Suspension cells

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6543 - 7
The 3' untranslated region of a rice alpha-amylase gene functions as a sugar-dependent mRNA stability determinant; Chan MT et al.; In plants, sugar feedback regulation provides a mechanism for control of carbohydrate allocation and utilization among tissues and organs . The sugar repression of alpha-amylase gene expression in rice provides an ideal model for studying the mechanism of sugar feedback regulation . We have shown previously that sugar repression of alpha-amylase gene expression in rice suspension cells involves control of both transcription rate and mRNA stability . The alpha-amylase mRNA is significantly more stable in sucrose-starved cells than in sucrose-provided cells . To elucidate the mechanism of sugar-dependent mRNA turnover, we have examined the effect of alphaAmy3 3' untranslated region (UTR) on mRNA stability by functional analyses in transformed rice suspension cells . We found that the entire alphaAmy3 3' UTR and two of its subdomains can independently mediate sugar-dependent repression of reporter mRNA accumulation . Analysis of reporter mRNA half-lives demonstrated that the entire alphaAmy3 3' UTR and the two subdomains each functioned as a sugar-dependent destabilizing determinant in the turnover of mRNA . Nuclear run-on transcription analysis further confirmed that the alphaAmy3 3' UTR and the two subdomains did not affect the transcription rate of promoter . The identification of sequence elements in the alpha-amylase mRNA that dictate the differential stability has very important implications for the study of sugar-dependent mRNA decay mechanisms.

J Biol Chem, 1998 Apr 24, 273(17), 10120 - 31
Sugar response sequence in the promoter of a rice alpha-amylase gene serves as a transcriptional enhancer; Lu CA et al.; Expression of alpha-amylase genes in both rice suspension cells and germinating embryos is repressed by sugars and the mechanism involves transcriptional regulation . The promoter of a rice alpha-amylase gene alphaAmy3 was analyzed by both loss- and gain-of-function studies and the major sugar response sequence (SRS) was located between 186 and 82 base pairs upstream of the transcription start site . The SRS conferred sugar responsiveness to a minimal promoter in an orientation-independent manner . It also converted a sugar-insensitive rice actin gene promoter into a sugar-sensitive promoter in a dose-dependent manner . Linker-scan mutation studies identified three essential motifs: the GC box, the G box, and the TATCCA element, within the SRS . Sequences containing either the GC box plus G box or the TATCCA element each mediated sugar response, however, they acted synergistically to give a high level glucose starvation-induced expression . Nuclear proteins from rice suspension cells binding to the TATCCA element in a sequence-specific and sugar-dependent manner were identified . The TATCCA element is also an important component of the gibberellin response complex of the alpha-amylase genes in germinating cereal grains, suggesting that the regulation of alpha-amylase gene expression by sugar and hormone signals may share common regulatory machinery.

Plant Physiol, 1998 May, 117(1), 43 - 53
Expression pattern of the carrot EP3 endochitinase genes in suspension cultures and in developing seeds
van Hengel AJ, Guzzo F, van Kammen A, de Vries SC.
Carrot (Daucus carota) extracellular protein 3 (EP3) class IV endochitinases were previously identified based on their ability to rescue somatic embryos of the temperature-sensitive cell line ts11 . Whole-mount in situ hybridization revealed that a subset of the morphologically distinguishable cell types in embryogenic and nonembryogenic suspension cultures, including ts11, express EP3 genes . No expression was found in somatic embryos . In carrot plants EP3 genes are expressed in the inner integumentary cells of young fruits and in a specific subset of cells located in the middle of the endosperm of mature seeds . No expression was found in zygotic embryos . These results support the hypothesis that the EP3 endochitinase has a "nursing" function during zygotic embryogenesis and that this function can be mimicked by suspension cells during somatic embryogenesis.

Plant Cell Physiol, 1998 Mar, 39(3), 275 - 84
Cloning and expression of the soybean chlH gene encoding a subunit of Mg-chelatase and localization of the Mg2+ concentration-dependent ChlH protein within the chloroplast; Nakayama M et al.; From the soybean cDNA library, we isolated and analyzed the chlH gene encoding a subunit of Mg-chelatase . The subunit was a polypeptide of 1,383 amino acids with a molecular mass of 153,491 Da, which shared 90% identity with the olive gene from Antirrhinum majus . The regulation of the expression of chlH was investigated in photomix-otrophic soybean suspension cells (SB-P) . The expression was light-inducible, and the induction was more rapid than those of chlI and cab2 . Furthermore, the levels of the transcripts and products of chlH appeared to be regulated by a circadian oscillation . The subchloroplastic localization of ChlH was investigated by immunoblot analyses with antiserum against recombinant ChlH . Depending on the concentration of Mg2+ in the lysis buffer, the localization of ChlH protein migrated between the stroma and the envelope membrane; ChlH was localized on the envelope membrane, a major site of chlorophyll biosynthesis, when the Mg2+ concentration of the lysis buffer was high (above 5 mM) . These results indicated that the activity of Mg-chelatase was regulated by modulation of the expression and subchloroplastic localization of ChlH protein.

Plant Cell, 1998 Mar, 10(3), 435 - 50
Activation of the tobacco SIP kinase by both a cell wall-derived carbohydrate elicitor and purified proteinaceous elicitins from Phytophthora spp; Zhang S et al.; Two purified proteinaceous fungal elicitors, parasiticein (an alpha elicitin) and cryptogein (a beta elicitin), as well as a fungal cell wall-derived carbohydrate elicitor all rapidly activated a 48-kD kinase in tobacco suspension cells . The maximum activation of this kinase paralleled or preceded medium alkalization and activation of the defense gene phenylalanine ammonia-lyase (PAL) . In addition, the two elicitins, which also induced hypersensitive cell death, activated a 44- and a 40-kD kinase with delayed kinetics . By contrast, the cell wall-derived elicitor only weakly activated the 44-kD kinase and failed to activate the 40-kD kinase . The size and substrate preference of the 48-kD kinase are reminiscent of the recently purified and cloned salicylic acid-induced protein (SIP) kinase, which is a member of the mitogen-activated protein kinase family . Antibodies raised against a peptide corresponding to the unique N terminus of SIP kinase immunoreacted with the 48-kD kinase activated by all three elicitors from Phytophthora spp . In addition, the cell wall elicitor and the salicylic acid-activated 48-kD kinase copurified through several chromatography steps and comigrated on two-dimensional gels . Based on these results, all three fungal elicitors appear to activate the SIP kinase . In addition, inhibition of SIP kinase activation by kinase inhibitors correlated with the suppression of cell wall elicitor-induced medium alkalization and PAL gene activation, suggesting a regulatory function for the SIP kinase in these defense responses.

Plant Cell, 1998 Jan, 10(1), 51 - 62
AtKUP1: an Arabidopsis gene encoding high-affinity potassium transport activity; Kim EJ et al.; Because plants grow under many different types of soil and environmental conditions, we investigated the hypothesis that multiple pathways for K+ uptake exist in plants . We have identified a new family of potassium transporters from Arabidopsis by searching for homologous sequences among the expressed sequence tags of the GenBank database . The deduced amino acid sequences of AtKUP (for Arabidopsis thaliana K+ uptake transporter) cDNAs are highly homologous to the non-plant Kup and HAK1 potassium transporters from Escherichia coli and Schwanniomyces occidentalis, respectively . Interestingly, AtKUP1 and AtKUP2 are able to complement the potassium transport deficiency of an E . coli triple mutant . In addition, transgenic Arabidopsis suspension cells overexpressing AtKUP1 showed increased Rb+ uptake at micromolar concentrations with an apparent K(m) of approximately 22 microM, indicating that AtKUP1 encodes a high-affinity potassium uptake activity in vivo . A small, low-affinity Rb+ uptake component was also detected in AtKUP1-expressing cells . RNA gel blot analysis showed that the various members of the AtKUP family have distinct patterns of expression, with AtKUP3 transcript levels being strongly induced by K+ starvation . It is proposed that plants contain multiple potassium transporters for high-affinity uptake and that the AtKUP family may provide important components of high- and low-affinity K+ nutrition and uptake into various plant cell types.

Biol Pharm Bull, 1998 Feb, 21(2), 163 - 6
The involvement of Ca2+-dependent protein kinase in the regeneration of rice cultured suspension cells; Karibe H et al.; Short-term cultured cells of rice (Oryza sativa) were found to be capable of regeneration, in contrast to those obtained from long-term cultures . For clarification of the mechanism of regeneration, it was first necessary to distinguish protein kinase activity in long-term and short-term cultured cells; this activity was found greater in the former than latter . The activity was dependent on calcium, not phospholipid, phorbol ester or calmodulin . The apparent Mr of both Ca2+-dependent protein kinases was 32 kDa according to gel phosphorylation . Phosphoserine was identified in serine residues in phosphorylated histone III-S by phosphoamino acid analysis . A Ca2+-dependent protein kinase having a relative Mr of 32 kDa is thus shown to be possibly essential to regeneration in rice cultured cells.

J Virol, 1998 Mar, 72(3), 2323 - 34
Anchorage-independent transcription of the cyclin A gene induced by the E7 oncoprotein of human papillomavirus type 16; Schulze A et al.; To develop an experimental model for E7-mediated anchorage-independent growth, we studied the ability of E7-expressing NIH 3T3 subclones to enter S phase when they were cultured in suspension . We found that expression of E7 prevents the inhibition of cyclin E-associated kinase and also triggers activation of cyclin A gene expression in suspension cells . A point mutation in the amino terminus of E7 prevented E7-driven rescue of cyclin E-associated kinase activity in suspension cells; however, cells with this mutation retained some ability to activate cyclin A gene expression and promote S-phase entry . Activation of cyclin A gene expression by E7 was correlated with an increased binding of free E2F to a regulatory element in the cyclin A promoter which mediates both repression of cyclin A upon loss of adhesion and its reactivation by E7 . Surprisingly, expression of E7 led to a nuclear accumulation of one species of free E2F, namely, an E2F-4-DP-1 heterodimer, that is exclusively cytoplasmic in the absence of E7 . Taken together, the data reported here indicate that several different E7-dependent changes of cellular-growth-regulating pathways can cooperate to allow adhesion-independent entry into S phase.

Plant Mol Biol, 1997 Nov, 35(5), 539 - 50
Isolation and characterization of an Arabidopsis biotin carboxylase gene and its promoter; Bao X et al.; In the plastids of most plants, acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is a multisubunit complex consisting of biotin carboxylase (BC), biotin-carboxyl carrier protien (BCCP), and carboxytransferase (alpha-CT, beta-CT) subunits . To better understand the regulation of this enzyme, we have isolated and sequenced a BC genomic clone from Arabidopsis and partially characterized its promoter . Fifteen introns were identified . The deduced amino acid sequence of the mature BC protein is highly conserved between Arabidopsis and tobacco (92.6% identity) . BC expression was evaluated using northern blots and BC/GUS fusion constructs in transgenic Arabidopsis . GUS activity in the BC/GUS transgenics as well as transcript level of the native gene were both found to be higher in silique and flower than in root and leaf . Analysis of tobacco suspension cells transformed with truncated BC promoter/GUS gene fusions indicated the region from -140 to +147 contained necessary promoter elements which supported basal gene expression . A positive regulatory region was found to be located between -2100 and -140, whereas a negative element was possibly located in the first intron . In addition, several conserved regulatory elements were identified in the BC promoter . Surprisingly, although BC is a low-abundance protein, the expression of BC/GUS fusion constructs was similar to 35S/GUS constructs.

J Cell Physiol, 1997 Dec, 173(3), 361 - 70
Arachidonate initiated protein kinase C activation regulates HeLa cell spreading on a gelatin substrate by inducing F-actin formation and exocytotic upregulation of beta 1 integrin; Chun J et al.; HeLa cell spreading on a gelatin substrate requires the activation of protein kinase C (PKC), which occurs as a result of cell-attachment-induced activation of phospholipase A2 (PLA2) to produce arachidonic acid (AA) and metabolism of AA by lipoxyginase (LOX) . The present study examines how PKC activation affects the actin- and microtubule-based cytoskeletal machinery to facilitate HeLa cell spreading on gelatin . Cell spreading on gelatin is contingent on PKC induction of both actin polymerization and microtubule-facilitated exocytosis, which is based on the following observations . There is an increase in the relative content of filamentous (F)-actin during HeLa cell spreading, and treating HeLa cells with PKC-activating phorbol esters such as 12-O-tetradecanoyl phorbol 13-acetate (TPA) further increases the relative content of F-actin and the rate and extent to which the cells spread . Conversely, inhibition of PKC by calphostin C blocked both cell spreading and the increase of F-actin content . The increased F-actin content induced by PKC activators also was observed in suspension cells treated with TPA, and the kinetics of F-actin were similar to that for PKC activation . In addition, PKC epsilon, which is the PKC isoform most involved in regulating HeLa cell spreading in response to AA production, is more rapidly translocated to the membrane in response to TPA treatment than is the increase in F-actin . Blocking the activities of either PLA2 or LOX inhibited F-actin formation and cell spreading, both of which were reversed by TPA treatment . This result is consistent with AA and a LOX metabolite of AA as being upstream second messengers of activation of PKC and its regulation of F-actin formation and cell spreading . PKC appears to activate actin polymerization in the entire body of the cell and not just in the region of cell-substrate adhesion because activated PKC was associated not only with the basolateral plasma membrane domain contacting the culture dish but also with the apical plasma membrane domain exposed to the culture medium and with an intracellular membrane fraction . In addition to the facilitation of F-actin formation, activation of PKC induces the exocytotic upregulation of beta 1 integrins from an intracellular domain to the cell surface, possibly in a microtubule-dependent manner because the upregulation is inhibited by Nocodazole . The results support the concept that cell-attachment-induced AA production and its metabolism by LOX results in the activation of PKC, which has a dual role in regulating the cytoskeletal machinery during HeLa cell spreading . One is through the formation of F-actin that induces the structural reorganization of the cells from round to spread, and the other is the exocytotic upregulation of collagen receptors to the cell surface to enhance cell spreading.

Photochem Photobiol, 1997 Oct, 66(4), 464 - 70
Action spectrum for induction of promoter activity of phenylalanine ammonia-lyase gene by UV in carrot suspension cells; Takeda J et al.; The full-length promoter (-2335) of the carrot (Daucus carota) phenylalanine ammonia-lyase gene (gDcPAL1) fused to the luciferase reporter gene was transiently transformed to carrot protoplasts by electroporation, and the promoter activity induced by monochromatic UV light of various wavelengths was examined . The action spectrum constructed from the fluence-response curves showed a single peak at around 280 nm, suggesting that the activation of the gDcPAL1 promoter is categorizable as one of the UVB light responses . The same assay system was applied to variously truncated gDcPAL1 promoters and to CaMV35S promoter fusion with various parts 5'-upstream of the gDcPAL1 promoter . The region from -396 to -190 (relative to the transcription start site) fused to the CaMV35S core (-90) promoter showed a 280 nm-dominant response . However, gDcPAL1 promoters truncated above -570 and -396, although they contain the region between -396 and -190, did not show such a typical UVB response, i.e . they responded to 260 nm light as much as to 280 nm light . The promoter truncated to below -190 also responded to 260 nm light as much as to 280 nm light . Therefore we assumed that the gDcPAL1 promoter is composed of three functionally different parts: the upstream above -570 (modulator), the region from -396 to -190 (UVB responsive) and the down-stream below -190 (UVB and C responsive) . The overall UVB response of the gDcPAL1 full-length promoter is explained as the result of interaction of these three components.

Leukemia, 1997 Sep, 11(9), 1453 - 8
Mechanical agitation induces gene expression of NOR-1 and its closely related orphan nuclear receptors in leukemic cell lines; Bandoh S et al.; NOR-1, NGFI-B and Nurr1 are closely related transcription factors that constitute a distinct subfamily within the nuclear receptor superfamily . Genes for these proteins are immediate-early genes, and are inducible in diverse cell types by various stimuli . In the present study, we investigated the effect of mechanical agitation on the gene expression of these transcription factors in cultured suspension cells by the quantitative reverse transcription-polymerase chain reaction . We found that mechanical agitation transiently induced NOR-1, NGFI-B and Nurr1 mRNAs in several leukemic cell lines in a dose-dependent manner . This induction was most pronounced in the HL-60 promyelocytic leukemia cell line, but also occurred to a lesser extent in other cell lines including KG-1, THP-1 and U937 cells . The induction was attenuated by serum or albumin, which are shear stress protectants for suspension culture cells . These reagents did not suppress forskolin-induced NOR-1 gene expression . These findings suggest the involvement of fluid shear stress in agitation-induced immediate-early gene expression . Since even moderate agitation could cause the induction, investigators should be cautious when evaluating the expression of immediate-early genes in some leukemic cell lines.

