 - 16
Green fluorescent protein as a secretory reporter and a tool for process optimization in transgenic plant cell cultures; Liu S et al.; Green fluorescent protein (GFP) is an attractive reporter for bioprocess monitoring . Although expression of GFP in plants has been widely reported, research on the use of GFP in plant cell cultures for bioprocess applications has been limited . In this study, the suitability of GFP as a secretory reporter and a useful tool in plant cell bioprocess optimization was demonstrated . GFP was produced and secreted from suspension cells derived from tobacco that was transformed with a binary vector containing mgfp5-ER cDNA, a modified GFP for efficient sorting to the endoplasmic reticulum, under control of the CaMV 35S promoter . For cell line gfp-13, extracellular and intracellular GFP accumulated to 15.4 and 29.4 mg x 1(-1), respectively . Extracellular GFP accounted for 30.9% of the total extracellular protein . The molecular mass of extracellular GFP was nearly identical to that of a recombinant GFP standard, indicating cleavage of the signal sequence . Neomycin phosphotransferase II, a cytosolic selection marker, was found almost exclusively in cellular extracts with less than 2% in the extracellular medium . These results suggest that extracellular GFP is most likely the result of secretion rather than nonspecific leakage from cells . Furthermore, medium fluorescence intensity correlated nicely with extracellular GFP concentration supporting the use of GFP as a quantitative secretory reporter . During the batch cultivation, culture GFP fluorescence also followed closely with cell growth . A medium feeding strategy was then developed based on culture GFP fluorescence that resulted in improved biomass as well as GFP production in a fed-batch culture.

J Cancer Res Clin Oncol, 2001 Feb, 127(2), 139 - 41
Transition from suspension to adherent growth is accompanied by tissue factor expression and matrix metalloproteinase secretion in a small cell lung cancer cell line; Salge U et al.; Small cell lung cancer (SCLC) is a very malignant tumor known to grow aggressively and to metastasize early . It is well established that metastasis generally involves both tumor cell adhesion and proteolytic degradation of the extracellular matrix . However, SCLC cells cultured in vitro, such as the classic SCLC cell line NCI-H69, grow in floating aggregates and express only negligible proteolytic activity . In this report, we show that NCI-H69 cells can be selected for adherent growth . In contrast to parental suspension cells, the adherent cells were found to express tissue factor as well as gelatinolytic activity, attributable to matrix metalloproteinases 2 and 9 . Such a switch of tumor cell characteristics, if it could occur in SCLC patients, might add to the understanding of the steps involved in the spreading of this highly metastatic type of lung cancer.

Plant Mol Biol, 2000 Nov, 44(5), 675 - 85
A Catharanthus roseus BPF-1 homologue interacts with an elicitor-responsive region of the secondary metabolite biosynthetic gene Str and is induced by elicitor via a JA-independent signal transduction pathway; van der Fits L et al.; Plants respond to pathogen attack by induction of various defence responses, including the biosynthesis of protective secondary metabolites . In Catharanthus roseus, the elicitor-induced expression of the terpenoid indole alkaloid biosynthetic gene Strictosidine synthase (Str) is mediated via the plant stress hormonejasmonate . In the promoters of several defence-related genes, cis-acting elements have been identified that are important for transcriptional regulation upon stress signals . Here we show that an upstream region in the Str promoter confers responsiveness to partially purified yeast elicitor and jasmonate . Yeast one-hybrid screening with this element as a bait identified a MYB-like protein, which shows high homology to parsley box P-binding factor-1 (PcBPF-1) . In vitro analyses showed that the Str promoter fragment contained a novel binding site for BPF-1-like proteins with higher binding affinity than the previously described box P . CrBPF-1 mRNA accumulated rapidly in elicitor-treated C . roseus suspension cells, whereas no induction was observed with jasmonate . Inhibitor studies indicated that CrBPF-1 plays a role in an elicitor-responsive but jasmonate-independent signal transduction pathway, acting downstream of protein phosphorylation and calcium influx.

Plant Cell Physiol, 2000 Oct, 41(10), 1143 - 8
Synthesis of phosphatidylserine in carrot cells cultured under carbon-source starvation; Manoharan K et al.; When carrot suspension cells were cultured on medium containing no carbon source (starvation), the levels of phosphatidylserine (PS) increased transiently 3-4 d after the initiation of starvation while levels of most other phospholipid (PL) species decreased . We previously reported that fatty acids of these PLs served as an alternative carbon source during starvation . The present study showed that cells possess two different biosynthetic pathways involving phosphatidylcholine (PC)/phosphatidylethanolamine (PE) exchange enzymes and PS synthase to synthesize PS . These activities peaked similarly 4 d after the initiation of starvation and coincided with the peak of PS level . The synthesis of serine was also significantly activated during starvation . The activity of phosphoserine aminotransferase (PSAT) which is involved in serine synthesis increased with a time course similar to that of the increase in the PS level . These observations suggest that the increase in PS level plays an important role in membranes which are degraded during starvation.

J Exp Bot, 2000 Dec, 51(353), 1991 - 9
Osmotic stress-induced changes of sucrose metabolism in cultured sweet potato cells; Wang HL et al.; The intra- and extracellular sugar contents, the activities of sucrose-metabolizing enzymes, and the metabolism of {U-(14)C} glucose in a pulse-chase experiment were compared between the normal and osmotically stressed (by 0.6 M sorbitol) sweet potato (Ipomoea batatas) suspension cells . The stress enhanced the levels of sucrose and sucrose phosphate synthase (SPS) activity . Northern blot analysis also showed that prolonged osmotic stress enhanced the SPS gene expression at the transcriptional level . Stressed cells also had higher activities of sucrose cleaving enzymes, such as alkaline invertase and sucrose synthase . The (14)C-sucrose isolated from normal and stressed cells had (14)C-fructose and (14)C-glucose ratios of 0.68 and 1, respectively . These data suggest the continual cycling of degradation and synthesis of sucrose in both types of cells . Among the enzymes used in constructing such futile cycling, besides invertase and SPS, sucrose synthase (SS) should be involved in normal cells, but not in stressed ones . It is apparent that the osmotic stress caused a significant change in the pattern of sucrose metabolism.

Plant J, 2000 Dec, 24(5), 693 - 701
Promotive effect of brassinosteroids on cell division involves a distinct CycD3-induction pathway in Arabidopsis; Hu Y et al.; Brassinosteroids (BRs) are steroid hormones that play an essential role in plant growth and development . However, the contradictory results of previous studies make their role in cell division unclear . Using a cDNA array, we identified genes that respond to BR in the det2 suspension culture of Arabidopsis, and found that epi-brassinolide upregulated transcription of the CycD3, a D-type plant cyclin gene through which cytokinin activates cell division . RNA gel-blot analysis and cell culturing showed that epi-brassinolide may promote cell division through CycD3, and can substitute cytokinin in culturing of Arabidopsis callus and suspension cells . The CycD3 induction by epi-brassinolide was further shown to involve de novo protein synthesis, but no protein phosphorylation or dephosphorylation . Induction was also found to occur in cells of a BR-insensitive mutant, bri1, suggesting that BR induces CycD3 transcription through a previously unknown signal pathway in plants.

J Virol, 2001 Jan, 75(1), 396 - 407
Genetic evidence of a role for ATM in functional interaction between human T-cell leukemia virus type 1 Tax and p53; Van PL et al.; Recent evidence from several investigators suggest that the human T-cell leukemia virus type 1 Tax oncoprotein represses the transcriptional activity of the tumor suppressor protein, p53 . An examination of published findings reveals serious controversy as to the mechanism(s) utilized by Tax to inhibit p53 activity and whether the same mechanism is used by Tax in adherent and suspension cells . Here, we have investigated Tax-p53 interaction simultaneously in adherent epithelial (HeLa and Saos) and suspension T-lymphocyte (Jurkat) cells . Our results indicate that Tax activity through the CREB/CREB-binding protein (CBP), but not NF-kappaB, pathway is needed to repress the transcriptional activity of p53 in all tested cell lines . However, we did find that while CBP binding by Tax is necessary, it is not sufficient for inhibiting p53 function . Based on knockout cell studies, we correlated a strong genetic requirement for the ATM, but not protein kinase-dependent DNA, protein in conferring a Tax-p53-repressive phenotype.

FEBS Lett, 2000 Dec 8, 486(2), 155 - 8
Upregulation of vacuolar H(+)-translocating pyrophosphatase by phosphate starvation of Brassica napus (rapeseed) suspension cell cultures; Palma DA et al.; The influence of phosphate (Pi) deprivation on the vacuolar H(+)-translocating pyrophosphatase (PPiase) and ATPase in tonoplast vesicles from Brassica napus suspension cells was assessed . Pi starvation significantly elevated the ratios of PPi-:ATP-dependent H(+) translocation rate and H(+)-PPiase:H(+)-ATPase hydrolytic activities . These increases were reversed 36 h following resupply of 2.5 mM Pi to the Pi-starved cells . Immunoblotting indicated that Pi starvation also induced a two-fold increase in the amount of H(+)-PPiase protein, whereas the amount of H(+)-ATPase remained unchanged . It is proposed that H(+)-PPiase facilitates the conservation of limited ATP pools, and Pi recycling during Pi stress.

Planta, 2000 Oct, 211(5), 656 - 62
Chemical-induced apoptotic cell death in tomato cells: involvement of caspase-like proteases; De Jong AJ et al.; 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.