Plant J, 1997 Aug, 12(2), 313 - 22
Identification of the peroxisomal targeting signal for cottonseed catalase; Mullen RT et al.; Catalase is a ubiquitous peroxisomal matrix enzyme, yet the molecular targeting signal(s) for sorting it in plant cells has not been defined . The most common peroxisome targeting signal (PTS) is a C-terminal tripeptide composed of a conserved SKL motif (type 1 PTS) . The PTS for cottonseed catalase (Ccat) was elucidated in this study from immunofluorescence microscopic analyses of tobacco BY-2 suspension cells serving as an in vivo import system . To distinguish biolistically introduced Ccat from endogenous tobacco catalase, Ccat was hemagglutinin (HA)epitope-tagged at its N-terminus . Bombardment with HA-Ccat resulted in the import of Ccat into glyoxysomes, the specialized type of peroxisome in BY-2 cells . The C-terminal tripeptide of Ccat, PSI, is necessary for import . Evidence for this were mislocalizations to the cytosol of PSI-truncated Ccat and AGV-substituted (for PSI) Ccat . PSI-COOH, however, was not sufficient to re-route chloramphenicol acetyltransferase (CAT) from the cytosol to glyoxysomes, whereas the Ccat tetrapeptide RPSI-COOH was sufficient . Surprisingly, substitution of K (common at the fourth position in other plant catalases) for the R (CAT-KPSI) decreased import efficiency . However, substitution of K did not affect import, when additional upstream residues in Ccat were included (e.g . CAT-NVKPSI) . Other evidence for the importance of upstream residues comprised abolishment of Ccat import due to substitutions with non-conserved residues (e.g . -AGVNVRPSI for -SRLNVRPSI) . These data indicate that Ccat is sorted to plant peroxisomes by a degenerate type 1 PTS (PSI-COOH) whose residues are functionally dependent on a strict context of adjacent C-terminal amino acid residues.

J Cell Sci, 1997 Aug, 110 ( Pt 16), 1947 - 56
Tetracycline regulated expression of vimentin in fibroblasts derived from vimentin null mice; Holwell TA et al.; Fibroblast cell lines were derived from vim-/- mice that express a mouse vimentin transgene in a tetracycline regulatable manner . Vimentin null mouse primary embryo fibroblasts were transformed with SV-40 early genes and vim- cell lines were isolated . A vim- cell line was then serially transfected with an expression plasmid encoding the tetracycline regulatable transactivator (tTA) and a mouse vimentin cDNA expression plasmid under the regulation of Escherichia coli tet operator and minimal CMV promoter sequences . Two stable cell lines were obtained that contained little or no vimentin in the presence of low concentrations of tetracycline but rapidly expressed abundant vimentin filaments after removal of tetracycline . The vimentin content of one cell line was similar to that of control vim+/+ fibroblasts . The level of transgene expression could be regulated by the concentration of tetracycline in a dose dependent fashion . Induction of vimentin expression in these cells did not observably affect cell growth, the distribution of microfilaments or microtubules, or the shape of the nucleus . Enucleation studies indicated that while disassembly of microfilaments significantly increased the sensitivity of the cells to enucleation, the presence or absence of vimentin had no detectable effect on the degree of enucleation with increasing sedimentation force . Monolayer wounding experiments demonstrated that vimentin expression did not alter the mobility of polarized cells at the edge of the wound . Experiments to more directly test the effect of vimentin expression on the capacity of these fibroblasts to survive mechanical trauma indicated that vimentin expression had no obvious effect on the survival of suspension cells subjected to nitrogen cavitation or the fraction of cells that survived the mechanical scraping of monolayer culture . These studies indicate that vimentin expression in a single population of cells does not have an obvious effect on cytoplasmic organization and provides a useful system to study the effects of IFs on the capacity of individual cells to resist mechanical injury.

Placenta, 1997 Sep, 18(7), 577 - 85
In vitro cultured human term cytotrophoblast: a model for normal primary epithelial cells demonstrating a spontaneous differentiation programme that requires EGF for extensive development of syncytium; Morrish DW et al.; Normal human term cytotrophoblast cells prepared by trypsin-DNAse I digestion with and without secondary immunological purification with CD9 antibodies were investigated for the expression of morphological and genetic markers of proliferation and differentiation . After 24 h of culture, the cell preparations demonstrated spontaneous formation of microvilli and formation of small syncytial units as assessed by desmoplakin staining and FITC-dextran microinjection . EGF was required for mature syncytial formation . Compared to log-phase proliferating HeLa cells, uptake of {3H}thymidine incorporation was low and quickly decreased to negligible levels . Expression of the proto-oncogenes c-myc, c-fos, and c-jun and histone 2A decreased rapidly in the first 24 h of culture in both cell preparations, followed by an increase in expression of c-fos and junB over the next 3 days of culture . Proto-oncogene changes were similar in attached and suspension cells . Spontaneous increases in alpha hCG, pregnancy-specific beta(1)-glycoprotein and 3 beta-hydroxysteroid dehydrogenase (3 beta OHSD) occurred within 1 day in both cell preparations . EGF receptor blocking antibodies did not inhibit minor degrees of spontaneous syncytial formation nor inhibit spontaneous expression of alpha hCG or 3 beta OHSD mRNA, but did prevent extensive synctialization induced by EGF . The results demonstrate that term cytotrophoblast cells even in serum-free conditions or suspension culture rapidly commit to a non-proliferative differentiation program in culture which includes limited syncytialization and marked hormone mRNA expression . However, EGF is required for extensive syncytial development.

FEBS Lett, 1997 Aug 11, 413(1), 181 - 4
Isolation and characterization of replication protein A (RP-A) from tobacco cells; Garcia-Maya MM et al.; Replication protein A (RP-A) was isolated from tobacco suspension cells and purified to near homogeneity by a procedure involving isolation of protoplasts, preparation of nuclei, nuclear lysis, binding to a column of single-stranded (ss) DNA cellulose and elution at different salt concentrations . The purified protein contained three subunits with molecular masses of 70, 34 and 14 kDa, and was free from nuclease activity . Tobacco RP-A had a high affinity for ssDNA . Binding competition experiments indicated only a weak affinity for double-stranded DNA and no detectable affinity for ssRNA . Photochemical cross-linking experiments indicated that the 70 kDa subunit has the ssDNA-binding activity . Tobacco RP-A was able to stimulate the activity of a tobacco alpha-like DNA polymerase about 4-fold . This is the first isolation of RP-A from a plant and the procedure may be generally applicable to other plant species.

Plant Cell, 1997 May, 9(5), 809 - 24
Salicylic acid activates a 48-kD MAP kinase in tobacco; Zhang S et al.; The involvement of phosphorylation/dephosphorylation in the salicylic acid (SA) signal transduction pathway leading to pathogenesis-related gene induction has previously been demonstrated using kinase and phosphatase inhibitors . Here, we show that in tobacco suspension cells, SA induced a rapid and transient activation of a 48-kD kinase that uses myelin basic protein as a substrate . This kinase is called the p48 SIP kinase (for SA-Induced Protein kinase) . Biologically active analogs of SA, which induce pathogenesis-related genes and enhanced resistance, also activated this kinase, whereas inactive analogs did not . Phosphorylation of a tyrosine residue(s) in the SIP kinase was associated with its activation . The SIP kinase was purified to homogeneity from SA-treated tobacco suspension culture cells . The purified SIP kinase is strongly phosphorylated on a tyrosine residue(s), and treatment with either protein tyrosine or serine/threonine phosphatases abolished its activity . Using primers corresponding to the sequences of internal tryptic peptides, we cloned the SIP kinase gene . Analysis of the SIP kinase sequence indicates that it belongs to the MAP kinase family and that it is distinct from the other plant MAP kinases previously implicated in stress responses, suggesting that different members of the MAP kinase family are activated by different stresses.

Biologicals, 1997 Mar, 25(1), 65 - 73
Phenotypic features of BHK-21 cells used for production of foot-and-mouth disease vaccine; Amadori M et al.; BHK-21 c13 monolayer and suspension cells were investigated with regard to some phenotypic features which could bear on the quality of foot-and-mouth disease virus (FMDV) antigen produced in them . Despite good viability, suspension cells differed from monolayer cells in fundamental features of susceptibility to FMDV . Most important, FMD virus particles grown in suspension cells at high passage levels were shown to be largely degraded following inactivation with an aziridine compound . Suspension cells were characterised by a downregulation in the surface expression of both alpha 5 and alpha V integrin chains . According to the results of binding assays, both integrins could act as FMDV receptors on BHK-21 c13 cells . Reduced surface expression of integrins was correlated with disappearance of actin stress fibres, which would play a role in regular encapsidation of viral RNA and hence in stability of virus particles . With regard to FMD vaccine production, practical suggestions are put forward to evaluate the quality of BHK-21 c13 cells and FMDV Ag, which must prove stable during downstream processing.

Plant J, 1997 Mar, 11(3), 613 - 21
The green fluorescent protein as a marker to visualize plant mitochondria in vivo; Kohler RH et al.; To determine how to utilize the green fluorescent protein (GFP) as a marker for subcellular localization and as a label for plant mitochondria in vivo, transgenic suspension cells and tobacco plants expressing GFP with and without a mitochondrial localization signal were generated . The first GFP form used, GFP1, is easily observable in cells with low autofluorescence, such as suspension cells or trichomes, but masked in green tissue . For the visualization of GFP in cells and tissues with high autofluorescence, such as leaf, the use of a very strong promoter (35S35SAMV), a highly expressed modified mGFP4 coding region and a brighter mutant form of GFP (S65T) was necessary . Confocal or two-photon laser scanning microscopy reveal a distinct subcellular localization of the fluorescence in cells expressing GFP or coxIVGFP . In cells expressing untargeted GFP, fluorescence accumulates in the nucleoplasm but is also distributed throughout the cytoplasm . It is excluded from vacuoles, nucleoli and from round bodies that are likely to be leucoplasts . In contrast, fluorescence is localized specifically to mitochondria in cells expressing coxIVGFP fusion protein as shown by co-localization with a mitochondrial-specific dye . This permits the direct observation of mitochondria and mitochondrial movements in living plant cells and tissues throughout plant development . Three-dimensional reconstruction of individual cells can give additional information about the distribution and numbers of mitochondria.

Plant Physiol, 1997 Mar, 113(3), 853 - 62
Sequences necessary for nitrate-dependent transcription of Arabidopsis nitrate reductase genes; Hwang CF et al.; Nitrate increases the transcription of the two Arabidopsis thaliana nitrate reductase genes . We demonstrated previously that 238 and 330 bp of the 5' flanking regions, designated as NP1 and NP2, of the two nitrate reductase genes NR1 and NR2, respectively, are sufficient for nitrate-dependent transcription (Y . Lin, C.-F . Hwang, J.B . Brown, C.-L . Cheng {1994} Plant Physiol 106: 477-484) . Here we identify the cis-acting elements of NP1 and NP2 that are necessary for nitrate-dependent transcription by linker-scanning (LS) analysis . In transgenic plants one LS mutant of NP1 and two LS mutants of NP2 exhibited significantly lower nitrate-induced reporter gene chloramphenicol acetyltransferase activity . To distinguish which of these three mutants lost nitrate inducibility, competitive reverse-transcriptase polymerase chain reaction was used to measure the chloramphenicol acetyltransferase mRNA levels before and after nitrate induction . The single LS mutant in NP1 lost its response to nitrate, whereas the two LS mutants in NP2 partially lost their response to nitrate . A 12-bp sequence is conserved between the NP1 site and the two NP2 sites . This sequence motif is also conserved in the 5' flanking regions of other nitrate-inducible plant genes . Gel mobility shift experiments indicate that these three regions bind to similar proteins . The binding is constitutive with respect to nitrate treatment and was observed in both nonphotosynthetic suspension cells and green leaves.

Plant J, 1997 Feb, 11(2), 263 - 76
Transcriptional repression by Oshox1, a novel homeodomain leucine zipper protein from rice; Meijer AH et al.; This paper describes the characterization of Oshox1, a cDNA clone from rice encoding a member of the homeodomain-leucine zipper (HD-Zip) class of putative transcription factors . Oshox1 maps to chromosome 10 and belongs to a family of related rice genes . Two-hybrid assays showed that Oshox1 protein can homodimerize, but can also form heterodimers with an Arabidopsis HD-Zip protein . This suggests that protein-protein interactions may also occur between different HD-Zip proteins in rice, which would provide enormous versatility for generating specific gene-control mechanisms . Oshox1 mRNA could be detected in various rice tissues at different developmental stages, with highest levels in embryos, shoots of seedlings, and leaves of mature plants . Transgenic expression of Oshox1 in Arabidopsis retarded growth and affected leaf size and shape, indicative of a role as developmental regulator . In vitro and in vivo DNA-binding studies revealed that Oshox1 interacts with the pseudopalindromic sequence CAAT(C/G)ATTG, confirming that the protein represents a transcription factor . Oshox1 was found to repress reporter gene activity in rice suspension cells, most likely by a mechanism of active transcriptional repression . Repression was strictly dependent on the presence of upstream Oshox1 binding sites in the reporter gene constructs and a function of the N-terminal region of Oshox1, preceding the homeodomain.

Plant J, 1997 Feb, 11(2), 181 - 90
Differential expression of a CAK (cdc2-activating kinase)-like protein kinase, cyclins and cdc2 genes from rice during the cell cycle and in response to gibberellin; Sauter M; Progress through the eukaryotic cell cycle is regulated by cyclin-dependent cdc2 protein kinases . In rice (Oryza sativa L), two cdc2 protein kinases, cdc2Os-1 and cdc2Os-2, and two cyclins, cycOs1 and cycOs2, have been described . In this study, we report on the cell-cycle phase-specific expression of these genes . Using partially synchronized suspension cells from rice, we found that cdc2Os-1 was expressed constitutively throughout the cell cycle . The cdc2Os-2 transcript level was elevated in G1 and S phase . The cycOs1 and cycOs2 transcripts increased steadily through G2 and dropped off rapidly in mitosis as is typical for mitotic cyclins . We hypothesize that the cdc2Os-2 gene product acts in G1/S and that the growth-promoting hormone gibberellin (GA) that induces expression of cdc2Os-2, cycOs1 and cycOs2 in the intercalary meristem of deepwater rice internodes accelerates G1/S phase progression through increased expression of cdc2Os-2 and G2/M phase progression through increased expression of the mitotic cyclins cycOs1 and cycOs2 . The R2 gene from rice has 55% sequence identity to the cdc2-activating kinase (CAK) family of protein kinases which have been shown to phosphorylate and thereby activate cdc2 protein kinases in animals and yeast . In partially synchronized suspension cells, R2 mRNA levels were elevated in G1 and S phase . In GA-treated rice internodes, R2 transcript levels were elevated in the meristem and part of the elongation zone . These results are consistent with a role for R2 in regulating G1/S phase progression.

Planta, 1997, 201(3), 349 - 58
Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L . cells; Smertenko A et al.; Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L . was analysed using a panel of specific antibodies . Polyglutamylated, tyrosinated, nontyrosinated, acetylated and delta 2-tubulin variants were detected on alpha-tubulin subunits; polyglutamylation was also found on beta-tubulin subunits . Modified tubulins were detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts . They were, however, located differently in the various microtubule structures . The antibodies against tyrosinated, acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and delta 2-tubulin provided dot-like staining of interphase microtubules . Additionally, immunoreactivity of antibodies against acetylated and delta 2-tubulins was strong in the pole regions of mitotic spindles . High-resolution isoelectric focusing revealed 22 tubulin charge variants in N . tabacum suspension cells . Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of alpha- and beta-tubulin molecules, respectively, revealed that 11 isoforms belonged to the alpha-subunit and 11 isoforms to the beta-subunit . Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several alpha-tubulin isoforms, antibodies against nontyrosinated and delta 2-tubulin reacted with only one . The combined data demonstrate that plant tubulin is extensively post-translationally modified and that these modifications participate in the generation of plant tubulin polymorphism.

Plant Mol Biol, 1997 Jan, 33(2), 359 - 66
Molecular cloning, characterization and expression of cDNA encoding phosphoserine aminotransferase involved in phosphorylated pathway of serine biosynthesis from spinach; Saito K et al.; Phosphoserine aminotransferase (PSA) catalyzes the conversion of phosphohydroxypyruvate to phosphoserine in the phosphorylated pathway of serine biosynthesis . A cDNA clone encoding PSA was isolated from the cDNA library of spinach (Spinacia oleracea L.) green leaves . Determination of the nucleotide sequence revealed the presence of an open reading frame encoding 430 amino acids, exhibiting 38-50% homology with the amino acid sequences of bacterial, yeast and animal PSA . It contains an N-terminal extension of ca . 60 amino acids in addition to the sequences from other organisms . The general features of plastidic transit peptide are observed in this N-terminal sequence, suggesting the plastid localization of the PSA protein encoded by this cDNA . The bacterial expression of the cDNA could functionally rescue the auxotrophy of serine in the serC- mutant, Escherichia coli KL282 . The enzymatic activity of PSA was demonstrated in vitro in the extracts of E . coli over-expressing the cDNA . Southern blot analysis indicated the presence of a couple of related genes (Psa) in the spinach genome . RNA blot hybridization suggested the preferential expression of the Psa gene in the roots of green seedlings and in the suspension cells cultured under a dark condition.