Cell Death Differ, 2000 Sep, 7(9), 795 - 803
Activation of protein kinase C relays distinct signaling pathways in the same cell type: differentiation and caspase-mediated apoptosis; Meinhardt G et al.; Activation of PKC with 5 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h in human U937 myeloid leukemia cells is associated with induction of adherence, followed by monocytic differentiation and G0/G1 cell cycle arrest . In this study, we demonstrate that in addition to these effects about 25% of U937 cells accumulated in an apoptotic subG1 phase after TPA treatment . The appearance of these apoptotic suspension cells was detectable throughout the time course of the culture and was independent of TPA concentrations between 0.5 and 500 nM . Experiments with cells synchronized by centrifugal elutriation revealed dominant susceptibility of G1-phase cells to TPA-mediated apoptosis . While adherent cells expressed differentiation markers including the integrin CD11c, this effect was less pronounced in the TPA-treated suspension fraction . Moreover, previous work has demonstrated cell cycle arrest in differentiating U937 cells . Accordingly, PKC activation by TPA treatment was associated with a significant expression of the cdk/cyclin inhibitor p21WAF/CIP/sdi-1 in the adherent population and subsequent G0/G1 cell cycle arrest . In contrast, suspension cells failed to induce significant levels of p21WAF/CIP/sdi-1 after TPA stimulation . Immunoblotting experiments demonstrated no difference in the expression of the pro-apoptotic factors Bax, Bad, and Bak in either control U937 and TPA-treated adherent or suspension cells, respectively . However, anti-apoptotic factors including Bcl-2, Bcl-xL, and Mcl-1 were significantly induced in the adherent population whereas no induction was detectable in the suspension cells . In this context, incubation with the caspase-3/caspase-7 specific tetrapeptide inhibitor DEVD prior to TPA treatment prevented an accumulation of cells in subG1, respectively, demonstrating an involvement of these caspases . Taken together, these data suggest that PKC activation can relay distinct signaling pathways such as induction of adherence coupled with monocytic differentiation and growth arrest, or induction of caspase-mediated apoptosis coupled with the failure to adhere and to differentiate.

Proc Natl Acad Sci U S A, 1997 Jun 24, 94(13), 7103 - 7108
Purified vesicles of tobacco cell vacuolar and plasma membranes exhibit dramatically different water permeability and water channel activity; Maurel C et al.; The vacuolar membrane or tonoplast (TP) and the plasma membrane (PM) of tobacco suspension cells were purified by free-flow electrophoresis (FFE) and aqueous two-phase partitioning, with enrichment factors from a crude microsomal fraction of >/=4- to 5-fold and reduced contamination by other cellular membranes . For each purified fraction, the mean apparent diameter of membrane vesicles was determined by freeze-fracture electron microscopy, and the osmotic shrinking kinetics of the vesicles were characterized by stopped-flow light scattering . Osmotic water permeability coefficients (P(f)) of 6.1 +/- 0.2 and 7.6 +/- 0.9 microm small middle dots(-1) were deduced for PM-enriched vesicles purified by FFE and phase partitioning, respectively . The associated activation energies (E(a); 13.7 +/- 1.0 and 13.4 +/- 1.4 kcal small middle dotmol(-1), respectively) suggest that water transport in the purified PM occurs mostly by diffusion across the lipid matrix . In contrast, water transport in TP vesicles purified by FFE was characterized by (i) a 100-fold higher P(f) of 690 +/- 35 microm small middle dots(-1), (ii) a reduced E(a) of 2.5 +/- 1.3 kcal small middle dotmol(-1), and (iii) a reversible inhibition by mercuric chloride, up to 83% at 1 mM . These results provide functional evidence for channel-mediated water transport in the TP, and more generally in a higher plant membrane . A high TP P(f) suggests a role for the vacuole in buffering osmotic fluctuations occurring in the cytoplasm . Thus, the differential water permeabilities and water channel activities observed in the tobacco TP and PM point to an original osmoregulatory function for water channels in relation to the typical compartmentation of plant cells.

FEBS Lett, 2000 Oct 13, 483(1), 67 - 70
Abscisic acid plasmalemma perception triggers a calcium influx essential for RAB18 gene expression in Arabidopsis thaliana suspension cells; Ghelis T et al.; Pretreatment of Arabidopsis thaliana suspension cells with impermeant calcium chelator EGTA inhibited the ABA-induced RAB18 gene expression . However, extracellular calcium alone, up to 10 mM, did not trigger RAB18 expression . Spectrofluorimetric extracellular Ca(2+) measurement with Fluo-3 showed a fast, within 1 min, Ca(2+) influx associated with outer plasmalemma ABA perception . In the presence of the Ca(2+) blockers Cd(2+) and Ni(2+), RAB18 expression was suppressed . Pimozide and fluspirilene inhibited Ca(2+) influx and ABA-induced RAB18 expression . Thus we demonstrated the involvement of specific Ca(2+) influx in the ABA signaling sequence leading to RAB18 expression.

Cell Biol Toxicol, 2000, 16(3), 185 - 200
Induction of apoptosis in normal cultured rat hepatocytes and in Hep3B, a human hepatoma cell line; Lamboley C et al.; The in vitro occurrence of apoptosis in hepatic cells has not been well characterized because it depends on apoptosis inducing-agents and culture conditions . Furthermore, for a given hepatic cell and the same agent, discrepant results have been reported depending on the technique used to evaluate the proportion of apoptotic cells . In this study, we compared the effects of several apoptosis-inducing agents - transforming growth factor beta1 (TGF-beta1), retinoic acid (RA), okadaic acid (OA), and cycloheximide (CY) - on two types of hepatic cells, the human hepatoma cell line Hep3B and normal rat hepatocytes, maintained either plated for 24 to 48 h or in suspension for 20 h . Chromatin condensation and/or nucleus fragmentation were investigated morphologically by DAPI staining . DNA fragmentation was investigated biochemically by agarose gel electrophoresis and poly(ADP-ribose) polymerase (PARP) cleavage was studied by western blot . Apoptotic cells were quantified either by counting cells on UV microscopy after DAPI staining or by flow cytometry . Nuclear changes, the ladder pattern on DNA electrophoresis and PARP cleavage were observed in plated cells, hepatoma cells and normal rat hepatocytes, with all inducers but especially with OA . Semiquantification confirmed that OA was a strong inducer in plated cells under the present conditions, since about 14% and 30% of Hep3B cells (with DAPI staining and flow cytometry, respectively) were apoptotic after 48 h treatment, while, with the other inducers, apoptosis was weaker and discrepancies were also observed between the two counting methods (TGF-beta1; 4% and 12%; RA, 7% and 12%; CY, 4% and 16%, with DAPI staining and flow cytometry, respectively) . OA induced a moderate apoptosis in cultured hepatocytes (13% with DAPI staining), while TGF-beta1, RA and CY were found to be weakly apoptotic (respectively 4% for the first two and 6% for the last ) after 48 h . In contrast, in suspension cells, apoptosis was observed neither in Hep3B cells nor in normal hepatocytes, whatever the apoptotic inducer and whatever the techniques used to detect apoptosis . In conclusion, our results show that induction of apoptosis in hepatic cells depends not only on the apoptosis-inducing agent but also on the culture conditions.

Plant Physiol, 2000 Oct, 124(2), 899 - 910
Hydrogen peroxide yields during the incompatible interaction of tobacco suspension cells inoculated with Phytophthora nicotianae; Able AJ et al.; Rates of H(2)O(2) production by tobacco suspension cells inoculated with zoospores from compatible or incompatible races of the pathogen Phytophthora nicotianae were followed by direct measurement of oxygen evolution from culture supernatants following catalase addition . Rates of HO(2)(*)/O(2)(-) production were compared by following the formation of the formazan of sodium, 3'-{1-{phenylamino-carbonyl}-3,4-tetrazolium}-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate . In the incompatible interaction only, both reactive oxygen species (ROS) were produced by the cultured host cells in a minor burst between 0 and 2 h and then in a major burst between 8 and 12 h after inoculation . Absolute levels of H(2)O(2) could not be accurately measured due to its metabolism by host cells, but results are consistent with the majority of H(2)O(2) being formed via dismutation of HO(2)(*)/O(2)(-) . The effects of inhibitors of endogenous Cu/Zn superoxide dismutase (diethyldithiocarbamate) and catalase (3-amino-1,2,4-triazole and salicylic acid) were also examined . Yields of ROS in the presence of the inhibitors diphenylene iodonium, allopurinol, and salicylhydroxamic acid suggest that ROS were generated in incompatible host responses by more than one mechanism.

Plant Cell, 2000 Sep, 12(9), 1679 - 88
Association of phosphatidylinositol 3-kinase with nuclear transcription sites in higher plants; Bunney TD et al.; The kinases responsible for phosphorylation of inositol-containing lipids are essential for many aspects of normal eukaryotic cell function . Genetic and biochemical studies have established that the phosphatidylinositol (PtdIns) 3-kinase encoded by the yeast VPS34 gene is essential for the efficient sorting and delivery of proteins to the vacuole; the kinase encoded by the human VPS34 homolog has been equally implicated in the control of intracellular vesicle traffic . The plant VPS34 homolog also is required for normal growth and development, and although a role for PtdIns 3-kinase in vesicle trafficking is likely, it has not been established . In this study, we have shown that considerable PtdIns 3-kinase activity is associated with the internal matrix of nuclei isolated from carrot suspension cells . Immunocytochemical and confocal laser scanning microscopy studies using the monoclonal antibody JIM135 (John Innes Monoclonal 135), raised against a truncated version of the soybean PtdIns 3-kinase, SPI3K-5p, revealed that this kinase appears to have a distinct and punctate distribution within the plant nucleus and nucleolus . Dual probing of root sections with JIM135 and anti-bromo-UTP antibodies, after in vitro transcription had been allowed to proceed in the presence of bromo-UTP, showed that SPI3K-5p associates with active nuclear and nucleolar transcription sites . These findings suggest a possible link between PtdIns 3-kinase activity and nuclear transcription in plants.