FEBS Lett, 1996 Dec 2, 398(2-3), 248 - 52
Arrest of mitochondrial biogenesis in copper-treated sycamore cells; Padua M et al.; Sycamore suspension cells (Acer pseudoplatanus L.) were grown in the presence of sublethal concentrations of copper (50 microM) . During the first 5-6 days of treatment, growth was not affected, but cell respiration (coupled and uncoupled) declined to approximately 60% of its normal value . This decline of respiration was attributed to a progressive diminution of the number of mitochondria in copper-treated cells, based on the demonstration of the concomitant decline of (1) cardiolipin (diphosphatidylglycerol) and cytochrome aa3 (cytochrome oxidase), two specific markers of mitochondrial inner membrane, and (2) fumarase activity, a specific marker of mitochondrial matrix space . In addition, the mitochondria extracted from copper-treated cells presented the same properties as those from control cells, concerning substrate oxidation, cardiolipin and cytochrome aa3 contents, and fumarase activity . These results strongly suggest that copper triggered an arrest of mitochondrial biogenesis, which preceded cell division arrest.

Plant J, 1996 Dec, 10(6), 1177 - 86
A plant in vitro system for the nuclear import of proteins; Merkle T et al.; This paper reports the development of an in vitro system that allows the direct assay of protein import into plant nuclei . In this assay the import of fluorescently labelled karyophilic protein substrates into nuclei isolated from evacuolated tobacco BY-2 suspension cells is monitored . It is demonstrated that import of the fluorescently labelled peptide conjugates is rapid, saturable and nuclear localization signal (NLS)-dependent . Exclusion of high molecular weight (70 kDa) dextran and substrates carrying mutated NLS sequences further underline the specificity of this system . Nuclear translocation of karyophilic import substrates in tobacco, similar to mammalian systems, is inhibited by the non-hydrolysable GTP analogue GTP-gamma-S . In contrast, protein uptake is not blocked by wheat germ agglutinin, N-ethyl-maleinimide and iodoacetic acid . Furthermore, it is shown that nuclear import of proteins is only partially inhibited by low temperature (0-4 degrees C) . The in vitro nuclear import assay does not depend on exogenously added ATP or cytosolic factors . However, a block of nuclear import with GTP-gamma-S could be overcome by the addition of cytosolic extract, suggesting the dependence on cytosolic factors or proteins . These data indicate that the characteristics of nuclear protein import in plant and mammalian cells are similar, but may be, at least in some respects, also different from each other.

Gene Ther, 1996 Nov, 3(11), 1010 - 7
An electron microscopy study into the mechanism of gene transfer with lipopolyamines; Labat-Moleur F et al.; Cationic amphiphiles have been shown to mediate gene transfer to eukaryotic cells, although the nature and fate of the lipid-DNA complexes is still a matter of debate . Negative staining transmission electron microscopy (TEM) of the complexes in physiological medium, as well as thin-section TEM of transfected cells has been used to visualize the particles and the possible pathways leading to transgene expression . Lipopolyamines form a network of tubular micelles into which plasmid DNA is intertwined and condensed; the cationic particles contain hundreds of plasmid molecules and are heterogeneous with respect to size (0.1-0.5 microgram) and shape . Adherent cells (293M, 3T3, MRC5, primary leptomeningeal cells) take them up readily within minutes by spontaneous endocytosis . Among suspension cells, lymphocytes only incidentally show cytoplasmic inclusions and monocytes degrade the particles by phagocytosis . The marked decrease in transfection efficiency generally observed between adherent and nonadherent cells is thus due to reduce cell binding . This suggests that cationic particles bind to membrane components responsible for Ca2+-mediated cell anchoring to the extracellular matrix . Cation/anion-mediated endocytosis leads to endosomes that are entirely filled with the particles . Consequently, two escape mechanisms may operate: disruption of the lamellar envelope in close contact with tubular micelles, and endosome buffering by the lipopolyamine in response to proton entry, leading to osmotic swelling and endosome rupture . Even for moderately transfected MRC5 cells, 10(2)-10(3) particles are found either free or in cytoplasmic vacuoles 24 h after transfection, highlighting a very inefficient nuclear translocation process . Such high numbers are also the clue to the small concentration window between transfection and cytotoxicity that is often observed with nonviral vectors . Nuclear particle inclusions are sometimes seen, yet it is unclear whether plasmid uncoating (before expression) takes place by anion exchange in the cytoplasm or in the nucleus . The still lower efficiency of free plasmid translocation to the nucleus suggests an active role for the cationic lipid during this step . Although the last stages of the transfection mechanism remain unclear, the present work shows that the major barrier which hampers in vitro gene delivery with cationic vectors is nuclear translocation (and cell entry for nonadherent cells), providing precise targets for the design of improved nonviral vectors.

Plant Physiol, 1996 Nov, 112(3), 931 - 8
Immunolocalization of mannitol dehydrogenase in celery plants and cells; Zamski E et al.; Immunolocalization of mannitol dehydrogenase (MTD) in celery (Apium graveolens L.) suspension cells and plants showed that MTD is a cytoplasmic enzyme . MTD was found in the meristems of celery root apices, in young expanding leaves, in the vascular cambium, and in the phloem, including sieve-element/companion cell complexes, parenchyma, and in the exuding phloem sap of cut petioles . Suspension cells that were grown in medium with mannitol as the sole carbon source showed a high anti-MTD cross-reaction in the cytoplasm, whereas cells that were grown in sucrose-containing medium showed little or no cross-reaction . Gel-blot analysis of proteins from vascular and nonvascular tissues of mature celery petioles showed a strong anti-MTD sera cross-reactive band, corresponding to the 40-kD molecular mass of MTD in vascular extracts, but no cross-reactive bands in nonvascular extracts . The distribution pattern of MTD within celery plants and in cell cultures that were grown on different carbon sources is consistent with the hypothesis that the Mtd gene may be regulated by sugar repression . Additionally, a developmental component may regulate the distribution of MTD within celery plants.

Blood, 1996 Nov 1, 88(9), 3465 - 73
Megakaryocytic cell line-specific hyperploidy by cytotoxic necrotizing factor bacterial toxins; Hudson KM et al.; Cytotoxic necrotizing factor (CNF) toxins, isolated from certain Escherichia coli strains known to cause intestinal and extra intestinal infections, induce reorganization of the actin cytoskeleton and generate hyperploidy in adherent cell lines . We have examined the effect of CNF toxin on one of the few cell types that naturally increase nuclear DNA content, megakaryocytes . Our studies show that only hematopoietic cells capable of differentiating along the megakaryocyte lineage responded to the CNF2 toxin by becoming polyploid and by reorganizing actin . The K562, HEL, and CHRF-288-11 cell lines can be induced with phorbol ester to differentiate along the megakaryocyte lineage, and these cells also respond to the toxin with increased DNA content and actin cytoskeletal rearrangements . Interestingly, treatment of the K562 and HEL cell lines with CNF2 does not result in an increase in production of the megakaryocytic marker glycoprotein IIIa, unlike phorbol ester treatment . Conversely, two T-cell leukemic cell lines, CEM and Molt4, and the promyelocytic HL-60 cell line, which do not differentiate along the megakaryocyte lineage in response to phorbol myristate acetate, do not respond to CNF2, by increased expression of gpIIIa, increased nuclear DNA content, or actin reorganization . A potential target of these toxins, RhoA, is expressed by both megakaryocytic and nonmegakaryocytic cell lines, as shown by reverse transcription-polymerase chain reaction and Western blot . Although it is clear that the CNF toxins can affect a wide variety of adherent nonhematopoietic cell lines, we propose that the response to CNF, in terms of reorganizing actin structure and increase in DNA content in hematologic suspension cells, correlates with the capability of these target cells to differentiate along the megakaryocytic lineage.

J Biol Chem, 1996 Oct 25, 271(43), 26998 - 7004
Carbohydrate starvation stimulates differential expression of rice alpha-amylase genes that is modulated through complicated transcriptional and posttranscriptional processes; Sheu JJ et al.; Expression of alpha-amylase genes in cultured rice suspension cells is induced by sucrose starvation . To study the mechanism of sugar metabolite regulation on the expression of individual alpha-amylase genes, DNA fragments specific to each of eight rice alpha-amylase genes were synthesized and used as gene-specific probes . Comparison of the relative abundance of mRNA revealed that expression of the eight alpha-amylase genes in rice cells was differentially regulated by sucrose starvation . Accumulation of all the alpha-amylase mRNAs increased in response to sucrose starvation; however, levels of the alphaAmy3 and alphaAmy8 mRNAs were distinctly higher and constituted 90% of total alpha-amylase mRNAs . RNA gel blot and nuclear run-on transcription analyses demonstrated a positive correlation between the increased transcription rates and the elevated steady-state levels of alpha-amylase mRNAs induced by sucrose starvation . The half-lives of alphaAmy3, alphaAmy7, and alphaAmy8 were prolonged by sucrose-starvation; however, the stability of the three mRNAs seems controlled by different mechanisms . The translation inhibitors cycloheximide and anisomycin preferentially blocked the sucrose-suppressed expression of alphaAmy3 but not that of alphaAmy7 and alphaAmy8 . These inhibitors also enhanced the sucrose starvation-induced accumulation of alphaAmy3 mRNA but not that of alphaAmy7 or alphaAmy8 mRNAs . Cycloheximide did not significantly alter the transcription rates of alpha-amylase genes, suggesting that labile proteins may selectively stabilize the alphaAmy7 and alphaAmy8 mRNAs but destabilize the alphaAmy3 mRNA.

J Biol Chem, 1996 Oct 18, 271(42), 25742 - 5
Histone H1 enhances the DNA binding activity of the transcription factor EmBP-1; Schultz TF et al.; Previous work indicated that nuclear extracts isolated from embryogenic rice suspension cells treated with the phytohormone abscisic acid (ABA) have enhanced binding activity to an ABA response element (Em1a) in the promoter of the Em gene from wheat . We identified an activity in wheat and maize nuclear extracts that enhances binding of the recombinant transcription factor EmBP-1 to Em1a by 80-fold . Fractionation of nuclear extracts led us to identify histone H1 and HMGb (but not HMGc or -d) as two factors that can enhance the ability of EmBP-1 to bind to Em1a and account for at least a part of this activity of nuclear extracts . Our results, which indicate for the first time that histone H1 possesses this type of activity, lend further support to the model that positively charged proteins can drastically affect the DNA binding activity of specific transcription factors . Furthermore, our study points to these chromosomal proteins as potential targets of an ABA-mediated modification (e.g . acetylation) that could affect the regulation of Em gene expression.

Plant Cell Physiol, 1996 Sep, 37(6), 748 - 53
Phosphorylation of a protein (pp56) is related to the regeneration of rice cultured suspension cells; Komatsu S et al.; Short-term cultured suspension cells of rice (Oryza sativa L.) are capable of regeneration, but not in long-term culture . For clarification of the mechanism of regeneration, protein phosphorylation in short-term and long-term cultured suspension cells was compared by two dimensional-polyacrylamide gel electrophoresis . A 56 kDa protein having an isoelectric point of 4.5 was phosphorylated in vitro in short-term cultured suspension cells, but was not phosphorylated after regeneration . This protein in long-term cultured suspension cells remained phosphorylated after transfer to the regeneration medium . However, using an antibody raised against this protein from short-term cultured suspension cells, it was always detected in long-term and short-term cultured suspension cells after transfer to the regeneration medium . The partial amino acid sequence of the HPLC-purified protein showed homology to a calcium-binding protein from maize . The phosphorylation of the 56 kDa protein (pp56) appears to be associated with the regeneration of cultured rice cells.

Plant Physiol, 1996 Sep, 112(1), 343 - 51
Purification and characterization of pyrophosphate-dependent phosphofructokinase from phosphate-starved Brassica nigra suspension cells; Theodorou ME et al.; Previously, we reported that inorganic phosphate (Pi) deprivation of Brassica nigra suspension cells or seedlings leads to a progressive increase in the alpha: beta-subunit ratio of the inorganic pyrophosphate (PPi)-dependent phosphofructokinase (PFP) and that this coincides with a marked enhancement in the enzyme's activity and sensitivity to its allosteric activator, fructose-2,6-bisphosphate (Fru-2,6-P2) . To further investigate the effect of Pi nutrition on B . nigra PFP, the enzyme was purified and characterized from Pi-starved B . nigra suspension cell cultures . Polyacrylamide gel electrophoresis, immunoblot, and gel-filtration analyses of the final preparation indicated that this enzyme exists as a heterooctamer of approximately 500 kD and is composed of a 1:1 ratio of immunologically distinct alpha (66 kD) and beta (60 kD) subunits . The enzyme's alpha subunit was susceptible to partial proteolysis during purification, but this was prevented by the presence of chymostatin and leupeptin . In the presence and absence of 5 microM Fru-2,6-P2, the forward activity of PFP displayed pH optima of pH 6.8 and 7.6, respectively . Maximal activation of the forward and reverse reactions by Fru-2,6-P2 occurred at pH 6.8 . The potent inhibition of the forward activity by Pi (concentration of inhibitor producing 50% inhibition of enzyme activity {I50} = 1.3 mM) was attributed to a marked Pi-dependent reduction in Fru-2,6-P2 binding . The reverse reaction was substrate-inhibited by Pi (I50 = 13 mM) and product-inhibited by PPi (I50 = 0.9 mM) . The kinetic data are consistent with the hypothesis that PFP may function to bypass the ATP-dependent PFP in Pi-starved B . nigra . The importance of the Pi nutritional status to the regulation and predicted physiological function of PFP is emphasized.

Mol Plant Microbe Interact, 1996 Sep, 9(7), 594 - 9
Mutational analysis of the putative nicking motif in the replication-associated protein (AC1) of bean golden mosaic geminivirus; Hoogstraten RA et al.; Geminiviruses are circular single-stranded DNA viruses that replicate by a rolling circle mechanism involving the viral-encoded AC1 protein . DNA nicking is necessary both for initiating replication of the covalently closed double-stranded DNA templates and for releasing unit-length monomers . The effects of mutations in a putative nicking motif (K101 A Y I D K106; E . V . Koonin and T . V . Ilyina, J . Gen . Virol . 73:2763-2766, 1992) of the AC1-derived protein for bean golden mosaic geminivirus isolate GA (BGMV-GA) were studied . The amino acids equivalent to Y103 and K106 of BGMV-GA are invariant in all whitefly-transmitted geminiviruses . Phaseolus vulgaris plant infectivity assays showed that the mutants K101-->H, K101-->A, and D105-->T produced symptoms, but mutants Y103-->A, Y103-->F, K106-->R, and K106-->H did not . A mutant with a stop codon in the N terminus of the AC4 open reading frame (ORF) produced the same symptoms as the wild-type BGMV-GA . Only those that were infectious replicated in NT-1 tobacco suspension cells . These results indicate that the Y103 and K106 residues are essential for replication, and that this putative DNA-nicking motif of the AC1 ORF may be functional in the rolling circle mechanism of replication for geminiviruses . The potential role of these mutants in the design of antiviral strategies is discussed.

Plant J, 1996 Aug, 10(2), 251 - 9
Isolation of microtubule-associated proteins from carrot cytoskeletons: a 120 kDa map decorates all four microtubule arrays and the nucleus; Chan J et al.; A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised . The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents . Depolymerization of cytoskeletal microtubules with calcium at 4 degrees C releases MAPs which are then isolated by association with taxol stabilized neurotubules . Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins . Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self-assemble . By adding it to rhodamine-conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol . In the electron microscope, these arrays are seen to be composed of mainly single microtubules . Blot-affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs . Antibodies to an antigenically related triplet of proteins about 60-68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules . Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti-MAP 65, the nuclear periphery is also stained . Both the anti-65 kDa and the anti-120 kDa antibodies stain cortical MTs in detergent-extracted, substrate-attached plasma membrane disks ('footprints') . Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs.

J Cell Biol, 1996 Jun, 133(6), 1251 - 63
Ultrastructural and biochemical characterization of autophagy in higher plant cells subjected to carbon deprivation: control by the supply of mitochondria with respiratory substrates; Aubert S et al.; Autophagy triggered by carbohydrate starvation was characterized at both biochemical and structural levels, with the aim to identify reliable and easily detectable marker(s) and to investigate the factors controlling this process . Incubation of suspension cells in sucrose-free culture medium triggered a marked degradation of the membrane polar lipids, including phospholipids and galactolipids . In contrast, the total amounts of sterols, which are mainly associated with plasmalemma and tonoplast membranes, remained constant . In particular, phosphatidylcholine decreased, whereas phosphodiesters including glycerylphosphorylcholine transiently increased, and phosphorylcholine (P-Cho) steadily accumulated . P-Cho exhibits a remarkable metabolic inertness and therefore can be used as a reliable biochemical marker reflecting the extent of plant cell autophagy . Indeed, whenever P-Cho accumulated, a massive regression of cytoplasm was noticed using EM . Double membrane-bounded vacuoles were formed in the peripheral cytoplasm during sucrose starvation and were eventually expelled into the central vacuole, which increased in volume and squeezed the thin layer of cytoplasm spared by autophagy . The biochemical marker P-Cho was used to investigate the factors controlling autophagy . P-Cho did not accumulate when sucrose was replaced by glycerol or by pyruvate as carbon sources . Both compounds entered the cells and sustained normal rates of respiration . No recycling back to the hexose phosphates was observed, and cells were rapidly depleted in sugars and hexose phosphates, without any sign of autophagy . On the contrary, when pyruvate (or glycerol) was removed from the culture medium, P-Cho accumulated without a lag phase, in correlation with the formation of autophagic vacuoles . These results strongly suggest that the supply of mitochondria with respiratory substrates, and not the decrease of sucrose and hexose phosphates, controls the induction of autophagy in plant cells starved in carbohydrates.

Anal Biochem, 1996 May 1, 236(2), 322 - 6
A chemiluminescence-based method for the detection and quantification of antigen-antibody interactions on the surface of eukaryotic cells; Lischke A et al.; Enhanced chemiluminescence was applied to detect the binding of monoclonal antibodies to surface antigens on intact cells . The fast and simple assay is performed in the microtiter scale and thus allows for the simultaneous processing of a large number of samples with a sensitivity comparable to conventionally used techniques such as cytometry or Western blot analysis . In two model experiments, we demonstrate (a) the detection of a heterologously expressed cytokine receptor subunit on the surface of suspension cells and (b) the screening of hybridoma clones for the production of antibodies specifically recognizing surface antigens on a tumor cell line . Moreover, the assay is shown to be suitable for the determination of antibody affinities and of antibody binding sites per cell.

Biotechnol Prog, 1996 May-Jun, 12(3), 398 - 402
Confocal laser scanning microscopy examination of cell distribution in macroporous microcarriers; Bancel S et al.; Macroporous microcarriers are often used to cultivate animal cells . The pores in the interior of the beads provide surface and space for cell growth . It is not clear how anchorage-dependent and suspension cells populated these microcarriers during cultivation . Confocal laser scanning microscopy was employed to perform time lapse observation of the cells in the interior . The structure of the bead was stained with fluorescein isothiocyanate for visualization, while the cells were stained with dialkyl indocarbocyanines for tracking over time . It was observed that mouse fibroblastic cells CRE BaG2 did not move extensively after initial attachment . Some cell divisions were observed during the course of the experiments, and essentially all cells remained viable throughout . Few hybridoma cells were deposited into the pores in the interior of the microcarriers . The results suggest that the occupancy of the internal volume by cells after prolonged cultivation is largely due to the growth of cells that are deposited in the interior as opposed to the migration of cells from the external surface into the interior . This method of observing cell behavior in a three-dimensional structure may find applications in other three-dimensional cell culture systems . The animation of time lapse sections is available on the worldwide web at approximately wshu_grp/acre/microcarrier.html.

Planta, 1996, 200(1), 2 - 12
Cytokinin controls the cell cycle at mitosis by stimulating the tyrosine dephosphorylation and activation of p34cdc2-like H1 histone kinase; Zhang K et al.; In excised pith parenchyma from Nicotiana tabacum L . cv . Wisconsin Havana 38, auxin (naphthalene-1-acetic acid) together with cytokinin (6-benzylaminopurine) induced a greater than 40-fold increase in a p34cdc2-like protein, recoverable in the p13suc1-binding fraction, that had high H1 histone kinase activity, but enzyme induced without cytokinin was inactive . In suspension-cultured N . plumbaginifolia Viv., cytokinin (kinetin) was stringently required only in late G2 phase of the cell division cycle (cdc) and cells lacking kinetin arrested in G2 phase with inactive p34cdc2-like H1 histone kinase . Control of the Cdc2 kinase by inhibitory tyrosine phosphorylation was indicated by high phosphotyrosine in the inactive enzyme of arrested pith and suspension cells . Yeast cdc25 phosphatase, which is specific for removal of phosphate from tyrosine at the active site of p34cdc2 enzyme, was expressed in bacteria and caused extensive in-vitro activation of p13suc1-purified enzyme from pith and suspension cells cultured without cytokinin . Cytokinin stimulated the removal of phosphate, activation of the enzyme and rapid synchronous entry into mitosis . Therefore, plants can control cell division by tyrosine phosphorylation of Cdc2 but differ from somatic animal cells in coupling this mitotic control to hormonal signals.

Plant Cell, 1996 Jan, 8(1), 119 - 132
A kinesin-like protein, KatAp, in the cells of arabidopsis and other plants; Liu B et al.; The kinesin-like proteins (KLPs) are a large family of plus- or minus-end-directed microtubule motors important in intracellular transport, mitosis, meiosis, and development . However, relatively little is known about plant KLPs . We prepared an antibody against two peptides in the microtubule binding domain of an Arabidopsis KLP (KatAp) encoded by the KatA gene, one of a family of genes encoding KLPs whose motor domain is located near the C terminus of the polypeptide . Such KLPs typically move materials toward the minus end of microtubules . An immunoreactive band (Mr of 140,000) corresponding to KatAp was demonstrated with this antibody on immunoblots of Arabidopsis seedling extracts . During immunofluorescence localizations, the antibody produced weak, variable staining in the cytoplasm and nucleus of interphase Arabidopsis suspension cells but much stronger staining of the mitotic apparatus during division . Staining was concentrated near the midzone during metaphase and was retained there during anaphase . The phragmoplast was also stained . Similar localization patterns were seen in tobacco BY-2 cells . The antibody produced a single band (Mr of 130,000) in murine brain fractions prepared according to procedures that enrich for KLPs (binding to microtubules in the presence of AMP-PNP but not ATP) . A similar fraction from carrot suspension cells yielded a cross-reacting polypeptide of similar apparent molecular mass . When dividing BY-2 cells were lysed in the presence of taxol and ATP, antibody staining moved rapidly toward the poles, supporting the presence of a minus-end motor . Movement did not occur without ATP, with AMP-PNP, or with ATP plus antibody . Our results indicate that the protein encoded by KatA, KatAp, is expressed in Arabidopsis and is specifically localized to the midzone of the mitotic apparatus and phragmoplast . A similar protein is also present in other species.

Plant Mol Biol, 1995 Dec, 29(6), 1267 - 77
Repair mechanisms of UV-induced DNA damage in soybean chloroplasts; Cannon GC et al.; In order to better understand the biochemical mechanisms of DNA metabolism in chloroplasts, repair of UV induced plastome damage in vivo was determined by exposure of soybean suspension cells to UV light and subsequent quantitation of the damage remaining in nuclear and chloroplast encoded genes with time by quantitative polymerase chain reaction (QPCR) . The kinetics of damage repair in the nuclear rbcS gene suggest that photoreactivation and dark mechanisms are active, while for the plastome encoded psbA gene only a light-dependent repair process was detected which is considerably slower than would be expected for photolyase-mediated photoreactivation.

J Biotechnol, 1995 Dec 1, 43(2), 103 - 10
Dextran as protectant against damage caused by sparging for hybridoma cells in a bubble column; van der Pol LA et al.; The effect of the addition of dextran as a protective polymer against sparging was examined with hybridoma suspension cells in a bubble column under standardized conditions . The protective effect of high concentrations of high molecular weight dextran showed a correlation with the bulk viscosity of the medium . A distinct protective effect occurs at viscosities greater than 20 x 10(-3) Pa s-1 . In contrast, low molecular weight dextrans that cause a minor increase in viscosity, also provide no protection against sparging . There is no strict correlation between surface tension and the protective effect of dextran against sparging . Oxygen transfer is strongly reduced by high concentrations of high molecular weight dextran . Therefore, addition of dextran as protective polymer against sparging for large-scale production processes with animal cells in stirred reactors does not seem feasible.

Nucleic Acids Res, 1995 Nov 11, 23(21), 4246 - 54
Only one of four possible secondary structures of the central conserved region of potato spindle tuber viroid is a substrate for processing in a potato nuclear extract; Baumstark T et al.; The influence of RNA secondary structure on the substrate activity of a longer-than-unit length transcript for processing to circular viroids was studied in a nuclear extract from potato suspension cells . The nuclear extract was prepared according to a modified procedure for a plant transcription extract . The transcript of the potato spindle tuber viroid (PSTVd) consists of a monomeric molecule with 17 additional nucleotides, thus doubling most of the central conserved region of viroids of the PSTVd-class . The transcript can assume four different secondary structures, which either co-exist as conformers in solution or can be kept as metastable structures after different treatments by temperature and/or ionic strength . The structures were analysed by thermodynamic calculations and temperature-gradient gel electrophoresis and were confirmed by oligonucleotide mapping . Only the so-called extended middle structure was processed to exact viroid circles . In this structure the 5'- and 3'-ends are branching out from the rod-like viroid structure at the loop starting with nucleotide 87 . The other structures were processed only if they could be rearranged into the active structure.

Plant Mol Biol, 1995 Nov, 29(3), 413 - 29
Promoter analysis of the auxin-regulated tobacco glutathione S-transferase genes Nt103-1 and Nt103-35; Droog F et al.; We have analysed the promoter regions of two closely related auxin-regulated glutathione S-transferase genes . All active deletion constructs tested showed expression of the reporter gene beta-glucuronidase (gusA) in root tips of young seedlings and newly developing lateral roots . Auxin treatment greatly enhanced the level of expression . The Nt103-1 promoter region -370/-276 was found to be necessary, at least as a quantitative element to confer auxin-responsiveness to a reporter gene, and sequences responsible for the auxin-responsiveness must be located downstream of -370 . The region -651/-370 contains sequence information necessary for uninduced expression . The Nt103-35 promoter manifested its auxin-responsiveness within the -504/-310 region . Electrophoretic mobility shift analysis, using nuclear extracts from tobacco leaves and suspension cells, identified a factor binding to a sequence (ap103, TGAGTCT) at position -560 of the Nt103-1 promoter, which shows homology to the mammalian AP-1 site . A second factor was found to bind a sequence (as103, ATAGCTAAGTGCTTACG) with homology to the CaMV 35S promoter as-1 element . The as103 element is present in both promoters and positioned around -360, so within the region determined to be indispensable for the response to auxin . A third factor was found binding to the -276/-190 region of both promoters . Combined, these data point to the relevance of a 90 bp region for auxin-induced activity of both tobacco genes . The ASF-1 like factor binding to the as103 element within this region might be involved in mediating the auxin response.

Virology, 1995 Aug 1, 211(1), 1 - 9
Mutational analysis of a putative NTP-binding domain in the replication-associated protein (AC1) of bean golden mosaic geminivirus; Hanson SF et al.; Bean golden mosaic virus (BGMV) is a whitefly-transmitted, ssDNA geminivirus with a bipartite genome . AC1 is the only ORF required for geminiviral replication . A putative NTP-binding motif, EGX4GKTX32DD, was present in the derived amino acid sequence of the replication-associated protein from the AC1 ORF for 13 geminiviruses including BGMV-GA (Guatemalan isolate, amino acids 221-263) . We analyzed the phenotypes of mutations within this domain using a rapid and sensitive PCR-based assay for geminiviral replication developed for these studies . Replication in tobacco cells (NT-1 suspension cells) and infection of beans were abolished when codons were changed from K228 to H or D262 to R within the putative NTP-binding site . A temperature-sensitive replication phenotype was conferred by changing E221 to R within the putative NTP-binding domain . Replication was unaffected by changing a nonconserved codon near the putative NTP-binding domain from 1190 to R . Our results demonstrate that the putative NTP-binding domain is required for geminiviral replication . The role of NTP hydrolysis and the possible value of these mutants in a trans-dominant interference scheme for virus-derived resistance are discussed.

Plant Mol Biol, 1995 Aug, 28(5), 885 - 900
Stress responses in alfalfa (Medicago sativa L.) XIX . Transcriptional activation of oxidative pentose phosphate pathway genes at the onset of the isoflavonoid phytoalexin response; Fahrendorf T et al.; We have isolated cDNA clones encoding the pentose phosphate pathway enzymes 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from alfalfa (Medicago sativa L.) . These exhibit extensive nucleotide and amino acid sequence similarity to the corresponding genes from bacteria, Drosophila and mammals . Transcripts encoding both enzymes are expressed at high levels in roots and nodules . Exposure of alfalfa suspension cells to an elicitor from yeast cell walls results in co-ordinated increases in transcription rates for both genes, followed by increased steady state transcript levels but only slightly increased extractable enzyme activities, at the onset of accumulation of isoflavonoid phytoalexins . Levels of NADPH and NADP remain relatively constant in alfalfa cells following elicitation . The rapid transcriptional activation of 6PGDH and G6PDH does not therefore appear to be a response to altered pyridine nucleotide redox state . These genes appear to respond to early events in elicitor-mediated signalling rather than to subsequent elicitor-induced changes in secondary metabolism . Hydrogen peroxide, a potential signal for elicitation of anti-oxidative genes in biologically stressed plant cells, did not induce 6PGDH or G6PDH transcripts or enzymatic activity.

Plant Mol Biol, 1995 Aug, 28(5), 859 - 70
Isolation of cDNA clones of genes with altered expression levels in phosphate-starved Brassica nigra suspension cells; Malboobi MA et al.; Differential gene expression at the transcriptional level was examined as an initial step in the investigation of the P(i) starvation response of Brassica nigra suspension cells . Total RNA was extracted from 7-day old cells grown in media containing either no P(i), 1.25 mM or 10 mM Pi . In vitro translation was carried out using their respective poly(A)+ RNA isolates and the resultant polypeptides were separated on a high-resolution SDS-PAGE gel . Scanning densitometry identified four polypeptides (ca . 31.7, 32.3, 52.5 and 64.8 kDa) present only in the P(i)-starved samples . Screening by differential hybridization was performed on a cDNA library constructed from mRNA isolated from P(i)-starved cells . Probes prepared from mRNA from P(i)-deficient and P(i)-sufficient cells identified a number of clones representing mRNA species that were preferentially transcribed under P(i) deficiency . These phosphate starvation-responsive (psr) clones were placed into eleven groups as determined by cross-hybridization . Northern blots showed that the corresponding genes are inducible in both mild and severe P(i) starvation conditions . Preliminary sequencing identified one of the clones as being homologous to beta-glucosidases from several plant species . The possible role of beta-glucosidase during Pi starvation and the identities of the other psr genes are discussed.

J Cell Physiol, 1995 Aug, 164(2), 334 - 43
THP-1 macrophage membrane-bound plasmin activity is up-regulated by transforming growth factor-beta 1 via increased expression of urokinase and the urokinase receptor; Falcone DJ et al.; Receptors for urokinase (uPA) and plasminogen provide a mechanism to direct the cellular activation of plasminogen . The regulation of these receptors is important for several macrophage functions . In these studies, the effect of transforming growth factor-beta 1 (TGF-beta 1) on uPA, uPA receptor, and plasminogen receptor expression by human THP-1 macrophage was examined . TGF-beta 1 induction of uPA expression by THP-1 cells was differentiation dependent . Suspension and adherent cultures expressed similar constitutive levels of uPA . Exposure of adherent cells to TGF-beta 1 led to a dose- and time-dependent increase in uPA activity which was paralleled by an increase in uPA antigen and uPA mRNA . In contrast, uPA expression by suspension cultures was unresponsive to TGF-beta 1 . The differential response exhibited by suspension and adherent THP-1 cells may reflect differences in their expression of TGF-beta 1 receptors, since when assayed by crosslinking techniques, suspension cells primarily expressed a 65 kDa receptor; whereas, the adherent cells expressed 65 and 100 kDa receptors . TGF-beta 1-induced alterations in uPA receptor expression by adherent THP-1 cells were examined by quantitating membrane-bound uPA activity . Membrane-bound uPA activity increased three-fold when cells were incubated with TGF-beta 1 . The increase in membrane-uPA activity expressed by TGF-beta 1-treated cells was not due to increased uPA receptor occupancy since incubation of either control or TGF-beta 1 primed cells with exogenous uPA did not lead to an increase in membrane-bound uPA activity . Furthermore, immunoreactive uPA receptor was increased in TGF-beta 1-treated cells . Following incubation with plasminogen, membrane-bound plasmin activity increased three-fold in TGF-beta 1-treated cells . However, no change in immunoreactive membrane-bound plasmin(ogen) was observed . In addition, binding of 125I-Lys-plasminogen to THP-1 cells was not affected by TGF-beta 1 treatment . We conclude that TGF-beta 1 stimulates membrane-bound plasmin activity, without affecting plasminogen receptor expression, through the up-regulation of uPA and the uPA receptor expression.