Biochem J, 2000 Sep 15, 350 Pt 3, 717 - 22
A new phosphospecific cell-based ELISA for p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and cAMP-response-element-binding protein; Versteeg HH et al.; Assaying activation of signal transduction is laborious and does not allow the study of large numbers of samples, essential for high-throughput drug screens or for large groups of patients . Using phosphospecific antibodies, we have developed ELISA techniques enabling non-radioactive semi-quantitative assessment of the activation state of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and the transcription factor cAMP-response-element-binding protein (CREB) in 96-well plates . This assay has been termed PACE (phosphospecific antibody cell-based ELISA) and was used successfully for both adherent and suspension cells . Various stimuli induced dose-dependent enzymic activity of which the kinetics closely correlated with those measured via classical methodology . Using PACE we have now characterized for the first time the concentration-dependent effects of various inflammatory prostaglandins on CREB phosphorylation in macrophages . PACE is a straightforward and novel technique enabling the large-scale analysis of signal transduction.

Plant Sci, 2000 Aug 22, 157(2), 209 - 216
Additional phosphate stabilises uninterrupted growth of a Dioscorea deltoidea cell culture; Kandarakov O et al.; Suspension cells of Dioscorea deltoidea Wall (strain D-1) were maintained in a semicontinuous culture (SCC) in shake flasks at a high growth rate . It was shown that continuous propagation growth of this culture is unstable on Murashige's and Skoog's (MS) medium due to P starvation . On a P-enriched MS-medium the SCC was stable even at mean specific growth rates >0.3 day(-1) . Highest volumetric concentrations of furostanol glycosides were obtained, when a P-enriched SCC was not further subcultivated but fed with sucrose . The investigated culture is able to control phosphate uptake and to prevent toxicity on media with excess P . High concentrations of cellular P(i) did not effect the ratio of furostanol to starch.

Plant Sci, 2000 Jul 28, 156(2), 201 - 211
Structure, expression and promoter activity of two polyubiquitin genes from rice (Oryza sativa L.); Wang J et al.; We have isolated two rice polyubiquitin genes designated as RUBQ1 and RUBQ2 by screening a Bacterial Artificial Chromosome (BAC) genomic library with a 32P-labeled ubiquitin cDNA probe . DNA sequence data revealed that both genes contained an open reading frame encoding a hexameric precursor ubiquitin and an intron immediate upstream of the initiation codon . The deduced amino acid sequences of both genes were identical to each other and to other plant ubiquitin sequences . Several putative regulatory elements such as enhancer core and heat shock consensus sequences were found in the 5'-upstream regions of both genes . Northern blot analyses using the 3'-untranslated region as gene specific probes showed that both genes were actively expressed in all rice plant tissues tested . Differential expression was observed in roots where RUBQ2 appeared to be predominantly expressed . Chimeric genes containing the 5'-upstream region including the intron of RUBQ1 or RUBQ2 and the beta-glucuronidase (GUS) coding region were constructed and transferred into rice suspension cells via particle bombardment . GUS activity from constructs containing RUBQ1 and RUBQ2 promoters in rice suspension cells was ten to 15-fold greater than those using the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter, and two to threefold greater than constructs with the maize polyubiquitin Ubi1 promoter . The results demonstrate the potential usefulness of the two rice polyubiquitin promoters in rice or other monocot transformation systems.

Eur J Biochem, 2000 Jul, 267(14), 4477 - 85
Purification and characterization of cytosolic pyruvate kinase from Brassica napus (rapeseed) suspension cell cultures: implications for the integration of glycolysis with nitrogen assimilation; Smith CR et al.; Cytosolic pyruvate kinase (PKc) from Brassica napus suspension cells was purified 201-fold to electrophoretic homogeneity and a final specific activity of 51 micromol phosphoenolpyruvate utilized per min per mg protein . SDS/PAGE and gel filtration analyses of the final preparation indicated that this PKc is a 220-kDa homotetramer composed of 56-kDa subunits . The enzyme was relatively heat-stable and displayed a broad pH optimum of pH 6.8 . PKc activity was absolutely dependent upon the simultaneous presence of a bivalent and univalent cation, with Mg2+ and K+ fulfilling this requirement . Hyperbolic saturation kinetics were observed for phosphoenolpyruvate, ADP, Mg2+ and K+ (apparent Km values = 0.12, 0.075, 0.21 and 0.48 mM, respectively) . Although the enzyme utilized UDP, CDP and IDP as alternative nucleotides, ADP was the preferred substrate . L-Glutamate, oxalate, and the flavonoids rutin and quercetin were the most effective inhibitors (I50 values = 4, 0.3, 0.07, and 0.10 mM, respectively) . L-Aspartate functioned as an activator (Ka = 0.31 mM) by causing a 40% increase in Vmax while completely reversing the inhibition of PKc by L-glutamate . Reciprocal control by L-aspartate and L-glutamate is specific for these amino acids and provides a rationale for the in vivo activation of PKc that occurs during periods of enhanced NH +4-assimilation . Allosteric features of B . napus PKc are compared with those of B . napus phosphoenolpyruvate carboxylase . A model is presented that highlights the pivotal role of L-aspartate and L-glutamate in the coordinate regulation of these key phosphoenolpyruvate utilizing cytosolic enzymes.

Eur J Biochem, 2000 Jul, 267(14), 4465 - 76
Purification and characterization of phosphoenolpyruvate carboxylase from Brassica napus (rapeseed) suspension cell cultures: implications for phosphoenolpyruvate carboxylase regulation during phosphate starvation, and the integration of glycolysis with nitrogen assimilation; Moraes TF et al.; Phosphoenolpyruvate carboxylase (PEPC) specific activity increased by 250% following 8 to 10 days of Pi starvation of Brassica napus suspension cells . Densitometric scanning of PEPC immunoblots revealed a close correlation between PEPC activity and the amount of the antigenic 104-kDa PEPC subunit . To further assess the influence of Pi deprivation on PEPC, the enzyme was purified from Pi-sufficient (+Pi) and Pi-starved (-Pi) cells to electrophoretic homogeneity and final specific activities of 37-40 micromol phosphoenolpyruvate utilized per min per mg protein . Gel filtration, SDS/PAGE, and CNBr peptide mapping indicated that the +Pi and -Pi PEPCs are both homotetramers composed of an identical 104-kDa subunit . Respective pH-activity profiles, phosphoenolpyruvate saturation kinetics, and sensitivity to L-malate inhibition were also indistinguishable . Kinetic studies and phosphatase treatments revealed that PEPC of the +Pi and -Pi cells exists mainly in its dephosphorylated (L-malate sensitive) form . Thus, up-regulation of PEPC activity in -Pi cells appears to be solely due to the accumulation of the same PEPC isoform being expressed in +Pi cells . PEPC activity was modulated by several metabolites involved in carbon and nitrogen metabolism . At pH 7.3, marked activation by glucose 6-phosphate and inhibition by L-malate, L-aspartate, L-glutamate, DL-isocitrate, rutin and quercetin was observed . The following paper provides a model for the coordinate regulation of B . napus PEPC and cytosolic pyruvate kinase by allosteric effectors . L-Aspartate and L-glutamate appear to play a crucial role in the control of the phosphoenolpyruvate branchpoint in B . napus, particularly with respect to the integration of carbohydrate partitioning with the generation of carbon skeletons required during nitrogen assimilation.

FEBS Lett, 2000 May 26, 474(1), 43 - 7
Abscissic acid specific expression of RAB18 involves activation of anion channels in Arabidopsis thaliana suspension cells; Ghelis T et al.; The abscissic acid (ABA) transduction cascade following the plasmalemma perception was analyzed in intact Arabidopsis thaliana suspension cells . In response to impermeant ABA, anion currents were activated and K(+) inward rectifying currents were inhibited . Anion current activation was required for the ABA specific expression of RAB18 . By contrast, specific inhibition of K(+) channels by tetraethylammonium or Ba(2+) did not affect RAB18 expression . Thus, outer plasmalemma ABA perception triggered two separated signaling pathways.

FEBS Lett, 2000 May 26, 474(1), 11 - 5
Involvement of poly(ADP-ribose) polymerase and activation of caspase-3-like protease in heat shock-induced apoptosis in tobacco suspension cells; Tian R et al.; The cleavage of poly(ADP-ribose) polymerase (PARP) by caspase (casp)-3 is an essential link in the apoptotic pathway in animal cells . In plant cells, however, there is no authentic evidence for the similar role that PARP may play during apoptosis . Using a heat shock (HS)-induced apoptosis system of tobacco cells, we found that immediately after a 4 h heat treatment, PARP was cleaved to form an 89 kDa signature fragment, while DNA laddering appeared only after a 20 h recovery following the HS . An activation of casp-3-like protease was also observed . The results suggest that apoptosis in plants and animals may share common mechanisms . On the other hand, when cells were preincubated with 4 mM 3-aminobenzamide or 2-8 mM nicotinamide, the specific inhibitors of PARP, before HS treatment, apoptotic cell death was reduced significantly . Our results thus imply that PARP may also be involved in apoptosis in a different way from the casp-related events.

J Biol Chem, 2000 May 19, 275(20), 15520 - 5
120- and 160-kDa receptors for endogenous mitogenic peptide, phytosulfokine-alpha, in rice plasma membranes; Matsubayashi Y et al.; Plant cells in culture secrete a sulfated peptide named phytosulfokine-alpha (PSK-alpha), and this peptide induces the cell division and/or cell differentiation by means of specific high and low affinity receptors . Putative receptor proteins for this autocrine type growth factor were identified by photoaffinity labeling of plasma membrane fractions derived from rice suspension cells . Incubation of membranes with a photoactivable (125)I-labeled PSK-alpha analog, {N(epsilon)-(4-azidosalicyl)Lys(5)}PSK-alpha (AS-PSK-alpha), followed by UV irradiation resulted in specific labeling of 120- and 160-kDa bands in SDS-polyacrylamide gel electrophoresis . The labeling of both bands was completely inhibited by unlabeled PSK-alpha and partially decreased by PSK-alpha analogs possessing moderate binding activities . In contrast, PSK-alpha analogs that have no biological activity showed no competition for (125)I-AS-PSK-alpha binding, confirming the specificity of binding proteins . Analysis of the affinity of (125)I incorporation into the protein by ligand saturation experiments gave apparent K(d) values of 5.0 nm for the 120-kDa band and 5.4 nm for the 160-kDa band, suggesting that both proteins correspond to the high affinity binding site . Treatment of (125)I-AS-PSK-alpha cross-linked proteins with peptide N-glycosidase F demonstrated that both proteins contained approximately 10 kDa of N-linked oligosaccharides . Specific cross-linking of (125)I-AS-PSK-alpha was also observed by using plasma membranes derived from carrot and tobacco cells, indicating the widespread occurrence of the binding proteins . Together, these data suggest that the 120- and 160-kDa proteins are PSK-alpha receptors that mediate the biological activities of PSK-alpha.

Plant Mol Biol, 2000 Mar, 42(5), 667 - 78
The rice R gene family: two distinct subfamilies containing several miniature inverted-repeat transposable elements; Hu J et al.; The R and B genes of maize regulate the anthocyanin biosynthetic pathway and constitute a small gene family whose evolution has been shaped by polyploidization and transposable element activity . To compare the evolution of regulatory genes in the distinct but related genomes of rice and maize, we previously isolated two R homologues from rice (Oryza sativa) . The Ra1 gene on chromosome 4 can activate the anthocyanin pathway, whereas the Rb gene, of undetermined function, maps to chromosome 1 . In this study, rice R genes have been further characterized . First, we found that an Rb cDNA can induce pigmentation in maize suspension cells . Second, another rice R homologue (Ra2) was identified that is more closely related to Ra1 than to Rb . Domesticated rice and its wild relatives harbor multiple Ra-like and Rb-like genes despite the fact that rice is a true diploid with the smallest genome of all the grass species analyzed to date . Finally, several miniature inverted-repeat transposable elements (MITEs) were found in R family members . Their possible role in hastening the divergence of R genes is discussed.

Plant Cell Physiol, 2000 Feb, 41(2), 209 - 17
Kinetic analysis of the K(+)-selective outward rectifier in Arabidopsis mesophyll cells: a comparison with other plant species; Miedema H et al.; This paper gives a kinetic analysis of the K(+)-selective outward-rectifier (IK,out) in the plasma membrane of Arabidopsis thaliana mesophyll cells in terms of the Hodgkin-Huxley formalism . We compared the kinetic characteristics of IK,out in Arabidopsis with IK,out channels in three other plant species that were subjected to a similar analysis: tobacco suspension cells, Vicia faba guard cells and Plantago media root cells . Because the activation kinetics of IK,out shows a clear voltage dependence, the time constant of half-activation (tau 1/2) and the elementary rate constant of channel opening (a) were calculated at the potential of half-activation (V1/2) . The Arabidopsis IK,out activates relatively slowly and this is reflected in a tau 1/2 of approximately 1 s . The reason for this slow activation is twofold . Firstly, the value of a is 1.5 s-1 falls at the lower end of the range of values obtained for tobacco, Vicia and Plantago: 1.1 to 3.0 s-1 . Secondly, IK,out in Arabidopsis has four closed states, while tobacco and Vicia have only two . As observed in other plant species, the activation kinetics of IK,out in Arabidopsis are sensitive to external K+: V1/2 shifts with EK but remains approximately 50 mV more positive than EK.

J Biol Chem, 2000 Apr 28, 275(17), 13118 - 25
The association of CRKII with C3G can be regulated by integrins and defines a novel means to regulate the mitogen-activated protein kinases; Buensuceso CS et al.; In studies to define mechanisms of ERK activation in Chinese hamster ovary cells, we have observed an inverse correlation between CRKII-C3G complex formation and ERK activity . That is, we were able to coprecipitate the guanine nucleotide exchange factor C3G with the adaptor protein CRKII in lysates from suspended cells that had low ERK activity, but we could not do so or could do so less efficiently in lysates of adherent cells with increased ERK activity . Consistent with the presence of a functional CRKII-C3G complex, we detected more GTP-loaded RAP1 in suspension than adherent lysates . Overexpression of cDNAs encoding B-RAF, CRKII W109L, and PTP1B C215S activated ERK in suspension cells, the latter two constructs also disrupting CRKII-C3G complex formation . Finally, we have also observed that certain integrin alpha subunit cytoplasmic splice variants differentially regulate ERK1/2 but also in a manner that correlated with levels of a CRKII-C3G complex . Thus, these data suggest the involvement of integrins in an ERK suppression pathway mediated by CRKII-C3G complex formation and downstream signaling from activated RAP1.

EMBO J, 2000 Apr 3, 19(7), 1672 - 80
Suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus; Lucy AP et al.; Post-transcriptional gene silencing (PTGS) is a homology-dependent RNA degradation process that may target RNA exclusively in the cytoplasm . In plants, PTGS functions as a natural defense mechanism against viruses . We reported previously that the 2b protein encoded by cucumber mosaic cucumovirus (CMV) is a virulence determinant and a suppressor of PTGS initiation in transgenic Nicotiana benthamiana . By fusion with the green fluorescent protein, we now show that the CMV 2b protein localizes to the nuclei of tobacco suspension cells and whole plants via an arginine-rich nuclear localization signal, (22)KRRRRR(27) . We further demonstrate that the nuclear targeting of the 2b protein is required for the efficient suppression of PTGS, indicating that PTGS may be blocked in the nucleus . In addition, our data indicate that the PTGS suppressor activity is important, but not sufficient, for virulence determination by the 2b protein.

Mol Gen Genet, 2000 Feb, 263(1), 30 - 7
Rapid systemic accumulation of transcripts encoding a tobacco WRKY transcription factor upon wounding; Hara K et al.; An immediate-early, transiently activated wound-responsive gene was identified in tobacco by fluorescent differential display screening . The full-length cDNA encodes a polypeptide of 356 amino acids with a relative molecular mass of 39,082 Da . The deduced amino acid sequence shows two characteristic features; a leucine-zipper motif found in the more N-terminal region and a WRKY domain containing a zinc-finger motif located in the central region . The gene was designated as wizz (wound-induced leucine zipper zinc finger) . Northern analysis showed that upon wounding wizz transcripts were locally and systemically accumulated within 10 min, reached a maximum level by 30 min, and decreased thereafter to the basal level . Analyses of a WIZZ-GFP fusion protein clearly indicated that WIZZ is a nuclear factor . WIZZ specifically binds to sequences containing two TTGAC core motifs that are separated by a spacer of appropriate length . The binding activity was dependent on bivalent cations, most probably zinc . In transient reporter assays, however, WIZZ did not show transactivation activity in tobacco suspension cells, suggesting that it functions together with other components . The results indicate that WIZZ is a new transcription factor which participates in early stages of the wound response.

Plant Sci, 2000 May 15, 154(1), 89 - 98
In vivo evaluation of the context sequence of the translation initiation codon in plants; Lukaszewicz M et al.; Statistical analysis of the AUG initiation codon context in several plant organisms identified a nucleotide preference in some positions around the AUG . Sixteen AUG contexts were studied using transient expression in tobacco, maize and Norway spruce . Besides the importance of A or G at position -3, we revealed the role of positions -2, -1 for which AA or CC were found to be the best for tobacco and maize, respectively . GC (positions +4, +5) were also found to be important in both tobacco and maize . Finally, we identified a variation in context efficiency according to cell type, since A was better than G at position -3 in tobacco leaf protoplasts, while both nucleotides were equally efficient in tobacco suspension cells.

Phytochemistry, 2000 Jan, 53(1), 1 - 5
Characterization and oxidative in vitro cross-linking of an extensin-like protein and a proline-rich protein purified from chickpea cell walls; Otte O et al.; Two cell wall proteins from chickpea, known to be rapidly insolubilised by an elicitor-stimulated oxidative burst in-vivo, were purified from suspension cells . N-terminal protein sequencing revealed them as a proline-rich protein and an extensin-like protein . Oxidative cross-linking could be modelled in an in vitro system utilising horseradish peroxidase, H2O2 and the substrate proteins.

Int J Artif Organs, 1999 Dec, 22(12), 816 - 22
Rotary cell culture system (RCCS): a new method for cultivating hepatocytes on microcarriers; Mitteregger R et al.; The Rotary Cell Culture System (RCCS) is a new technology for growing anchorage dependent or suspension cells in the laboratory . The RCCS is a horizontally rotated, bubble free disposable culture vessel with diffusion gas exchange . The system provides a reproducible, complex 3D in vitro culture system with large cell masses . During cell growing the rotation speed can be adjusted to compensate for increased sedimentation rates . The unique environment of low shear forces, high mass transfer, and microgravity, provides very good cultivating conditions for many cell types, cell aggregates or tissue particles in a standard tissue culture laboratory . The system enables to culture HepG2 cells on Cytodex 3 microcarriers (mcs) to high densities . We inoculated 2 x 10(5)/ml HepG2 cells and 200 mg Cytodex 3 mcs in 50 ml Williams E medium (incl . 10% FCS) allowing them to attach to the mcs in the rotating vessel (rotation rate 14-20 rpm) . HepG2 cells readily attached to the mcs while the vessel was rotating . Attachment of HepG2 to the mcs was about 50% after 24 hrs and 100 % within 48 hrs . After 72 hrs of rotary culturing small aggregates of Hep G2 on mcs were built . HepG2 cells and the aggregates rotated with the vessel and did not settle within the vessel or collide with the wall of the vessel . We conclude that this new RCCS is an excellent technology for culturing HepG2 cells on Cytodex 3 mcs . The system is easy to handle and enables to culture anchorage dependent cells to high densities in a short period.