Immunopharmacology, 1995 Jun, 30(1), 89 - 101
High susceptibility of U937-derived subclones to infection with human immunodeficiency virus type 1 is correlated with virus-induced cell differentiation and superoxide generation; Kameoka M et al.; The promonocytic human leukemic cell line U937, when infected with lymphotropic human immunodeficiency virus type 1 (HIV-1), becomes a continuous virus producer . A total of 46 U937-derived subclones in suspension was isolated and classified into three (2 high, 42 middle, and 2 low) types based on their susceptibility to the infection . By analyzing subclones before infection, we found that the high-type subclones expressed LFA-1 antigens at a relatively low level . In addition, the ability of these subclones to induce adherence after exposure to phorbol 12-myristate 13-acetate (PMA) was reduced . In contrast, a transition by HIV-1 infection to adherent macrophage-like cells was induced only in the high-type, but not in the low-type subclones . The high-type adherent cells obtained by HIV-1 infection were followed by further lineage to become retrodifferentiated suspension cells showing reduced syncytia formation ability . Superoxide was generated in the high-type subclones, without PMA-mediated differentiation, from the early stage of infection before HIV-1 replication, as well as during undifferentiated, differentiated and retrodifferentiated stages . In contrast, it was only transiently generated at acute phase of HIV-1 replication in low-type subclones . Long-term culture of the low-type subclones decreased the expression of major structural viral protein Gag and also virus production . Thus, the mechanism by which PMA differentiates U937 cells is not the same as that induced by HIV-1 infection . The latter mechanism results in high susceptibility to infection . The HIV-1 phenotypes of finally obtained persistently infected cells were also affected by the cell stages at the time of infection.

Mol Biol Cell, 1995 Jun, 6(6), 637 - 47
Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase; Hildebrand JD et al.; Focal adhesion kinase (pp125FAK or FAK) and paxillin colocalize with integrins in structures called focal adhesions . pp125FAK plays an important role in the transmission of integrin-induced cytoplasmic signals . Paxillin has also been implicated in cell signaling by virtue of its association with the protein tyrosine kinases pp60src and Csk (C-terminal Src kinase) as well as with the adapter/oncoprotein p47gag-crk . In this report we show that endogenous pp125FAK and paxillin form a stable complex both in vivo and in vitro and that this interaction is direct, requiring only pp125FAK and paxillin . The paxillin binding site on pp125FAK has been localized to the carboxy-terminal 148 residues of pp125FAK, but appears to be distinct from the previously identified focal adhesion-targeting sequence also present in the carboxy-terminal domain of pp125FAK . The interaction of paxillin and pp125FAK is independent of the adhesion of cells to the extracellular matrix, as the association can be detected in suspension cells as well as those attached to fibronectin.

Mol Biotechnol, 1995 Jun, 3(3), 181 - 90
Rapid optimization of electroporation conditions for plant cells, protoplasts, and pollen; Saunders JA et al.; The optimization of electroporation conditions for maximal uptake of DNA during direct gene transfer experiments is critical to achieve high levels of gene expression in transformed plant cells . Two stains, trypan blue and fluorescein diacetate, have been applied to optimize electroporation conditions for three plant cell types, using different square wave and exponential wave electroporation devices . The different cell types included protoplasts from tobacco, a stable mixotrophic suspension cell culture from soybean with intact cell walls, and germinating pollen from alfalfa and tobacco . Successful electroporation of each of these cell types was obtained, even in the presence of an intact cell wall when conditions were optimized for the electroporation pulse . The optimal field strength for each of these cells differs, protoplasts having the lowest optimal pulse field strength, followed by suspension cells and finally germinating pollen requiring the strongest electroporation pulse . A rapid procedure is described for optimizing electroporation parameters using different types of cells from different plant sources.

J Biol Chem, 1995 Mar 3, 270(9), 4368 - 74
The plant inorganic pyrophosphatase does not transport K+ in vacuole membrane vesicles multilabeled with fluorescent probes for H+, K+, and membrane potential; Ros R et al.; It has been claimed that the inorganic pyrophosphatase (PPase) of the plant vacuolar membrane transports K+ in addition to H+ in intact vacuoles (Davies, J . M., Poole, R . J., Rea, P . A., and Sanders, D . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 11701-11705) . Since this was not confirmed using the purified and reconstituted PPase consisting of a 75-kDa polypeptide (Sato, M.H., Kasahara, M., Ishii, N., Homareda, H., Matsui, H., and Yoshida, M . (1994) J . Biol . Chem . 269, 6725-6728), these authors proposed that K+ transport by the PPase is dependent on its association with other membrane components lost during purification . We have examined the hypothesis of K+ translocation by the PPase using native vacuolar membrane vesicles from Vitis vinifera suspension cells, multilabeled with fluorescent probes for K+, H+, and membrane potential . This material contained a high proportion of right-side-out, tightly sealed vesicles, exhibiting high PPase activity which was strongly stimulated by uncouplers and K+ . Proton pumping occurred in response to pyrophosphate addition in the absence of K+ . No K+ incorporation into the vesicles could be observed after PPase energization in the presence of K+, although H+ transport was highly stimulated . The hydrolytic activity was stimulated by a protonophore and by a H+/K+ exchanger but not by the K+ ionophore valinomycin . No evidence could be obtained supporting the operation of an endogenous K+/H+ exchanger capable to dissipate the putative active K+ flux generated by the PPase . We conclude that PPase in native vacuolar membrane vesicles does not transport K+.

Chin J Biotechnol, 1995, 11(3), 207 - 11
Protoplast culture and plant regeneration from the suspension cells of Gynostemma pentaphyllum (Thumb) Mak; Zhang H et al.; From the suspension cultures of Gynostemma pentaphyllum (Thumb) Mak . protoplasts were isolated and cultured in a different medium with liquid and nurse culture method . Cell division was observed within 4 days and microcalli were formed within 4 weeks . On an MS solid medium with 1 mg/L of KT and 0.5 mg/L of IAA, protoplast-derived calli differentiated into embryos . Stems and leaves were formed on the MS medium with 1 mg/L of 6-BA and 0.5 mg/L of IAA . Finally, complete plantlets were obtained on a hormone-free MS solid medium.

Cell Motil Cytoskeleton, 1995, 31(2), 113 - 29
Experimental manipulation of gamma-tubulin distribution in Arabidopsis using anti-microtubule drugs; Liu B et al.; gamma-Tubulin-specific antibodies stain the microtubule (Mt) arrays of Arabidopsis suspension cells in a punctate or patchy manner . During division, staining of kinetochore fibers and the phragmoplast is extensive, except in the vicinity of the plus ends at the metaphase plate and cell plate . gamma-Tubulin localization responds to low levels of colchicine, with staining receding farther toward the minus (pole) ends of kinetochore fibers . At higher drug concentrations, gamma-tubulin also associates with abnormal Mt foci as well as with the surface of the daughter nuclei facing the phragmoplast . During UV-induced recovery from colchicine, gamma-tubulin increases along the presumptive minus ends of mitotic Mts as well as the phragmoplast near the daughter nuclei . With CIPC, immunostaining is concentrated around the centers of focal Mt arrays in multipolar spindles . In the presence of taxol, Mts are more prominent but the mitotic apparatus and phragmoplast are abnormal . As with CIPC, gamma-tubulin is concentrated at focal arrays . Increased punctate staining is also present in interphase arrays, with fluorescent dots often located at the ends of Mts . These results support a preferential association between gamma-tubulin and Mt minus ends, but are also consistent with more general binding along the walls of Mts . Thus, minus ends (and Mt nucleation sites) may be present throughout plant Mt arrays, but gamma-tubulin may also serve another function, such as in structural stabilization.

Brain Res Bull, 1995, 38(3), 215 - 20
Rat pineal cell aggregates: ultrastructural and functional characteristics; Shacoori V et al.; The aggregates were obtained by constant gyratory shaking of suspension cells freshly isolated from adult rat pineal glands . Their sizes ranged from 60 to 120 microns . Within 4-5 days, the aggregates formed by pinealocytes, astrocytes, and other unidentified cells became organized in a tissue-like configuration . There was no proliferation of the fibroblast cells . Ultrastructural characteristics of the aggregates were revealed by the presence of granular lysosomes, which are typical of pinealocytes, and are actively involved in the secretion . Functional characteristics were studied in static incubation . The aggregates secreted melatonin and other indole amines in culture medium . Basal melatonin release was detected until Day 24 of culture . This secretion was stimulated 230% with Isoproterenol (beta-adrenergic agonist), 725% with Epinephrine (alpha- and beta-adrenergic agonists), and 140% with Vasoactive Intestinal Peptide after 5 days in culture, then > 1200% with Forskolin 9 days later (14-day-old aggregates) . The results indicate that three-dimensional aggregates obtained from isolated pineal gland cells were the functional multicellular structures with in vivo characteristics.

Plant Mol Biol, 1994 Dec, 26(6), 1701 - 10
Coordinate expression of antibody subunit genes yields high levels of functional antibodies in roots of transgenic tobacco; van Engelen FA et al.; To explore the feasibility of employing antibodies to obtain disease resistance against plant root pathogens, we have studied the expression of genes encoding antibodies in roots of transgenic plants . A model monoclonal antibody was used that binds to a fungal cutinase . Heavy and light chain cDNAs were amplified by PCR, fused to a signal sequence for secretion and cloned behind CaMV 35S and TR2' promoters in a single T-DNA . The chimeric genes were cloned both in tandem and in a divergent orientation . The roots of tobacco plants transformed with these constructs produced antibodies that were able to bind antigen in an ELISA . Immunoblotting showed assembly to a full-size antibody . In addition, a F(ab')2-like fragment was observed, which is probably formed by proteolytic processing . Both antibody species were properly targeted to the apoplast, but the full-size antibody was partially retained by the wall of suspension cells . The construct with divergent promoters showed a better performance than the construct with promoters in tandem . It directed the accumulation of functional antibodies to a maximum of 1.1% of total soluble protein, with half of the plants having levels higher than 0.35% . The high efficiency of this construct probably results from coordinated and balanced expression of light and heavy chain genes, as evidenced by RNA blot hybridization.

Plant J, 1994 Nov, 6(5), 625 - 36
Expression of alpha-amylases, carbohydrate metabolism, and autophagy in cultured rice cells is coordinately regulated by sugar nutrient; Chen MH et al.; A rice suspension cell culture system has been established to study how sugar depletion regulates alpha-amylase expression, carbohydrate metabolism, and other physiological and cellular changes . It is shown here that a group of 44 kDa alpha-amylases are constitutively expressed whether or not the cells are starved of sucrose . However, expression of a new group of alpha-amylases of 46 kDa is dramatically induced when cells are starved of sucrose . Cellular sugar and starch were rapidly consumed and metabolic activity was decreased in the starved cells . Extensive autophagy also occurred in the starved cells, which caused an increase in vacuolar volume and degradation of cytoplasmic constituents including amyloplasts . Immunocytochemical studies revealed that alpha-amylases are localized in starch granules within amyloplasts, in cell walls, and in some of the vacuoles . The presence of putative signal sequences in the N-termini of nine rice alpha-amylases suggests hitherto unidentified pathways for import of alpha-amylases into amyloplasts . The studies show that differential alpha-amylase expression, carbohydrate metabolism, metabolic activity, and vacuolar autophagy are coordinately regulated by the sugar level in the medium . As the starved suspension cells exhibit some sugar-regulated characteristics of alpha-amylase expression in germinating rice embryos as well as physiological changes similar to those in senescing cells, this system represents an ideal tool for studying cellular, biochemical, and molecular biological aspects of alpha-amylase gene regulation, carbohydrate metabolism, senescence, and protein targeting in plants.

Mol Cell Biol, 1994 Sep, 14(9), 6125 - 34
Evidence for trans regulation of apoptosis in intertypic somatic cell hybrids; Gourdeau H et al.; The genetic components required for glucocorticoid induction of apoptosis were studied by using somatic cell hybridization . Intertypic whole-cell hybrids were generated by crossing the glucocorticoid-resistant rat liver cell line Fado-2 with the glucocorticoid-sensitive mouse thymoma cell line BW5147.3 . Morphological and biochemical criteria were used to assess sensitivity or resistance to glucocorticoid-induced cell death . Both phenotypes were observed, and all of the hybrids retained a functional glucocorticoid receptor as judged by their abilities to induce the metallothionein gene in response to dexamethasone (Dex) . Sensitivity to apoptosis did not correlate with morphological phenotype in that not all suspension cells were sensitive . The effect of glucocorticoids on the expression of apoptosis-linked genes was analyzed in a subset of Dex-sensitive and Dex-resistant hybrids . p53 and c-myc mRNAs were present in parental cells as well as sensitive and resistant hybrid cells, and their levels were not affected by glucocorticoid treatment . bcl-2 expression was restricted to the thymoma cell line and was also not affected by glucocorticoids . We did not detect any bcl-2 mRNA in the hepatoma cell line and the hybrids, suggesting that, as with most tissue-specific genes, bcl-2 is regulated in trans . Furthermore, while the majority of hybrids analyzed retained a full complement of mouse chromosomes, sensitive hybrids were missing some rat chromosomes (preferentially chromosomes 16 and 19), indicating that apoptosis is subject to trans repression . Resistant cells thus appear to repress the activity or synthesis of a nuclear factor that interacts with a glucocorticoid-dependent gene(s) to activate the cell death pathway.

Zhongguo Zhong Yao Za Zhi, 1994 Sep, 19(9), 529 - 31, 573
{Studies on immobilization of suspension cells of peltate yam (Dioscorea zingiberensis C.H . Wright)}; Ren JW et al.; The suspension cells of D . zingiberensis were immobilized with 3% sodium alginate, and then cultured in MS+2, 4-D1.0 + 6-BA 0.1 at 25 degrees C for a long period of time . The culture fluid free from cells was extracted and analyzed by TLC . The result showed that the immobilized cells could secrete the main component of D . zingiberensis--diosgenin, but not consecutively.

J Virol, 1994 Sep, 68(9), 5925 - 32
Vitronectin receptor antibodies inhibit infection of HeLa and A549 cells by adenovirus type 12 but not by adenovirus type 2; Bai M et al.; The penton base gene from adenovirus type 12 (Ad12) was sequenced and encodes a 497-residue polypeptide, 74 residues shorter than the penton base from Ad2 . The Ad2 and Ad12 proteins are highly conserved at the amino- and carboxy-terminal ends but diverge radically in the central region, where 63 residues are missing from the Ad12 sequence . Conserved within this variable region is the sequence Arg-Gly-Asp (RGD), which, in the Ad2 penton base, binds to integrins in the target cell membrane, enhancing the rate or the efficiency of infection . The Ad12 penton base was expressed in Escherichia coli, and the purified refolded protein assembled in vitro with Ad2 fibers . In contrast to the Ad2 penton base, the Ad12 protein failed to cause the rounding of adherent cells or to promote attachment of HeLa S3 suspension cells; however, A549 cells did attach to surfaces coated with either protein and pretreatment of the cells with an integrin alpha v beta 5 monoclonal antibody reduced attachment to background levels . Treatment of HeLa and A549 cells with integrin alpha v beta 3 or alpha v beta 5 monoclonal antibodies or with an RGD-containing fragment of the Ad2 penton base protein inhibited infection by Ad12 but had no effect on and in some cases enhanced infection by Ad2 . Purified Ad2 fiber protein reduced the binding of radiolabeled Ad2 and Ad12 virions to HeLa and A549 cells nearly to background levels, but the concentrations of fiber that strongly inhibited infection by Ad2 only weakly inhibited Ad12 infection . These data suggest that alpha v-containing integrins alone may be sufficient to support infection by Ad12 and that this pathway is not efficiently used by Ad2.

EMBO J, 1994 Jul 1, 13(13), 2970 - 5
Voltage-dependent calcium-permeable channels in the plasma membrane of a higher plant cell; Thuleau P et al.; Numerous biological assays and pharmacological studies on various higher plant tissues have led to the suggestion that voltage-dependent plasma membrane Ca2+ channels play prominent roles in initiating signal transduction processes during plant growth and development . However, to date no direct evidence has been obtained for the existence of such depolarization-activated Ca2+ channels in the plasma membrane of higher plant cells . Carrot suspension cells (Daucus carota L.) provide a well-suited system to determine whether voltage-dependent Ca2+ channels are present in the plasma membrane of higher plants and to characterize the properties of putative Ca2+ channels . It is known that both depolarization, caused by raising extracellular K+, and exposure to fungal toxins or oligogalacturonides induce Ca2+ influx into carrot cells . By direct application of patch-clamp techniques to isolated carrot protoplasts, we show here that depolarization of the plasma membrane positive to -135 mV activates Ca(2+)-permeable channels . These voltage-dependent ion channels were more permeable to Ca2+ than K+, while displaying large permeabilities to Ba2+ and Mg2+ ions . Ca(2+)-permeable channels showed slow and reversible inactivation . The single-channel conductance was 13 pS in 40 mM CaCl2 . These data provide direct evidence for the existence of voltage-dependent Ca2+ channels in the plasma membrane of a higher plant cell and point to physiological mechanisms for plant Ca2+ channel regulation . The depolarization-activated Ca(2+)-permeable channels identified here could constitute a regulated pathway for Ca2+ influx in response to physiologically occurring stimulus-induced depolarizations in higher plant cells.