Plant J, 2000 Jan, 21(1), 91 - 6
The ethylene-inducible PK12 kinase mediates the phosphorylation of SR splicing factors; Savaldi-Goldstein S et al.; The tobacco PK12 is induced by the plant hormone ethylene and is a member of the LAMMER family of protein kinases . Members of this family contain in their C-terminus a unique 'EHLAMMERI/VLGPLP' motif of unknown function, and are related to cyclin- and mitogen-activated protein (MAP)-dependent kinases . The animal members of this class play a role in differentiation . They phosphorylate and physically interact with serine/arginine-rich (SR) splicing factors in vivo to alter their activity and the splicing of target mRNAs . SR proteins have been recently described in plants . The capability of PK12 LAMMER kinase to bind and phosphorylate SR proteins was tested in vitro by kinase and binding assays . The tobacco PK12 phosphorylated both animal and plant SR proteins and specifically interacted with the plant splicing factor atSRp34/SR1 . In addition, by site-directed mutagenesis, the LAMMER motif was found to be required for PK12 kinase activity but was not necessary for substrate binding . Consistent with a role in phosphorylation of splicing factors, PK12 was found to localize to the nucleus when transiently over-expressed in suspension cells.

Eur J Biochem, 2000 Feb, 267(3), 737 - 45
Molecular cloning and characterization of calreticulin, a calcium-binding protein involved in the regeneration of rice cultured suspension cells; Li Z et al.; A full-length cDNA clone encoding a phosphoprotein (pp56) involved in the regeneration of rice (Oryza sativa L.)-cultured suspension cells was isolated by screening a rice cultured suspension cell cDNA library . The 1558-bp cDNA sequence contains an ORF encoding an acidic (pI 4.38) protein of 424 amino acids (47.9 kDa), sharing 70-93% and 50-53% homology with other plant and mammalian calreticulins, respectively . Sequence analysis of the cDNA clone revealed several significant conserved motifs, including a calreticulin family repeat motif in the central domain and two calreticulin family motifs in the N-domain, indicating that this gene is a rice calreticulin (CRO1) . The CRO1 gene in long-term rice cultured suspension cells shows constitutive expression in both suspension culture and regeneration media . In contrast, expression of the CRO1 gene in short-term rice cultured suspension cells, which possess regeneration potential, is increased dramatically when these cells are transferred to the regeneration medium . After approximately 2 weeks in the regeneration medium, the expression of the CRO1 gene reverts to constitutive levels . These results demonstrate the presence of calreticulin in rice cultured suspension cells and its developmental regulation during the regeneration of rice cultured suspension cells.

Planta, 1999 Nov, 210(1), 143 - 9
Characterization of two fungal-elicitor-induced rice cDNAs encoding functional homologues of the rab-specific GDP-dissociation inhibitor; Kim WY et al.; By using the mRNA differential display approach to isolate defense signaling genes active at the early stage of fungal infection two cDNA fragments with high sequence homology to rab-specific GDP-dissociation inhibitors (GDIs) were identified in rice (Oryza sativa L.) suspension cells . Using polymerase-chain-reaction products as probes, two full-length cDNA clones were isolated from a cDNA library of fungal-elicitor-treated rice, and designated as OsGDI1 and OsGDI2 . The deduced amino acid sequences of the isolated cDNAs exhibited substantial homology to Arabidopsis rab-GDIs . Northern analysis revealed that transcripts detected with the 3'-gene-specific DNA probes accumulated to high levels within 30 min after treatment with a fungal elicitor derived from Magnaporthe grisea . The functionality of the OsGDIs was demonstrated by their ability to rescue the Sec19 mutant of Saccharomyces cerevisiae which is defective in vesicle transport . The proteins, expressed in Escherchia coli, cross-reacted with a polyclonal antibody prepared against bovine rab-GDI . Like bovine rab-GDI, the OsGDI proteins efficiently dissociated rab3A from bovine synaptic membranes . Using the two-hybrid system, it was shown that the OsGDIs specifically interact with the small GTP-binding proteins belonging to the rab subfamily . The specific interaction was also demonstrated in vitro by glutathione S-transferase resin pull-down assay.

J Cell Sci, 2000 Jan, 113 ( Pt 1), 81 - 9
Plastid tubules of higher plants are tissue-specific and developmentally regulated; Kohler RH et al.; Green fluorescent stroma filled tubules (stromules) emanating from the plastid surface were observed in transgenic plants containing plastid-localized green fluorescent protein (GFP) . These transgenic tobacco plants were further investigated by epifluorescence and confocal laser scanning microscopy (CSLM) to identify developmental and/or cell type specific differences in the abundance and appearance of stromules and of plastids . Stromules are rarely seen on chlorophyll-containing plastids in cell types such as trichomes, guard cells or mesophyll cells of leaves . In contrast, they are abundant in tissues that contain chlorophyll-free plastids, such as petal and root . The morphology of plastids in roots and petals is highly dynamic, and plastids are often elongated and irregular . The shapes, size, and position of plastids vary in particular developmental zones of the root . Furthermore, suspension cells of tobacco exhibit stromules on virtually every plastid with two major forms of appearance . The majority of cells show a novel striking 'octopus- or millipede-like' structure with plastid bodies clustered around the nucleus and with long thin stromules of up to at least 40 (micro)m length stretching into distant areas of the cell . The remaining cells have plastid bodies distributed throughout the cell with short stromules . Photobleaching experiments indicated that GFP can flow through stromules and that the technique can be used to distinguish interconnected plastids from independent plastids.

Plant Physiol, 1999 Nov, 121(3), 793 - 803
In Organello and in Vivo Evidence of the Importance of the Regulatory Sulfhydryl/Disulfide System and Pyruvate for Alternative Oxidase Activity in Tobacco; Vanlerberghe GC et al.; After isolation of tobacco (Nicotiana tabacum) leaf mitochondria, alternative oxidase (AOX) is predominantly present as the disulfide-linked, less-active "oxidized" form . In an in organello assay, significant AOX activity was dependent upon both the reduction of the regulatory disulfide bond (such as occurs by dithiothreitol) and upon the presence of the activator pyruvate . However, AOX activity in these assays was substantially affected when mitochondria were isolated in the presence of pyruvate . First, pyruvate protects against the oxidation of the regulatory sulfhydryl during isolation, such that subsequent in organello AOX activity is not dependent upon dithiothreitol . Second, pyruvate stabilizes AOX activity, such that mitochondria kept in the presence of pyruvate have higher maximum rates of AOX activity than mitochondria kept for some time in the absence of pyruvate . The ability of pyruvate to protect against AOX oxidation was exploited to assess the in vivo status of the regulatory sulfhydryl/disulfide system . In both tobacco suspension cells and tobacco leaves with high levels of AOX protein, the protein is predominantly present as the "reduced" active form in vivo under a range of respiratory conditions . Experiments also indicate that, while the presence of reduced protein may be a necessary prerequisite for significant AOX activity, it is not sufficient for activity and other factors must also be critical.

Plant Mol Biol, 1999 Aug, 40(6), 935 - 44
Negative effect of the 5'-untranslated leader sequence on Ac transposon promoter expression; Scortecci KC et al.; Transposable elements are used in heterologous plant hosts to clone genes by insertional mutagenesis . The Activator (Ac) transposable element has been cloned from maize, and introduced into a variety of plants . However, differences in regulation and transposition frequency have been observed between different host plants . The cause of this variability is still unknown . To better understand the activity of the Ac element, we analyzed the Ac promoter region and its 5'-untranslated leader sequence (5' UTL) . Transient assays in tobacco NT1 suspension cells showed that the Ac promoter is a weak promoter and its activity was localized by deletion analyses . The data presented here indicate that the core of the Ac promoter is contained within 153 bp fragment upstream to transcription start sites . An important inhibitory effect (80%) due to the presence of the 5' UTL was found on the expression of LUC reporter gene . Here we demonstrate that the presence of the 5' UTL in the constructs reduces the expression driven by either strong or weak promoters.

Biochem Biophys Res Commun, 1999 Aug 11, 261(3), 798 - 801
Maize transposable element Ds is differentially spliced from primary transcripts in endosperm and suspension cells; Lal SK et al.; The process by which transposable elements are spliced from the host gene transcripts remains poorly understood . We previously reported that a maize transposable element Ds (dissociation) and a copy of its host site duplication are perfectly spliced from the shrunken-2 transcript in the endosperm . Here, we have monitored splicing of the Ds element and its flanking Sh2 sequence following transient expression in maize suspension cells . The pattern of Ds splicing in suspension cells differs dramatically from that in the endosperm . In contrast to splicing in the endosperm, Ds in suspension cells was completely spliced from the transcripts using multiple donor and acceptor splice sites outside the element . In addition, noncanonical splice sites were utilized in suspension cells . Our results indicate that this difference in splicing is due to the context of Ds placement in the construct and/or to tissue specific differences in splicing .

Fiziol Zh, 1999, 45(3), 92 - 7
{The effect of ultraviolet on the structural-functional characteristics of mouse thymocytes}; Paranich AV et al.; The changes of peculiarities thymocytes membranes after the UV actions on the suspension cells were investigated . The POL intensivity; content the carotene, vitamins A,E and its metabolites were determined . The state of membrane lipids and proteins was observed by fluorescent zondes . After 15 min of UV action form of the humoral activity products of POL was increased; the redox system of vitamin E was mobilized; the membranes proteins was opened; the neutral lipid fraction in membranes was decreased, and its polarity was increased . After 25 min of UV action was observed: the stimulation of the first and second steps of POL; decreased of AO activity; more opened of membranes proteins; more increased of polarity lipids and reform of lipofility of membranes . This changes lay in the base of activity and passivity of thymocytes by UV.