Mol Cell Biol, 1994 Jul, 14(7), 4350 - 9
Identification of a transcriptional activator-binding element in the 27-kilodalton zein promoter, the -300 element; Ueda T et al.; By utilizing a homologous transient-expression system, we have shown that a 58-bp sequence from the gamma-class 27-kDa zein promoter, spanning from -307 to -250 relative to the transcription start site, confers a high level of transcriptional activity on a truncated plant viral promoter . The transcriptional activity mediated by the 58-bp sequence is orientation independent, and it is further enhanced as a result of its multimerization . A similarly high level of transcriptional activity was also observed in protoplasts isolated from leaf tissue-derived maize suspension cells . In vitro binding and DNase I footprinting assays with nuclear protein prepared from cultured endosperm cells revealed the sequence-specific binding of a nuclear factor(s) to a 16-nucleotide sequence present in the 58-bp region . The nuclear factor binding sequence includes the -300 element, a cis-acting element highly conserved among different zein genes and many other cereal storage protein genes . A 23-bp oligonucleotide sequence containing the nuclear factor binding site is sufficient for binding the nuclear factor in vitro . It also confers a high level of transcriptional activity in vivo, but in an orientation-dependent manner . Four nucleotide substitutions in the -300 element drastically reduced binding and transcriptional activation by the nuclear factor . The same nuclear factor is abundant in the developing kernel endosperm and binds to the -300 element region of the 27-kDa or the alpha-class zein promoter . These results suggest that the highly conserved -300 element is involved in the common regulatory mechanisms mediating the coordinated expression of the zein genes.

Biochim Biophys Acta, 1994 Jun 1, 1192(1), 79 - 87
Voltage- and Ca(2+)-dependence of the K+ channel in the vacuolar membrane of Chenopodium rubrum L . suspension cells; Reifarth FW et al.; Voltage- and Ca(2+)-dependence of the slow-activating SV-K+ channel in the vacuolar membrane of Chenopodium rubrum suspension cells has been analyzed using the patch clamp technique in the vacuole-attached, outside-out and whole-vacuolar configuration . Patch-pipette perfusion was applied to measure Ca2+ dependence of single channels in the attached-configuration . Using the PCLAMP-software (Axon Instruments), an algorithm was developed to extract reliable individual channel data from multi-channel activity records, including open probability, mean open and closed times, as well as time constants for open and closed distributions . The channel conductance of the major open state was about 83 pS (seal resistance > 8 G omega) at 30 mV (transmembrane voltage Vm, vacuole negative), and symmetrical 100 mM KCl . the channel exhibited a strong voltage- and a weak Ca(2+)-activation: increasing Vm from 40 to 100 mV is equivalent to a Ca2+ concentration change from 10(-7) to 10(-4) M . Mean open probabilities at Vm = 30 mV were 0.03 with 1 microM and 0.09 with 100 microM Ca2+ . Mean open times were approx . 7 ms, and almost independent of both, voltage and Ca2+ . Mean closed times, however, varied in a strongly voltage- and Ca(2+)-dependent manner, e.g., at Vm = 30 mV dropped from 205 to 67 ms, if Ca2+ was raised from 10(-6) to 10(-4) M . Open and closed distributions of events within bursts could be fitted by the sum of two exponentials with time constants between 0.3 and 11 ms.

Plant Mol Biol, 1994 Jun, 25(3), 401 - 12
Isolation of a monocot 3-hydroxy-3-methylglutaryl coenzyme A reductase gene that is elicitor-inducible; Nelson AJ et al.; The rice (Oryza sativa) phytoalexins, momilactones and oryzalexins, are synthesized by the isoprenoid pathway . An early step in this pathway, one that is rate-limiting in mammalian systems, is catalyzed by the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) . A gene that encodes this enzyme has been isolated from rice, and found to contain an open reading frame of 1527 bases . The encoded protein sequence of the rice HMGR appears to be conserved with respect to other HMGR proteins, and 1 or 2 membrane-spanning domains characteristic of plant HMGRs are predicted by a hydropathy plot of the amino acid sequence . The protein is truncated at its 5' end, and shows reduced sequence conservation in this region as compared to other plant sequences . The rice genome contains a small family of HMGR genes . The isolated gene, HMGR I, is expressed at low levels in both vegetative and floral organs of rice plants . It is not induced in plants by wounding, but is strongly and rapidly induced in suspension cells by a fungal cell wall elicitor from the pathogen Magnaporthe grisea, causal agent of rice blast disease . This suggests that HMGR I may be important in the induction of rice phytoalexin biosynthesis in response to pathogen attack, and therefore may play a key role as a component of the inducible defense mechanism in rice.

Vaccine, 1994 Feb, 12(2), 159 - 66
Immunogenicity of foot-and-mouth disease virus grown in BHK-21 suspension cells . Correlation with cell ploidy alterations and abnormal expression of the alpha 5 beta 1 integrin; Amadori M et al.; BHK-21 suspension cells were characterized with regard to genetic and phenotypic features which might adversely affect the immunogenic properties of foot-and-mouth disease virus (FMDV) grown therein . A positive correlation was found between number of passages in suspension culture and both prevalence of polyploid cells and reduced cell growth on surfaces . Suspension cells also revealed differences in the expression of RGD-specific integrins and, in particular, of alpha 5 beta 1, which was shown to work as an FMDV receptor structure . These features, along with the notable instability of a few non-structural FMDV A5 proteins in infected cells, outline a new scenario, in which the reduced immunogenicity of FMDV might be accounted for by defined negative influences of the cell environment on viral replication.

Plant J, 1993 Nov, 4(5), 855 - 62
The carrot secreted glycoprotein gene EP1 is expressed in the epidermis and has sequence homology to Brassica S-locus glycoproteins; van Engelen FA et al.; Non-embryogenic carrot suspension cells secrete the EP1 glycoprotein . A cDNA clone encoding EP1 was isolated and sequenced . The EP1 sequence revealed a region of homology with Brassica S-locus glycoprotein genes, an Arabidopsis S-like gene and putative S-like receptor protein kinases from maize and Arabidopsis . EP1 gene expression, analysed by in situ mRNA localization, was detected in cells located at the surface of the seedling: in the epidermis of the root, the hypocotyl and the cotyledons, in the root cap, and in a crescent of cells in the apical dome of the shoot . In developing seeds, expression was most pronounced in both the inner and outer integument epidermis.

Int J Hyperthermia, 1993 Nov-Dec, 9(6), 799 - 802
Thermal response of synchronous CHO cells with different shapes; Smith NN et al.; This study examines the differential heat sensitivity of rounded suspension cells versus flattened monolayer cells . G1 populations of two different Chinese hamster ovary lines were used to eliminate possible cell cycle artifacts . The cell populations were heated at 43 and 45 degrees C . In all cases, cells treated in suspension were less sensitive to heat killing than cells treated as monolayers.

J Membr Biol, 1993 Oct, 136(1), 43 - 54
Pharmacology of the SV channel in the vacuolar membrane of Chenopodium rubrum suspension cells; Weiser T et al.; Single channel performance and deactivation currents have been analyzed in the presence of cation channel blockers to reveal pharmacological properties of the slow-activating (SV) cation-selective ion channel in the vacuolar membrane (tonoplast) isolated from suspension cells of Chenopodium rubrum L . At a holding potential of -100 mV, the SV channel showed half-maximal inhibition with 20 mM tetraethylammonium (TEA), 7 microM 9-amino-acridine, 6 microM (+)-tubocurarine, 300 nM quinacrine, and 35 microM quinine, respectively . The SV channel is also blocked by charybdotoxin (20 nM at -80 mV) but not by apamine . 9-Amino-acridine, (+)-tubocurarine and quinacrine act in a voltage-dependent fashion, binding to the open channel and to different sites along the transmembrane voltage profile according to Woodhull (J . Gen . Physiol . 61:687-708, 1973) . No binding site could be specified for charybdotoxin, which binds to the closed channel, and for quinine . Except for quinine, all tested blockers were effective only if added to the cytoplasmic side of the tonoplast . A structural relationship between the SV channel and Maxi-K channels in animal systems is inferred.

Plant J, 1993 Sep, 4(3), 545 - 54
Molecular characterization of the gene for carrot cell wall beta-fructosidase; Ramloch-Lorenz K et al.; Carrot cell wall beta-fructosidase, previously purified and cloned, is encoded by a single, wound- and pathogen-inducible gene . The developmental regulation of the gene was studied by determining the steady-state mRNA levels in different organs during carrot development: cell wall beta-fructosidase mRNA was detected in roots and leaves of young plants but not during tap root development . A genomic clone was isolated and characterized . The transcription start site was determined by primer extension analysis . Inspection of the promoter sequence (1488 bp) revealed the presence of sequences with high homology to cis-acting elements for the regulation of plant genes by wounding and infection . The 5'-regulatory sequence was fused to the reporter gene beta-glucuronidase (GUS) and tested in a transient expression assay with carrot suspension cells and wounded carrot root tissue (aged disks of carrot roots) . The expression of the GUS gene in the transfected cells proved that the isolated promoter was functional . In transgenic tobacco plants containing the cell wall beta-fructosidase promoter fused to GUS, the reporter gene was predominantly expressed in the shoot and root meristems of young seedlings . No GUS expression was detected in mature tobacco plants, showing that the development-specific regulation of the cell wall beta-fructosidase promoter seen in carrot was maintained in tobacco plants . In contrast, expression of the GUS reporter gene in transgenic tobacco was not wound inducible . To analyze the functional organization of the cell wall beta-fructosidase promoter, a 5'-deletion series was generated and tested in a transient expression assay in protoplasts of Nicotiana plumbaginifolia . Two regions containing putative silencer elements were identified . A comparison of these regions with known silencer elements identified in both regions one copy of the negative dominant cis-acting element found in a chalcone synthase promoter of petunia.

Plant J, 1993 Aug, 4(2), 265 - 78
Growth-related gene expression in Nicotiana tabacum mesophyll protoplasts; Marty I et al.; Eight cDNAs whose genes are more strongly expressed in suspension cells in growth phase than in stationary phase and at a low level in mature leaves have been isolated . The corresponding mRNAs are abundantly accumulated in young plant organs and in germinating seeds but are almost undetectable in mature plant tissues and dry seeds . Six of these cDNAs were characterized by comparison of nucleotide and protein sequences to the EMBL and SWISSPROT databanks . These eight growth-related genes are expressed in protoplasts isolated from Nicotiana tabacum mesophyll cells shortly after preparation (4 h) . Two of them are expressed in freshly isolated protoplasts (early genes), while the other six are detected after 4 h of culture (late genes) . Seven are more abundantly expressed in protoplasts than in growing plant organs while one growth-related gene is weakly expressed in protoplasts, as is the histone H4 gene . They seem to be induced in protoplasts by a synergistic effect of wounding and maceration . Sustained expression of the early genes is dependent on the presence of sucrose in the culture medium.

Mol Gen Genet, 1993 Jul, 240(1), 1 - 8
Low-temperature-dependent expression of a rice gene encoding a protein with a leucine-zipper motif; Aguan K et al.; We have isolated from rice suspension cells three non-sequence-related cDNAs the expression of which is markedly induced by low, non-freezing temperature . Here we further characterize one of the cDNA clones, lip19 . Expression of lip19 is positively regulated by low temperature, but not affected by high (40 degrees C) temperature . Sequencing and primer extension analyses showed that lip19 has a long (552 bp) 5' non-coding sequence followed by a single open reading frame specifying a protein of 148 amino acids . The deduced amino acid sequence of the protein, Lip19, shows at its amino-terminus a conserved basic region followed by a "leucine-zipper" domain . The reported sequence most similar to Lip19 is maize OCSBF-1, which is a bZip-type DNA binding protein . The possibility is suggested that Lip19 is a transcriptional factor that is positively controlled by low temperature.

Plant Cell, 1993 Jun, 5(6), 603 - 13
Scaffold attachment regions increase reporter gene expression in stably transformed plant cells; Allen GC et al.; The yeast ARS-1 element contains a scaffold attachment region (SAR) that we have previously shown can bind to plant nuclear scaffolds in vitro . To test effects on expression, constructs in which a chimeric beta-glucuronidase (GUS) gene was flanked by this element were delivered into tobacco suspension cells by microprojectile bombardment . In stably transformed cell lines, GUS activity averaged 12-fold higher (24-fold on a gene copy basis) for a construct containing two flanking SARs than for a control construct lacking SARs . Expression levels were not proportional to gene copy number, as would have been predicted if the element simply reduced position effect variation . Instead, the element appeared to reduce an inhibitory effect on expression in certain transformants containing multiple gene copies . The effect on expression appears to require chromosomal integration, because SAR constructs were only twofold more active than the controls in transient assays.

Plant J, 1993 Apr, 3(4), 619 - 26
Identification of cis elements involved in Commelina yellow mottle virus promoter activity; Medberry SL et al.; Commelina yellow mottle virus (CoYMV) is a double-stranded DNA virus that infects a monocot host . A promoter fragment isolated from CoYMV is a strong promoter when assayed after transient introduction into monocot and dicot suspension cells and is highly active in vascular cells of flowers, leaves, stems and roots of stably transformed tobacco plants . Here it is reported that in stably transformed maize calli and transgenic tobacco leaves the CoYMV and CaMV 35S promoters exhibit similar amounts of activity . Deletion of the sequences located distal to nucleotide -230 relative to the start of transcription has no significant effect on promoter strength or tissue specificity . The region between -230 and -200 shares sequence similarity with the as-1 promoter element of the CaMV 35S promoter . Deletion of this as-1-like motif decreases promoter activity in maize suspension cells by 85% . Analysis of deletions affecting the -200 to -52 region indicates that sequences located between -159 and -84 are required for activity in vascular tissues . In addition, this region exhibits properties of a vascular tissue-specific enhancer since it confers vascular expression in an orientation-independent manner when fused to promoters that are not normally active in vascular tissues.

Cytotechnology, 1993, 11(1), 35 - 40
A new type porous carrier and its application to culture of suspension cells; Gotoh T et al.; A new type porous carrier was fabricated from a mixture of sodium alginate, bovine serum albumin and sodium bicarbonate . The porous space of the carrier is an assembly of void spaces . The carrier was successfully applied to the cultivation of suspension animal cells . In the culture, while both cells and carriers were held in suspension, the cells were entrapped hydrodynamically into the void spaces in the carriers . A culture of hybridoma cells using this carrier resulted in a cell density up to 5.7 x 10(7) cells per ml-carrier.

Cytotechnology, 1993, 11 Suppl 1, S106 - 8
Genotypic and phenotypic changes of BHK-21 cells grown in suspension cultures; Amadori M et al.; The propagation of foot-and-mouth disease virus on BHK-21 suspension cells, although economically convenient, may yield a scarcely immunizing antigen . Helpful insights were obtained by investigating a few genotypic and phenotypic features of the cell cultures . The appearance of polyploid populations, higher cell concentrations at the end of culturing, the progressive reduction of spreading on surfaces and an abnormal expression of the alpha 5 beta 1 integrin were found to be correlated with the number of passages in suspension culture . The observed modifications in the normal course of the cell-cycle and in the expression of some surface proteins point at a possible mechanism of virus damages arising from defective cellular functions.

Gene, 1992 Dec 15, 122(2), 247 - 53
Regulation of alpha-amylase-encoding gene expression in germinating seeds and cultured cells of rice; Yu SM et al.; Four alpha-amylase-encoding cDNA (alpha Amy-C) clones were isolated from a cDNA library derived from poly(A)+RNA of gibberellic acid (GA3)-treated rice aleurone layers . Nucleotide sequence analysis indicates that the four cDNAs were derived from different alpha Amy genes . Expression of the individual alpha Amy gene in germinating seeds and cultured suspension cells of rice was studied using gene-specific probes . In germinating seeds, expression of the alpha Amy genes is positively regulated by GA3 in a temporally coordinated but quantitatively distinct manner . In cultured suspension cells, in contrast, expression of the alpha Amy genes is negatively and differentially regulated by sugars present in the medium . In addition, one strong and one weak carbohydrate-starvation-responsive alpha Amy genes have been identified . Interactions between the promoter region (HS501) of a rice alpha Amy gene and GA3-inducible DNA binding proteins in rice aleurone cells were also studied . A DNA mobility-shift assay showed that the aleurone proteins interact with two specific DNA fragments within HS501 . One fragment is located between nt -131 to -170 and contains two imperfect directly repeated pyrimidine elements and a putative GA3-response element . The other fragment is located between nt -92 to -130 that contains a putative enhancer sequence . The interactions between aleurone proteins and these two fragments are sequence-specific and GA-responsive.

Biochem Biophys Res Commun, 1992 Nov 30, 189(1), 304 - 8
Methyl jasmonate conditions parsley suspension cells for increased elicitation of phenylpropanoid defense responses; Kauss H et al.; Pre-incubation of suspension-cultured parsley cells with methyl jasmonate greatly enhances their ability to respond to fungal elicitors by secretion of coumarin derivatives . The effect is most pronounced at relatively low elicitor concentration and also observed for the incorporation of esterified hydroxycinnamic acids and of "lignin-like" polymers into the cell wall . These three responses correspond to defense reactions induced locally when a fungal pathogen attacks plant cells . In contrast, the conditioning of parsley cells by the signal substance methyl jasmonate is reminiscent of the developmental nature of systemic acquired resistance and renders the cells more effective for the elicitor-induced local defense reactions.

J Biol Chem, 1992 Nov 25, 267(33), 23515 - 9
Sequence-specific interaction between S1F, a spinach nuclear factor, and a negative cis-element conserved in plastid-related genes; Zhou DX et al.; The nuclear gene rps1 coding for the spinach plastid ribosomal protein CS1 exhibits both a constitutive and leaf-specific expression pattern . In contrast to other chloroplast-related genes like rbcS and cab, the leaf induction of rps1 expression is light-independent . These unique features of rps1 expression provide good models to study the mechanisms regulating plastid development and differentiation in higher plants . We report on the identification of a spinach leaf nuclear factor, designated S1F, interacting with the rps1 promoter . The S1F binding site is conserved in the promoter region of many plastid-related genes, including rbcS, cab, and rpl21 . A binding activity similar to S1F was detected in nuclear extract from dark-grown de-differentiated soybean suspension cells . Through site-specific mutagenesis and transient expression in soybean cell protoplasts, we show that the S1F binding site is a negative element down-regulating the promoter activity of rps1 . A ligated tetramer of S1F site was able to repress activity of the cauliflower mosaic virus 35 S promoter extending the negative function of the S1F binding site on promoter activity.

J Biol Chem, 1992 Oct 25, 267(30), 21901 - 5
Phosphate starvation-inducible synthesis of the alpha-subunit of the pyrophosphate-dependent phosphofructokinase in black mustard suspension cells; Theodorou ME et al.; PP(i)-dependent phosphofructokinase (PFP) activity, measured in the forward direction, increased approximately 19-fold when suspension cell cultures of black mustard (Brassica nigra) were subjected to 18 days of P(i) deprivation . Fructose 2,6-bisphosphate (2 microM) elicited a 10-fold activation of PFP from P(i)-deficient cells, compared to only a 2-fold activation of the enzyme from nutrient-sufficient cells . Also, PFP from P(i)-starved cells exhibited a greater affinity for the activator (Ka = 0.09 microM) than the enzyme from nutrient-sufficient cells (Ka = 0.32 microM) . Western blots of extracts from P(i)-deficient cells were probed with rabbit anti-(potato tuber PFP) immune serum and revealed equal intensity staining immunoreactive polypeptides of M(r) 66,000 (alpha-subunit) and 60,000 (beta-subunit) that co-migrated with the alpha- and beta-subunits of homogeneous potato tuber PFP . By contrast, only the M(r) 60,000 beta-subunit was observed on immunoblots of extracts prepared from nutrient-sufficient cells . Quantification of immunoblots indicated that in black mustard cells experiencing transition from P(i) sufficiency to deficiency or vice versa, the relative amount of immunoreactive alpha-subunit correlated with the degree of activation of PFP by fructose 2,6-bisphosphate . These observations provide additional evidence that (i) plant PFP is an adaptive enzyme that may function in glycolysis during P(i) deprivation, and (ii) the alpha-subunit acts as a regulatory protein in controlling the catalytic activity of the beta-subunit and its regulation by fructose 2,6-bisphosphate.

Cancer Res, 1992 Sep 15, 52(18), 4948 - 53
Cell surface accessibility of individual gangliosides in malignant melanoma cells to antibodies is influenced by the total ganglioside composition of the cells; Lloyd KO et al.; The reactivity of a panel of antiganglioside monoclonal antibodies with a number of melanoma cell lines having different ganglioside composition profiles was studied . One cell line synthesized only GM3, one produced both GM3 and GD2, 2 had GM3 and GD3 as their major gangliosides, and 2 others synthesized approximately equal amounts of GM3, GM2, GD3, and GD2 gangliosides . Antibody reactivity with viable cells was analyzed by: (a) flow cytometry on suspension cells; and (b) mixed hemagglutination assays or immune adherence assays on monolayer cells in culture . GM3 was efficiently detected only in the cell line having GM3 as its sole ganglioside . In the other cell lines, GM3 was difficult to detect even in cells in which it made up a high proportion (up to 50%) of the total ganglioside content . GM2 was easily detectable only in JB-RH melanoma cells (which contain only GM3 and GM2) . GD3 was the most reactive ganglioside in 2 cell lines and GD2 in 2 other lines . In general, the most complex ganglioside present in a cell was the one most accessible to antibody . The differential exposure at the cell surface of specific gangliosides may have implications for antibody-directed tumor detection and therapy and for cell-protein or cell-cell interactions that involve glycolipids.

Arerugi, 1992 Sep, 41(9), 1367 - 79
{The culture of mouse bone marrow-derived mast cells (BMMC) and the anaphylactic release of chemical mediators}; Yamamura H et al.; Bone marrow cells from BALB/c, C3H/He and WBB6F1+/+ mice were cultured for 5 wks in the presence of the culture supernatant from prokoweed mitogen-stimulated spleen cells to assess and compare the degree of growth, proliferation and chemical mediator release of the mast cells (BMMC) derived from them . BMMC, which were positive to alcian blue staining, were found in the suspension cells on the culture of the bone marrow cells of either species of mice after 2 wk culture . The percentages of BMMC in the suspension cells were increased with time of culture, reaching more than 90% after 5 wks . No differences in the growth and proliferation rate among BMMC from these three species were observed . However, in regard to the amount of anaphylactic leukotriene (LT) and histamine release . BMMC from BALB/c mice were superior to those from other species . From the above results, subsequent experiments were executed with BMMC from BALB/c mice . There was no obvious difference in the releasability of anaphylactic mediators among BMMC obtained at any stages of the passage during 4-12 wk culture . On the other hand, although BMMC cultured for 4 and 5 wks well responded to Ca ionophore A23187 for these mediator release, those for 6 to 12 wks obviously deteriorated with prolongation of the culture . The time course of the anaphylactic release of immunoreactive (i-) LTB4, i-LTC4 and histamine from BMMC revealed that almost maximum release was reached at 10, 20 and 5 min, respectively, after antigen challenge . Several drugs including antiallergics and beta-stimulants had no effect on their release . From these results, it is suggested that present BMMC may be inadequate cells for evaluation of antiallergic drugs that can inhibit the anaphylactic mediator release, but may be useful for the research of the mechanism of the release because the cells likely release the mediators without occurrence of complicated subordinate reactions.

Plant Mol Biol, 1992 Aug, 19(5), 715 - 23
Site-specific oligodeoxynucleotide binding to maize Adh1 gene promoter represses Adh1-GUS gene expression in vivo; Lu G et al.; There is a 36 bp tract of extreme homopurine/homopyrimidine (PuPy) asymmetry in the maize Adh1 gene promoter (from -44 to -79) that is S1-hypersensitive in plasmids under supercoil tension . Oligodeoxynucleotides corresponding to the PuPy tract were designed to examine the secondary structure of the region and address the possible role of the tract in gene regulation . On the basis of oligodeoxynucleotide band-shift and DNase I footprinting analyses, it was concluded that the homopyrimidine oligodeoxynucleotide can form a triple helix with the duplex PuPy tract in vitro . Transient assays in protoplasts, suspension cells, and seedling roots show that the homopyrimidine oligodeoxynucleotide is also capable of repressing Adh1-GUS gene expression during co-transformation, presumably by the formation of a triple helix with the PuPy tract in vivo . The complementary homopurine oligodeoxynucleotide would not form a triple helix in vitro, nor would it repress Adh1-GUS in vivo . We propose that triple helix formation is a potential regulatory phenomenon in vivo, and that an intraregion triple helix could occur within the Adh1 promoter via the formation of H-DNA.

Plant Cell, 1992 Feb, 4(2), 185 - 92
The Commelina yellow mottle virus promoter is a strong promoter in vascular and reproductive tissues; Medberry SL et al.; Commelina yellow mottle virus (CoYMV) is a double-stranded DNA virus that infects the monocot Commelina diffusa . Although CoYMV and cauliflower mosaic virus (CaMV; another double-stranded DNA virus) probably replicate by a similar mechanism, the particle morphology and host range of CoYMV place it in a distinct group . We present evidence that a prompter fragment isolated from CoYMV confers a tissue-specific pattern of expression that is different from that conferred by the CaMV 35S promoter . When the CoYMV promoter is used to drive expression of the beta-glucuronidase reporter gene in stably transformed tobacco plants, beta-glucuronidase activity occurs primarily in the phloem, the phloem-associated cells, and the axial parenchyma of roots, stems, leaves, and flowers . Activity is also detected throughout the anther, with highest activity in the tapetum . In contrast, the CaMV 35S promoter is active in most cell types . The CoYMV promoter is a strong promoter, and when the activity of the CoYMV promoter is compared with that of a duplicated CaMV 35S promoter, it is 30% as active in tobacco suspension cells and up to 25% as active in maize suspension cells . These properties of the CoYMV promoter make it potentially useful for high-level expression of engineered genes in vascular cells.

J Gen Virol, 1992 Jan, 73 ( Pt 1), 27 - 37
Fusion activity and inactivation of influenza virus: kinetics of low pH-induced fusion with cultured cells; Duzgunes N et al.; The kinetics of fusion of influenza virus (A/PR/8/34) with human promyelocytic leukaemia (HL-60), human T lymphocytic leukaemia (CEM) and murine lymphoma (S49) cells were investigated . Fusion was demonstrated by electron microscopy, and monitored by fluorescence dequenching of octadecylrhodamine incorporated in the virus membrane . Rapid fusion was induced upon mild acidification of the medium . At pH 5, all virus particles were capable of fusing with the cells . The initial rate and the extent of fusion were maximal between pH 4.9 and 5.2 and declined sharply below and above this range . The rate constants of adhesion of influenza virus to cells or erythrocyte ghosts were large, indicating a diffusion-controlled process . The rate constants of fusion of the virus with cells were smaller than those found previously for fusion with various liposomes . Although preincubation of the virus at acidic pH in the absence of target membranes almost completely inactivated the virus in its ability to fuse with erythrocyte ghosts, it reduced the extent of fusion with cultured cells by only 20 to 40% . Kinetic analysis of fusion revealed a mode of inactivation of the virus bound to erythrocyte ghosts or suspension cells, below pH 5.4, different from that of the virus preincubated at low pH without target membranes.

J Recept Res, 1992, 12(1), 71 - 100
The effects of receptor density and cell shape on epidermal growth factor binding; Berkers JA et al.; In this paper we describe the effects of receptor density and cell shape on the binding of epidermal growth factor (EGF) to its receptor . Association kinetics of EGF binding to cells with a high receptor density was done using A431 cells . The association rate of EGF binding was apparently independent of the EGF concentration, most likely due to diffusion limited EGF binding as result of high receptor density . The effect of receptor density on EGF association rate was examined by reducing the number of functional EGF receptors on A431 cells . Preincubation of the cells with a monoclonal antibody directed against the EGF receptor and isolation of the cytoskeletons of A431 cells which both leaves only EGF binding to high affinity receptors revealed an EGF concentration dependent association rate . These results were confirmed in HeLa cells with 40 times less receptor numbers than A431 cells demonstrating the effect of receptor density on EGF binding . The influence of shape of the cell on EGF binding was examined by comparing the EGF association to monolayer cells with that of suspension cells . EGF association to suspended A431 cells was EGF concentration dependent . In conclusion we have shown that binding of EGF to A431 cells is dependent not only on the intrinsic rate constants but in addition on both receptor numbers per cell and the shape of cells . These results are in agreement with the hypothesis that EGF binding can be restricted by limited diffusion.

Enzyme Microb Technol, 1992 Jan, 14(1), 68 - 75
Influence of lactate, ammonia, and osmotic stress on adherent and suspension BHK cells; Wentz D et al.; Adherent and suspension Baby Hamster Kidney (BHK) 21c13 cells were cultivated in a 2.5-1 stirred-tank reactor with indirect aeration . Cell concentration and viability as well as glucose, lactate, ammonia, and protein concentrations in the medium and intracellular and extracellular activities of the intracellular enzymes were determined off-line . The concentrations of glucose, lactate, ammonia, and the activity of lactate dehydrogenase in the culture medium were monitored on-line . The cell/cell fragment size distribution was determined by laser flow cytometer off-line . In several runs, the size distributions were ascertained on-line by a laser flow cytometer . The influence of lactate, ammonia, and osmotic pressure on the viability and biological parameters of the suspension cells was evaluated . In Roux flasks, lactate and ammonia had considerable influence on the cell properties; in stirred tank reactors, these influences were negligible up to 9.5 g l-1 lactate and 150 mg l-1 NH+4 ion concentrations . The influence of high osmolarity on the biological parameters of the cells was much less in the stirred-tank than in the Roux flasks . The adhesion of adherent cells on a surface was impeded neither by the lactate (up to 6 g l-1) nor by the ammonia concentration (up to 150 mg l-1) . However, with increasing osmolarity, the fraction of the cells adhered to a surface reduced to below 5% (at 680 mOsmol l-1).

Nouv Rev Fr Hematol, 1992, 34 Suppl, S9 - 13
Cellular and molecular aspects of PECAM-1; Newman PJ et al.; PECAM-1 is a surface membrane glycoprotein of 130,000 daltons found on platelets, endothelial cells, monocytes, neutrophils, and certain T-cell subsets . Except for bone marrow precursor cells, it does not seem to be found outside the vasculature . Previous studies have shown that PECAM-1 is a member of the immunoglobulin gene superfamily that localizes to the intercellular junction in cells, like endothelial cells, that grow in monolayers . We and other laboratories have been involved in studies designed to define the role of PECAM-1 in various cell types . Mammalian expression vectors have been constructed that express all, or selected domains, of the PECAM-1 protein . These vectors have been used to induce PECAM-1 expression in a variety of both monolayer and suspension cell types . Domain-specific anti-PECAM-1 monoclonal antibodies, synthetic peptides, and heparin were also used to define structurally and functionally important domains of this membrane glycoprotein . Protein synthesis, subcellular localization, and association with the cytoskeletons of various cells expressing PECAM-1 were examined . From these studies we have obtained evidence that: (1) PECAM-1 redistributes to the border of monolayer cells when they contact each other . (2) Border localization may result from homophilic interactions, i.e . PECAM-1 on one cell binds PECAM-1 on the adjacent cell . (3) Suspension cells such as L-cells or neutrophils may also interact via PECAM-1 mediated interactions, but an as yet unidentified ligand probably participates in a heterophilic, rather than homophilic, type of interaction . (4) Intracellular movement of PECAM-1 in both monolayer and suspension cell types requires active participation of the cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1991 Jul, 227(3), 348 - 55
Transcription of the Petunia mitochondrial CMS-associated Pcf locus in male sterile and fertility-restored lines; Pruitt KD et al.; Transcripts of the Petunia mitochondrial cytoplasmic male sterility (CMS)-associated S-Pcf locus, which consists of three co-transcribed genes (pcf, NADH dehydrogenase subunit 3, and ribosomal protein S12), have been characterized in reproductive tissues of CMS and fertility restored (Rf) Petunia lines by nuclease protection experiments and by RNA blot hybridization . Three 5' transcript termini have been previously described . Two 3' transcript termini and an additional S-Pcf locus transcript have now been identified . The relative abundance of the three 5' transcript termini is influenced by the presence of the nuclear Rf gene . A decrease in the abundance of the -121 5' transcript terminus relative to the -266 and -522 termini is consistently seen in Petunia lines which are restored to fertility by the Rf gene, compared to CMS Petunia lines . An additional transcript with a 5' terminus within the urf-s region of pcf is much more abundant in immature bud and anther tissue than in leaf or suspension cells . The total abundance of pcf transcripts varies greatly between plants of different nuclear backgrounds which lack the nuclear Rf allele, indicating that other nuclear genes also influence expression of the S-Pcf locus.