Plant J, 1999 Jun, 18(6), 577 - 87
Aquaporin Nt-TIPa can account for the high permeability of tobacco cell vacuolar membrane to small neutral solutes; Gerbeau P et al.; Members of the major intrinsic protein (MIP) family, described in plants as water-selective channels (aquaporins), can also transport small neutral solutes in other organisms . In the present work, we characterize the permeability of plant vacuolar membrane (tonoplast; TP) and plasma membrane (PM) to non-electrolytes and evaluate the contribution of MIP homologues to such transport . PM and TP vesicles were purified from tobacco suspension cells by free-flow electrophoresis, and membrane permeabilities for a wide range of neutral solutes including urea, polyols of different molecular size, and amino acids were investigated by stopped-flow spectrofluorimetry . For all solutes tested, TP vesicles were found to be more permeable than their PM counterparts, with for instance urea permeabilities from influx experiments of 74.9 +/- 9.6 x 10(-6) and 1.0 +/- 0.3 x 10(-6) cm sec-1, respectively . Glycerol and urea transport in TP vesicles exhibited features of a facilitated diffusion process . This and the high channel-mediated permeability of the same TP vesicles to water suggested a common role for MIP proteins in water and solute transport . A cDNA encoding a novel tonoplast intrinsic protein (TIP) homologue named Nicotiana tabacum TIPa (Nt-TIPa) was isolated from tobacco cells . Immunodetection of Nt-TIPa in purified membrane fractions confirmed that the protein is localized in the TP . Functional expression of Nt-TIPa in Xenopus oocytes showed this protein to be permeable to water and solutes such as urea and glycerol . These features could account for the transport selectivity profile determined in purified TP vesicles . These results support the idea that plant aquaporins have a dual function in water and solute transport . Because Nt-TIPa diverges in sequence from solute permeable aquaporins characterized in other organisms, its identification also provides a novel tool for investigating the molecular determinants of aquaporin transport selectivity.

Hokkaido Igaku Zasshi, 1999 Mar, 74(2), 157 - 66
Analysis of hematopoietic cell specific protein, M-DOCK; Nishihara H; Full length cDNA of M-DOCK was isolated at Kazusa DNA Institute and its sequence has been deposited as KIAA0209 . The cDNA sequence of M-DOCK contains a 5490 bp open reading frame that encodes a 1830-aa protein with a calculated molecular mass of 212 kDa . The amino acid sequence of M-DOCK has 62.3% identity with DOCK180, excluding the carboxyl terminal variable regions . DOCK180, which was originally identified as a major protein bound to Crk oncogene product, is an archetype of a family of proteins including Ced-5 of C . elegans and Mbc of D . melanogaster . DOCK180 is localized at focal adhesions and regulates cell morphology through the activation of Rac . Expression of DOCK180 is ubiquitous except in hematopoietic cells . Here, the author reports on the characterization of M-DOCK protein, which is closely related to DOCK180 but expressed only in the hematopoietic cells . We immunized rabbits with bacterially-expressed GST-M-DOCK protein, and obtained an anti-serum specific to M-DOCK . Tissue distribution of M-DOCK was examined by Northern blotting and Western blotting . M-DOCK was expressed abundantly in peripheral blood leukocytes . M-DOCK expression was detected also in the cell lines derived from T-cells, B-cells, and monocytes, but never in the adherent cells . Most of the lymphocytes and macrophages in human organs expressed M-DOCK diffusely in the cytoplasm, which was analyzed by immunohistochemistry using an anti-M-DOCK antibody . We previously reported that membrane-targeted form of DOCK180 induced spreading of NIH 3T3 cells . By contrast, overexpression of M-DOCK rounded up flat NRK cells . This suggests that M-DOCK may regulate cell detachment of adherent cells . These results demonstrate that M-DOCK is a new member of DOCK180-family proteins and regulates cell shape in suspension cells.

J Cell Sci, 1999 Jul, 112 ( Pt 13), 2223 - 32
Induction of apoptosis by overexpression of the DNA-binding and DNA-PK-activating protein C1D; Rothbarth K et al.; Apoptosis is induced in various tumor cell lines by vector-dependent overexpression of the conserved gene C1D that encodes a DNA-binding and DNA-PK-activating protein . C1D is physiologically expressed in 50 human tissues tested, which points to its basic cellular function . The expression of this gene must be tightly regulated because elevated levels of C1D protein, e.g . those induced by transient vector-dependent expression, result in apoptotic cell death . Cells transfected with C1D-expressing constructs show terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of DNA ends . Transfections with constructs in which C1D is expressed in fusion with the (enhanced) green fluorescent protein from A . victoria (EGFP) allow the transfected cells to be identified and the morphological changes induced to be traced . Starting from intense nuclear spots, green fluorescence reflecting C1D expression increases dramatically at 12-24 hours post-transfection . Expression of C1D-EGFP protein is accompanied by morphological changes typical of apoptotic cell death, e.g . cytoplasmic vacuolation, membrane blebbing and nuclear disintegration . Cell shrinkage and detachment from extracellular matrix are observed in monolayer cultures while suspension cells become progressively flattened . The facility to differentiate between transfected and non-transfected cells reveals that non-transfected cells co-cultured with transfected cells also show the morphological changes of apoptosis, which points to a bystander effect . C1D-dependent apoptosis is not induced in cells with non-functional p53 . Accordingly, C1D-induced apoptosis is discussed in relation to its potential to activate DNA-PK, which has been considered to act as an upstream activator of p53.

Plant Mol Biol, 1999 Mar, 39(5), 1063 - 71
A transcript encoding translation initiation factor eIF-5A is stored in unfertilized egg cells of maize; Dresselhaus T et al.; Differential screening of cDNA libraries of unfertilized egg cells and in vitro zygotes of maize resulted in the isolation of more than 50 different genes whose expression is up- or down-regulated after in vitro fertilization (IVF) . Among these genes, we identified a cDNA encoding the eukaryotic translation initiation factor eIF-5A . This highly conserved factor is thought to be necessary for selective mRNA stabilization and translation . It is also the only known protein that contains the unusual amino acid hypusine which is required for biological activity . High transcript amounts are stored in the egg cell, which is, in terms of metabolism, relatively inactive . Upon fertilization transcript amounts decrease, in contrast to metabolically inactive embryos in which the transcript cannot be detected and transcript levels increase upon germination . The expression pattern during the first embryonic cell cycle is also different from that observed during the somatic cell cycle: egg cells in the G0 phase contain high transcript levels, while arrested suspension cells contain few transcripts . In the somatic cell cycle, eif-5A is strongly induced during the G1 phase and transcripts are continuously degraded during the S, G2 and M phases until new induction during the G1 phase of the next cycle . eif-5A, a member of a small gene family in maize, is expressed in most maize tissues investigated . Based on our results, we suggest that the unfertilized egg cell of maize, although relatively inactive regarding its metabolism, is prepared for selective mRNA translation that is quickly triggered after fertilization . We also suggest that the regulation of eif-5A in the first embryonic cell cycle is different from the somatic cell cycle.

Plant J, 1999 Apr, 18(1), 13 - 22
Induction of RAB18 gene expression and activation of K+ outward rectifying channels depend on an extracellular perception of ABA in Arabidopsis thaliana suspension cells; Jeannette E et al.; Important progress has been made regarding the characterization of the ABA signalling components using genetic and molecular approaches (Leung and Giraudat, 1998) . However, we do not yet know the mechanism of ABA perception . Conflicting results concerning the site of ABA perception have been published . The prevailing view is that since ABA controls many responses, different sites of perception for ABA might exist . In order to establish the cellular localisation of the ABA receptors in Arabidopsis thaliana suspension cells, we developed two physiological tests based upon the capacity of impermeant ABA-BSA conjugate to mimic permeant free ABA effects . We show that purified ABA-BSA conjugate is able to trigger RAB18 gene expression and that this response is strictly due to the natural (+)-ABA enantiomer . The rate of RAB18 gene expression was independent of the level of ABA uptake by the cells . Using the voltage-clamp technique we show that ABA-BSA, similarly to ABA, evokes a membrane depolarization and activates time- and voltage-dependent outward rectifying currents (ORC) . We demonstrate that these ORC are due to a K+ efflux as assessed by tail currents and specific inhibition by both tetraethylammonium (TEA) and Ba2+ . These observations provide evidence in favour of an extracellular site for ABA perception.

Nucleic Acids Res, 1999 Apr 1, 27(7), 1709 - 18
Plant cell-directed control of virion sense gene expression in wheat dwarf virus; Gooding PS et al.; We have used particle bombardment (biolistics) to deliver replication-competent wheat dwarf virus (WDV)-based constructs, carrying reporter gene sequences fused to the virion sense promoter (Pv) or the CaMV 35S promoter, to suspension culture cells and immature zygotic embryos of wheat . While the replication of WDV double-stranded DNA forms (replicons) was equivalent between wheat suspension culture cells and embryos, GUS reporter gene activity was 20-40 times higher in the embryo cultures . Maximum expression of WDV replicons occurred in the embryonic axis tissue of wheat embryos but their expression in suspension cells was compromised, compared with transiently maintained input plasmid DNA containing the same sequences . From these studies, we propose that WDV replicons are subject to a host cell-controlled competency for virion sense transcription . The term competency is used to distinguish between the phenomenon described here and control of gene expression by specific transcription factors . Control of competency is independent of Pv, the replacement 35S promoter and of the complementary sense control of virion sense expression involving specific sequences in Pv . We propose that factors controlling the competency for replicon expression may be present in cells which, as well as maintaining high rates of DNA synthesis, are totipotent . Cell type control of active chromatin, methylation of specific sequences in WDV minichromosomes and/or interaction of virus-encoded proteins with specific host factors are considered as possible mechanisms.

J Cell Sci, 1999 Mar, 112 ( Pt 5), 695 - 706
Integrin and cytoskeletal regulation of growth factor signaling to the MAP kinase pathway; Aplin AE et al.; Integrin-mediated anchorage of NIH3T3 fibroblasts to the extracellular matrix component fibronectin permits efficient growth factor signaling to the p42 and p44 forms of mitogen-activated protein kinase (MAPK) . Since integrins bridge the extracellular matrix to focal adhesion sites and to the actin cytoskeleton, we analyzed the role of these integrin-associated structures in efficient growth factor activation of p42 and p44-MAPKs . Use of specific reagents that disrupt actin stress fiber and focal adhesion formation demonstrated that upon readhesion of NIH3T3 cells to fibronectin, cells that were poorly spread and lacked prominent focal adhesions but that formed cortical actin structures, efficiently signaled to p42 and p44-MAPKs upon EGF stimulation . In contrast, failure to form the cortical actin structures, despite attachment to fibronectin, precluded effective EGF signaling to p42 and p44-MAPKs . Actin cytoskeletal changes induced by expression of dominant-negative and constitutively active forms of Rho GTPases did not alter EGF activation of MAPK in adherent cells . However, active Cdc42, but not active Rac1 or RhoA, partially rescued EGF signaling to p44-MAPK in cells maintained in suspension . These data indicate that a limited degree of adhesion-mediated cytoskeletal organization and focal adhesion complex formation are required for efficient EGF activation of p42 and p44-MAPKs . Our studies exclude a major role for the GTPases RhoA and Rac1 in the formation of cytoskeletal structures relevant for signaling, but indicate that structures regulated by Cdc42 enhance the ability of suspension cells to activate MAPK in response to growth factors.

Leuk Res, 1999 Jan, 23(1), 13 - 8
Neuroleukin mediated differentiation induction of myelogenous leukemia cells; Chiao JW et al.; Leukemia cells enriched from peripheral blood of a patient with myelogenous leukemia were induced to differentiate with a purified T cell lymphokine neuroleukin . With sufficient neuroleukin concentrations, cells with macrophage-like morphology were identified among the developing adherent cells . After 2-5 days, approximately 38-50% of the suspension cells became macrophage-like and acquired CD21, alpha-naphthyl acetate reactivity and immune adherence capability . The amount of these nonproliferating cells increased along with cells containing fragmented DNA . Induction with insufficient neuroleukin quantity or with patient plasma alone developed few or no mature cells, indicating differentiation to mature cells is dose-dependent . The possibility of insufficient quantity of neuroleukin in regulation of patient plasma for differentiation was discussed.

Plant J, 1998 Nov, 16(3), 393 - 401
A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes; Kost B et al.; The C-terminus of mouse talin (amino acids 2345-2541) is responsible for all of the protein's f-actin binding capacity . Unlike full-length talin, the C-terminal f-actin binding domain is unable to nucleate actin polymerization . We have found that transient and stable expression of the talin actin-binding domain fused to the C-terminus of the green fluorescent protein (GFP-mTn) can visualize the actin cytoskeleton in different types of living plant cells without affecting cell morphology or function . Transiently expressed GFP-mTn co-localized with rhodamine-phalloidin in permeabilized tobacco BY-2 suspension cells, showing that the fusion protein can specifically label the plant actin cytoskeleton . Constitutive expression of GFP-mTn in transgenic Arabidopsis thaliana plants visualized actin filaments in all examined tissues with no apparent effects on plant morphology or development at any stage during the life cycle . This demonstrates that in a number of different cell types GFP-mTn can serve as a non-invasive marker for the actin cytoskeleton . Confocal imaging of GFP-mTn labeled actin filaments was employed to reveal novel information on the in vivo organization of the actin cytoskeleton in transiently transformed, normally elongating tobacco pollen tubes.

Eur J Immunol, 1998 Nov, 28(11), 3719 - 29
Low level of mixing of partner cells seen in extrathymic T cells in the liver and intestine of parabiotic mice: its biological implication; Suzuki S et al.; c-kit+ stem cells have recently been found in the liver and intestine of adult mice . We examined whether such stem cells give rise to extrathymic T cells in these organs in situ . To this end, we used parabiotic B6.Ly5.1 and B6.Ly5.2 mice, i.e . mice sharing the circulation . The origin of lymphocytes was identified by anti-Ly5.1 and anti-Ly5.2 monoclonal antibodies in conjunction with immunofluorescence assays . Lymphocytes in the blood, spleen, lymph nodes and liver had become a half-and-half mixture of Ly5.1+ and Ly5.2+ cells in both individuals by day 14 . However, this level of mixing decreased in extrathymic T cells in the liver (i.e . NK T cells) and intestine by day 14 and thereafter . The same was observed in T cells of the thymus . The data from immunohistochemical staining supported the results of immunofluorescence assays for suspension cells . The present results raise the possibility that extrathymic T cells in the liver and intestine may arise from their own pre-existing precursor cells, possibly from their own stem cells . Another important finding was that the composition pattern of lymphocyte subsets in one individual was quite similar to that in its partner at various sites . This result was interpreted to mean that only selected partner cells migrate to specific sites in the other partner individual.

Mol Biol Cell, 1998 Dec, 9(12), 3429 - 43
Extracellular calcium regulates HeLa cell morphology during adhesion to gelatin: role of translocation and phosphorylation of cytosolic phospholipase A2; Crawford JR et al.; Attachment of HeLa cells to gelatin induces the release of arachidonic acid (AA), which is essential for cell spreading . HeLa cells spreading in the presence of extracellular Ca2+ released more AA and formed more distinctive lamellipodia and filopodia than cells spreading in the absence of Ca2+ . Addition of exogenous AA to cells spreading in the absence of extracellular Ca2+ restored the formation of lamellipodia and filopodia . To investigate the role of cytosolic phospholipase A2 (cPLA2) in regulating the differential release of AA and subsequent formation of lamellipodia and filopodia during HeLa cell adhesion, cPLA2 phosphorylation and translocation from the cytosol to the membrane were evaluated . During HeLa cell attachment and spreading in the presence of Ca2+, all cPLA2 became phosphorylated within 2 min, which is the earliest time cell attachment could be measured . In the absence of extracellular Ca2+, the time for complete cPLA2 phosphorylation was lengthened to <4 min . Maximal translocation of cPLA2 from cytosol to membrane during adhesion of cells to gelatin was similar in the presence or absence of extracellular Ca2+ and remained membrane associated throughout the duration of cell spreading . The amount of total cellular cPLA2 translocated to the membrane in the presence of extracellular Ca2+ went from <20% for unspread cells to >95% for spread cells . In the absence of Ca2+ only 55-65% of the total cPLA2 was translocated to the membrane during cell spreading . The decrease in the amount translocated could account for the comparable decrease in the amount of AA released by cells during spreading without extracellular Ca2+ . Although translocation of cPLA2 from cytosol to membrane was Ca2+ dependent, phosphorylation of cPLA2 was attachment dependent and could occur both on the membrane and in the cytosol . To elucidate potential activators of cPLA2, the extracellular signal-related protein kinase 2 (ERK2) and protein kinase C (PKC) were investigated . ERK2 underwent a rapid phosphorylation upon early attachment followed by a dephosphorylation . Both rates were enhanced during cell spreading in the presence of extracellular Ca2+ . Treatment of cells with the ERK kinase inhibitor PD98059 completely inhibited the attachment-dependent ERK2 phosphorylation but did not inhibit cell spreading, cPLA2 phosphorylation, translocation, or AA release . Activation of PKC by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) induced and attachment-dependent phosphorylation of both cPLA2 and ERK2 in suspension cells . However, in cells treated with the PKC inhibitor Calphostin C before attachment, ERK2 phosphorylation was inhibited, whereas cPLA2 translocation and phosphorylation remained unaffected . In conclusion, although cPLA2-mediated release of AA during HeLa cell attachment to a gelatin substrate was essential for cell spreading, neither ERK2 nor PKC appeared to be responsible for the attachment-induced cPLA2 phosphorylation and the release of AA.

Plant J, 1998 Sep, 15(5), 685 - 95
The 3' untranslated region of a rice alpha-amylase gene mediates sugar-dependent abundance of mRNA; Chan MT et al.; A decrease in transcript stability is one of the important mechanisms that control the sugar repression of alpha-amylase gene expression in rice suspension cells . In this study, we investigated the function of the 3' untranslated region (3'UTR) of a rice alpha-amylase gene, alpha Amy3, in relation to sugar-dependent accumulation of mRNA . By examining the transient expression of chimeric genes in rice protoplasts, we were able to demonstrate that the alpha Amy3 3'UTR mediated the sugar-dependent repression of fused heterologous gene expression . The same kinetics of accumulation of alpha Amy3 mRNA and reporter mRNA carrying the alpha Amy3 3'UTR in response to glucose deprivation were observed, suggesting that the alpha Amy3 3'UTR is sufficient, and probably the major determinant for controlling the abundance of these transcripts . Functional analysis of two subdomains of alpha Amy3 3'UTR by insertion into a sugar-inducible chimeric gene confirmed their roles in sugar repressibility . The regulatory sequences in the alpha Amy3 3'UTR may act as potent determinants of mRNA stability in response to sugar availability . This finding has important implications for studying the regulatory mechanism of sugar repression in eukaryotes.

Gene, 1998 Aug 31, 216(2), 233 - 43
Genomic organization and promoter activity of the maize starch branching enzyme I gene; Kim KN et al.; Starch branching enzymes (SBE) which catalyse the formation of alpha-1,6-glucan linkages are of crucial importance for the quantity and quality of starch synthesized in plants . In maize (Zea mays L.), three SBE isoforms (SBEI, IIa and IIb) have been identified and shown to exhibit differential expression patterns . As a first step toward understanding the regulatory mechanisms controlling their expression, we isolated and sequenced a maize genomic DNA (-2190 to +5929) which contains the entire coding region of SBEI (Sbe1) as well as 5'-and 3'-flanking sequences . Using this clone, we established a complete genomic organization of the maize Sbe1 gene . The transcribed region consists of 14 exons and 13 introns, distributed over 5.7kb . A consensus TATA-box and a G-box containing a perfect palindromic sequence, CCACGTGG, were found in the 5'-flanking region . Genomic Southern blot analysis indicated that two Sbe1 genes with divergent 5'-flanking sequences exist in the maize genome, suggesting the possibility that they are differentially regulated . A chimeric construct containing the 5'-flanking region of Sbe1 (-2190 to +27) fused to the beta-glucuronidase gene (pKG101) showed promoter activity after it was introduced into maize endosperm suspension cells by particle bombardment . 1998 Elsevier Science B.V.

Plant J, 1998 Feb, 13(3), 317 - 29
Carrot DNA-methyltransferase is encoded by two classes of genes with differing patterns of expression; Bernacchia G et al.; In the present study, the isolation and characterization of two distinct cDNAs that code for carrot DNA (cytosine-5)-methyltransferase (DNA-METase) are reported . The screening of a cDNA library with a carrot genomic DNA fragment, previously obtained by PCR using degenerate primers, has led to the isolation of clones that belong to two distinct classes of genes (Met1 and Met2) which differ in sequence and size . Met1-5 and Met2-21 derived amino acid sequences are more than 85% identical for most of the polypeptide and completely diverge at the N-terminus . The larger size of the Met2-21 cDNA is due to the presence of nearly perfect fivefold repeat of a 171 bp sequence present only once in the Met1-5 cDNA . Northern and in situ hybridization analyses with young carrot plants and somatic embryos indicate that both genes are maximally expressed in proliferating cells (suspension cells, meristems and leaf primordia), but differ quantitatively and spatially in their mode of expression . Polyclonal antibodies were raised in rabbit using fusion proteins corresponding to the regulatory and catalytic regions of the most highly expressed gene (Met1-5) . In nuclear carrot extracts, both antibodies were found to recognize a band of about 200 kDa along with some additional bands of lower size . These results provide the first direct demonstration that DNA-METases of a higher eukaryote are encoded by a gene family.

Mol Cells, 1998 Jun 30, 8(3), 366 - 9
Role of jasmonic acid in biotransformation of (--)-isopiperitenone in suspension cell culture of Mentha piperita; Son JS et al.; The role of jasmonic acid was studied in biotransformation of (--)-isopiperitenone to (--)-7-hydroxyisopiperitenone using alpha suspension cells were treated with (--)-isopiperitenone, mRNA of a cytochrome P450 was induced in a similar time-course pattern as the biotransformation shown in a previous study (Park et al., 1997) . The induction of P450 mRNA and the biotransformation of (--)-isopiperitenone were increased by methyl jasmonate, but decreased by salicylhydroxamic acid, and inhibitor of jasmonic acid synthesis . These results suggest that the biotransformation involves the induction of P450 which is mediated by jasmonic acid as a signaling molecule.

Plant Physiol, 1998 Jun, 117(2), 491 - 9
Use of a new tetrazolium-based assay to study the production of superoxide radicals by tobacco cell cultures challenged with avirulent zoospores of phytophthora parasitica var nicotianae
Able AJ, Guest DI, Sutherland MW.
The relationship between the production of reactive oxygen species and the hypersensitive response (HR) of tobacco (Nicotiana tabacum L . ) toward an incompatible race of the Oomycete Phytophthora parasitica var nicotianae has been investigated . A new assay for superoxide radical (O2-) production based on reduction of the tetrazolium dye sodium,3'-(1-{phenylamino-carbonyl}-3, 4-tetrazolium)-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) has enabled the quantitative estimation of perhydroxyl/superoxide radical acid-base pair (HO2./O2-) production during the resistant response . Tobacco suspension cells were inoculated with zoospores from compatible or incompatible races of the pathogen . Subsequent HO2./O2- production was monitored by following the formation of XTT formazan . In the incompatible interaction only, HO2./O2- was produced in a minor burst between 0 and 2 h and then in a major burst between 8 and 10 h postinoculation . During this second burst, rates of XTT reduction equivalent to a radical flux of 9.9 x 10(-15) mol min-1 cell-1 were observed . The HO2./O2- scavengers O2- dismutase and Mn(III)desferal each inhibited dye reduction . An HR was observed in challenged, resistant cells immediately following the second burst of radical production . Both scavengers inhibited the HR when added prior to the occurrence of either radical burst, indicating that O2- production is a necessary precursor to the HR.

Biotechnology (N Y), 1995 May, 13(5), 481 - 5
Genetic transformation of banana and plantain (Musa spp.) via particle bombardment; Sagi L et al.; We have developed a simple protocol to allow the production of transgenic banana plants . Foreign genes were delivered into embryogenic suspension cells using accelerated particles coated with DNA . Bombardment parameters were optimized for a modified particle gun resulting in high levels of transient expression of the beta-glucuronidase gene in both banana and plantain cells . Bombarded banana cells were selected with hygromycin and regenerated into plants . Molecular and histochemical characterization of transformants revealed the stable integration of the transferred genes into the banana genome.

J Biol Chem, 1998 May 15, 273(20), 12606 - 11
Novel hydrogen peroxide metabolism in suspension cells of Scutellaria baicalensis Georgi; Morimoto S et al.; We identified a rapid and novel system to effectively metabolize a large amount of H2O2 in the suspension cells of Scutellaria baicalensis Georgi . In response to an elicitor, the cells immediately initiate the hydrolysis of baicalein 7-O-beta-D-glucuronide by beta-glucuronidase, and the released baicalein is then quickly oxidized to 6,7-dehydrobaicalein by peroxidases . Hydrogen peroxide is effectively consumed during the peroxidase reaction . The beta-glucuronidase inhibitor, saccharic acid 1,4-lactone, significantly reduced the H2O2-metabolizing ability of the Scutellaria cells, indicating that beta-glucuronidase, which does not catalyze the H2O2 degradation, plays an important role in the H2O2 metabolism . As H2O2-metabolizing enzymes, we purified two peroxidases using ammonium sulfate precipitation followed by sequential chromatography on CM-cellulose and hydroxylapatite . Both peroxidases show high H2O2-metabolizing activity using baicalein, whereas other endogenous flavones are not substrates of the peroxidase reaction . Therefore, baicalein predominantly contributed to H2O2 metabolism . Because beta-glucuronidase, cell wall peroxidases, and baicalein pre-exist in Scutellaria cells, their constitutive presence enables the cells to rapidly induce the H2O2-metabolizing system.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 7225 - 30
The tobacco wounding-activated mitogen-activated protein kinase is encoded by SIPK
Zhang S, Klessig DF.
It has been demonstrated that both salicylic acid and fungal elicitors activate a 48-kDa mitogen-activated protein kinase termed salicylic acid-induced protein kinase (SIPK) in tobacco suspension cells . Here, we show that infiltration of these agents into tobacco leaves also activates SIPK . Of particular interest, infiltration of water alone activated a kinase of the same size, possibly because of wounding and/or osmotic stresses . The kinetics of kinase activation, however, differ for these different treatments . Various mechanical stresses, including cutting and wounding by abrasion, also activated a 48-kDa kinase . By using an immune-complex kinase assay with antibodies specific for SIPK or wounding-induced protein kinase, we demonstrate that this wounding-activated 48-kDa kinase is SIPK, rather than wounding-induced protein kinase, as reported {Seo, S., Okamoto, M., Seto, H., Ishizuka, K., Sano, H . & Ohashi, Y . (1995) Science 270, 1988-1992} . Activation of SIPK after wounding was associated with tyrosine phosphorylation but not with increases in SIPK mRNA or protein levels . Thus, the same mitogen-activated protein kinase, SIPK, appears to facilitate signaling for two distinct pathways that lead to disease resistance responses and wounding responses.

Phytochemistry, 1998 May, 48(1), 93 - 102
Involvement of protein kinase and G proteins in the signal transduction of benzophenanthridine alkaloid biosynthesis; Mahady GB et al.; We have previously reported that elicitor-induced benzophenanthridine alkaloid biosynthesis in suspension-cell cultures of Sanguinaria canadensis L . (SCP-GM) is mediated by a signal transduction system that involves calcium and possibly protein kinase(s) . In this work, a number of exogenous agents were employed to further investigate the components of the signal transduction pathway involved in the induction of alkaloid biosynthesis by a fungal elicitor and abscisic acid (ABA) . SCP-GM suspension-cells were treated with compounds that modify protein kinase activity, including phorbol esters, and 1-oleoyl-2-acetyl-rac-glycerol (OAG), a synthetic diacylglycerol analogue . Phorbol-12-myristate-13-acetate induced alkaloid accumulation by as much as 65-fold over control values, while the negative control, phorbol-13-monoacetate, had no effect . OAG also increased alkaloid production by approximately 25-fold as compared to controls . Likewise, pretreatment of the suspension-cell cultures with H-7 or staurosporine, significantly suppressed ABA- or fungal-induction of benzophenanthridine alkaloid biosynthesis . Modulators of GTP-binding protein activity were also active in this system . Treatment of the suspension-cells with cholera toxin (CHX) induced alkaloid accumulation by 25-fold, which increased to 34-fold when CHX was combined with a fungal elicitor derived from Penicillium expansum (PE), and 32-fold when CHX was combined with ABA . Treatment of SCP-GM cells with CHX also enhanced the activities of two N-methyltransferases in the benzophenanthridine biosynthetic pathway namely, tetrahydroberberine-N-methyltransferase and tetrahydrocoptisine-N-methyltransferase, by six and seven fold, respectively . Furthermore, benzophenanthridine alkaloid biosynthesis was induced by treating the suspension-cells with the G-protein activators, mastoparan, mas-7 or melittin, while the inactive homologue, mas-17, did not . Suppression of alkaloid accumulation occurred when the suspension-cells were treated with GDP beta S or pertussis toxin prior to treatment of the SCP-GM cells with either PE or ABA . The results support the hypothesis that one or more protein kinases, and putative G proteins are involved in the signal transduction pathway that mediates ABA and fungal-induced benzophenanthridine alkaloid biosynthesis.

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