Biochim Biophys Acta, 1991 Jul 1, 1066(1), 109 - 10
Charybdotoxin blocks cation-channels in the vacuolar membrane of suspension cells of Chenopodium rubrum L; Weiser T et al.; Using the patch-clamp technique, we studied the action of charybdotoxin which blocks Ca(2+)-activated large-conductance K+ channels in animal tissue on the slow-activating (SV), Ca(2+)-activated cation channel in the vacuolar membrane of suspension-cells of Chenopodium rubrum L . The toxin reversibly reduced the vacuolar current with EC50 approximately 20 nM suggesting structural similarities between ion channels in animal and plant membranes.

Planta Med, 1991 Jun, 57(3), 232 - 6
Ribosome-inhibiting proteins from in vitro cultures of Phytolacca dodecandra; Thomsen S et al.; Phytolacca dodecandra (L'Herit) grown in cell cultures was investigated for content of ribosome-inhibiting proteins, which was evaluated by measuring inhibition of protein synthesis in a cell-free rat liver extract . Calli initiated from leaf, cotyledon, radicle, and hypocotyl and suspension cells initiated from leaf and cotyledon exhibited protein synthesis-inhibiting activity . Ribosome-inhibiting proteins were purified at least 14 times from suspension cells of P . dodecandra . The purified protein fraction contained two proteins as seen by sodium dodecyl sulphate polyacrylamide gel electrophoresis . The relative molecular masses were 30,000 and 31,000 and they showed a pI greater than 9.3 . These new RIP's were shown to be different from dodecandrin with respect to molecular mass.

Biochim Biophys Acta, 1991 May 31, 1065(1), 8 - 14
Lipophilic polylysines mediate efficient DNA transfection in mammalian cells; Zhou XH et al.; Low molecular weight (Mr approximately 3000) poly(L-lysine) (PLL) conjugated to N-glutarylphosphatidylethanolamine is an effective carrier to promote DNA-mediated transfection in cultured mammalian cells . The conjugates, named 'lipopolylysines', contained an average of two phospholipid groups per molecule of PLL . Similar conjugates of the non-degradable poly(D-lysine) also had a similar transfection activity, indicating that the degradation of the carrier is not required for the activity . Unconjugated polylysines had little activity . The transfection activity of the lipopolylysine has been optimized with respect to the DNA concentration, DNA/carrier ratio, incubation time and the presence of serum in the incubation medium . The binding of lipopolylysine with DNA was measured by the degree of retardation of DNA in agarose gel electrophoresis . It was found that at the optimal DNA/lipopolylysine ratio for transfection, all DNA were found in large complexes which did not enter the gel . The transfection activity of the lipopolylysine, under optimal conditions, was approximately 3-fold higher than that of lipofectin, a widely used commercial reagent . Moreover, lipopolylysine mediated transfection even in the presence of 10% calf serum; whereas the lipofectin lost about 70% of its activity under the same condition . However, unlike lipofectin the transfection activity of the lipopolylysine depended on scraping the treated cells . Furthermore, lipopolylysine only transfected attached monolayer cells, and not suspension cells.

Biochim Biophys Acta, 1991 May 7, 1064(2), 199 - 204
The electrical response of maize to auxins; Felle H et al.; The electrical response of Zea mays coleoptiles and suspension cultured cells to several growth-promoting auxins (IAA, IBA, 2,4-D, 2,4,5-T, 1-NAA) and some of their structural analogues (2,3-D, 2-NAA) has been tested . In coleoptile two typical electrical responses to IAA are observed: an immediated rapid depolarization, and a hyperpolarization following 7-10 minutes after the first external addition of IAA . Of the other tested compounds only 1-NAA significantly depolarized the cells, whereas all auxins as well as the analogues evoked delayed hyperpolarizations . In contrast, the suspension cells were not hyperpolarized by any of the tested compounds, but were strongly depolarized by IAA, 1-NAA, and to a lesser extent by 2-NAA . In these cells IAA and 1-NAA induced inwardly directed currents of positive charge which both saturated around 12 mA/m2 . The strong pH-dependence together with the half-maximal currents 0.49 microM IAA and 0.76 microM 1-NAA point to a symport of the anions with at least 2H+ . The delayed plasma membrane hyperpolarization is a different response, and seems to be initiated by the protonated auxin species . In accordance with the current literature, it is interpreted as consequence of a stimulated proton extrusion . The finding that all tested compounds evoked a hyperpolarization, makes this response unspecific . It is concluded that a stimulation of proton extrusion is a necessary, but not sufficient step to induce elongation growth.

J Cell Physiol, 1991 May, 147(2), 242 - 7
Evidence for cooperativity of protein dissolution in Brij 58 permeabilized L929 cells; Ridsdale JA et al.; Mouse L929 cells were exposed to the nonionic detergent Brij 58 . As has been shown in some other cell types, protein leaked from Brij 58 exposed cells only after a lag phase . In the current study we have extended the observations of the kinetics of protein efflux using cultured L cells subjected to treatment with buffers containing Brij 58 . The results show that while the cells become permeable essentially at first exposure to the detergent, proteins do not escape immediately . This lag in efflux is at least partly dependent on the concentration of detergent such that a greater lag is seen in cells exposed to the lowest concentrations of Brij . Data are presented that are most readily interpreted as protein leakage having occurred fairly rapidly from individual cells and that show that the time course of protein efflux results, to a large extent, from different sensitivities of individual cells to the detergent . The permeabilized suspension cells consist of only two types, whereas the conversion of cells from one type to the other occurs through the loss of protein to the permeabilization medium . Only two bands are seen in continuous density gradients and there is a conversion of the more dense type to the less dense with longer exposure to detergent . Moreover, the less dense cells contained about half of the protein per cell as the bottom banding cells, and the proteins of the more dense cells appear to be the sum of those released into the permeabilization medium plus those found in the less dense cells.

J Membr Biol, 1991 Apr, 121(1), 11 - 22
Whole-cell and single-channel currents across the plasmalemma of corn shoot suspension cells; Fairley K et al.; Whole-cell sealed-on pipettes have been used to measure electrical properties of the plasmalemma surrounding protoplasts isolated from Black Mexican sweet corn shoot cells from suspension culture . In these protoplasts the membrane resting potential (Vm) was found to be -59 +/- 23 mV (n = 23) in 1 mM Ko+ . The mean Vm became more negative as {K+}o decreased, but was more positive than the K+ equilibrium potential . There was no evidence of electrogenic pump activity . We describe four features of the current-voltage characteristic of the plasmalemma of these protoplasts which show voltage-gated channel activity . Depolarization of the whole-cell membrane from the resting potential activates time- and voltage-dependent outward current through K(+)-selective channels . A local minimum in the outward current-voltage curve near Vm = 150 mV suggests that these currents are mediated by two populations of K(+)-selective channels . The absence of this minimum in the presence of verapamil suggests that the activation of one channel population depends on the influx of Ca2+ into the cytoplasm . We identify unitary currents from two K(+)-selective channel populations (40 and 125 pS) which open when the membrane is depolarized; it is possible that these mediate the outward whole-cell current . Hyperpolarization of the membrane from the resting potential produces time- and voltage-dependent inward whole-cell current . Current activation is fast and follows an exponential time course . The current saturates and in some cases decreases at membrane potentials more negative than -175 mV . This current is conducted by poorly selective K+ channels, where PCl/PK = 0.43 +/- 0.15 . We describe a low conductance (20 pS) channel population of unknown selectivity which opens when the membrane is hyperpolarized . It is possible that these channels mediate inward whole-cell current . When the membrane is hyperpolarized to potentials more negative than -250 mV large, irregular inward current is activated . A third type of inward whole-cell current is briefly described . This activates slowly and with a U-shaped current-voltage curve over the range of membrane potentials -90 less than Vm less than 0 mV.

Plant Mol Biol, 1991 Apr, 16(4), 647 - 61
Acid phosphatase-1(1), a tightly linked molecular marker for root-knot nematode resistance in tomato: from protein to gene, using PCR and degenerate primers containing deoxyinosine; Aarts JM et al.; With a view to cloning the root-knot nematode resistance gene Mi in tomato by chromosome walking, we have developed a molecular probe for the tightly linked acid phosphatase-1 (Aps-1) locus . The acid phosphatase-1 allozyme (APS-1(1}, encoded by the Aps-1(1) allele originating from Lycopersicon peruvianum, was purified to apparent homogeneity from tomato roots and suspension cells . Microsequencing of CNBr and tryptic peptides generated from APS-1(1) provided a partial amino acid sequence, which accounted for approximately 23% of the protein and revealed two stretches of homology with soybean proteins KSH3 and VSP27, comprising 22 matches within 26 amino acid residues . The partial amino acid sequence information enabled us to isolate a 2.4 kb genomic Aps-1(1) sequence by means of the polymerase chain reaction (PCR), primed by degenerate pools of oligodeoxyribonucleotides, synthesized on the basis of the amino acid sequences . Synthesis of the 2.4 kb PCR product was specific for genomic templates carrying the L . peruvianum Aps-1(1) allele . Crucial to the priming specificity and the synthesis of the 2.4 kb genomic sequence was the use of degenerate primer pools in which the number of different primer species was limited by incorporating deoxyinosine phosphate residues at three and four base ambiguities . In using cDNA as a template, a 490 bp sequence was obtained, indicating a high proportion of intron sequences in the 2.4 kb genomic Aps-1(1) sequence . The Aps-1(1) origin of the PCR product was confirmed by RFLP (restriction fragment length polymorphism) analysis, using both a chromosome 6 substitution line and a pair of nearly isogenic lines, differing for a small chromosomal region around the Aps-1/Mi loci.

Mol Gen Genet, 1991 Jan, 225(1), 65 - 71
Structural and functional analysis of promoter from gliadin, an endosperm-specific storage protein gene of Triticum aestivum L; Aryan AP et al.; To identify cis-regulatory elements of the gliadin gene, a study of the gliadin gene promoter was conducted by transient expression analysis of plasmid DNAs which were introduced into plant protoplasts by electroporation . The promoter region (-592 bp to +18 bp from the translational start) of this developmentally regulated gene, when fused upstream to the chloramphenicol acetyl transferase (CAT) reporter cassette was unable to direct significant CAT expression in wheat or tobacco suspension cells . Because this monocot gene promoter appeared to be under stringent tissue-specific control, a hybrid promoter approach using a nopaline synthase (nos) promoter was employed . A series of 3' deletions of the gliadin promoter were placed upstream of either a nonfunctional -101 nos or a nearly wild-type -155 nos promoter fused in turn to a CAT reporter gene cassette . Transient expression analysis of these plasmid DNAs in tobacco cells showed that the gliadin fragment could either restore the activity of the non-functional nos promoter (series I) or enhance the activity of the functional nos promoter (series II) . The degree of restoration of the promoter function conferred by gliadin fragments of the first series was proportional to the enhancing effect of the same fragments in the second series of constructs . The transcriptional activity of the gliadin (-592 bp to -77 bp) -nos hybrid promoter was reduced by 26% upon 3' deletion of sequences in the region -141 bp to -77 bp, which contains both the TATA and CCAAT boxes.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1990 Dec 15, 265(35), 21603 - 11
The mechanism of cysteine conjugate cytotoxicity in renal epithelial cells . Covalent binding leads to thiol depletion and lipid peroxidation; Chen Q et al.; Nephrotoxic cysteine conjugates kill cells after they are metabolized by the enzyme cysteine conjugate beta-lyase to reactive fragments which bind to cellular macromolecules . We have investigated the cellular events which occur after the binding and lead ultimately to cell death in renal epithelial cells . Using S-(1,2-dichlorovinyl)-L-cysteine (DCVC) as a model conjugate, we found that the phenolic antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD), butylated hydroxyanisole, butylated hydroxytoluene, propyl galate, and butylated hydroxyquinone, and the iron chelator deferoxamine inhibited the cytotoxicity significantly . Among the five antioxidants, DPPD was most potent . DPPD blocked DCVC toxicity over an extended time period, and the rescued cells remained functional as measured by protein synthetic activity . DPPD was able to block the toxicity of two other toxic cysteine conjugates S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine . In addition to LLC-PK1 cells, DPPD also protected freshly isolated rat kidney epithelial cells in suspension and in primary culture . In suspension cells, DPPD was effective at low doses of DCVC (25-50 microM) but not at high concentrations (250-500 microM) . DPPD inhibition was not due to an inactivation of beta-lyase or a decrease in the binding of {35S}DCVC metabolites to cellular macromolecules and occurred at a step after the activation of the toxins . During DCVC treatment, lipid peroxidation products were detectable prior to cell death . DPPD blocked lipid peroxidation over the whole time course . Depletion of nonprotein thiols also occurred prior to cell death . DPPD did not prevent the loss of nonprotein thiols . However, the sulfhydryl-reducing agent DTT blocked lipid peroxidation and toxicity at a step after the activation of DCVC . Therefore, it appears that cysteine conjugates kill renal epithelial cells by a combination of covalent binding, depletion of nonprotein thiols, and lipid peroxidation.

Plant Mol Biol, 1990 Dec, 15(6), 809 - 19
Optimization of delivery of foreign DNA into higher-plant chloroplasts; Ye GN et al.; We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of beta-glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells . Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity . No GUS activity was detected in cells bombarded with pUC118 or pBI101.3 . Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants . pHD203-GUS was expressed exclusively in chloroplasts . We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation . Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device . Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate . Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium . This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before.

Glycobiology, 1990 Sep, 1(1), 71 - 82
Purification and properties of arylmannosidases from mung bean seedlings and soybean cells; Pastuszak I et al.; Two arylmannosidases (signified as A and B) were purified to homogeneity from soluble and microsomal fractions of mung bean seedlings . Arylmannosidase A from the microsomes appeared the same on native gels and on SDS gels as soluble arylmannosidase A, the same was true for arylmannosidase B . Sedimentation velocity studies indicated that both enzymes were homogeneous, and that arylmannosidase A had a molecular mass of 237 kd while B had a molecular mass of 243 kd . Arylmannosidase A showed two major protein bands on SDS gels with molecular masses of 60 and 55 kd, and minor bands of 79, 39 and 35 kd . All of these bands were N-linked since they were susceptible to digestion by endoglucosaminidase H . In addition, at least the major bands could be detected by Western blots with antibody raised against the xylose moiety of N-linked plant oligosaccharides, and they could also be labeled in soybean suspension cells with {2-3H}mannose . Arylmannosidase B showed three major bands with molecular masses of 72, 55 and 45 kd, and minor bands of 42 and 39 kd . With the possible exception of the 45 and 42 kd bands, all of these bands are glycoproteins . Arylmannosidases A and B showed somewhat different kinetics in terms of mannose release from high-mannose oligosaccharides, but they were equally susceptible to inhibition by swainsonine and mannostatin A . Polyclonal antibody raised against the arylmannosidase B cross-reacted equally well with arylmannosidase A from mung bean seedlings and with arylmannosidase from soybean cells . However, monoclonal antibody against mung bean arylmannosidase A was much less effective against arylmannosidase B . Antibody was used to examine the biosynthesis and structure of the carbohydrate chains of arylmannosidase in soybean cells grown in {2-3H}mannose . Treatment of the purified enzyme with Endo H released approximately 50% of the radioactivity, and these labeled oligosaccharides were of the high-mannose type, i.e . mostly Man9GlcNAc . The precipitated protein isolated from the Endo H treatment still contained 50% of the radioactivity, and this was present in modified structures that probably contain xylose residues.

Cell Regul, 1990 Jul, 1(8), 605 - 14
Lateral diffusion of nerve growth factor receptor: modulation by ligand-binding and cell-associated factors; Venkatakrishnan G et al.; We compared the properties in human melanoma cell line A875 and rat pheochromocytoma cell line PC12 of nerve growth factor receptor (NGFr) . We also analyzed NGFr and a truncated NGFR lacking the cytoplasmic domain, which were transiently expressed in COS cells . The full-length NGFR expressed in COS cells bound nerve growth factor (NGF) with positive cooperativity, but A875 NGFr and truncated NGFr in COS cells did not display positive cooperativity . The anti-human NGFr monoclonal antibody NGFR5 was characterized and found not to compete with NGF for binding to NGFr . Fabs were prepared from NGFR5 and 192, an anti-rat NGFR monoclonal antibody that was previously shown not to compete with NGF for binding . Fluorescein-labeled Fabs were used to measure the distribution and lateral diffusion of the NGFr . NGFr expressed on COS and A875 cells are diffusely distributed, but NGFr on the surface of PC12 cells appeared, for some cells, to be patched . In A875 cells, 51% of the NGFr was free to diffuse with diffusion coefficient (D) approximately 7 X 10(-10) cm2/s . In COS cells, 43% diffused with D approximately 5 X 10(-10) cm2/s . There was no significant difference in diffusibility between the full-length NGFr and the truncated NGFr . We compared NGFr diffusion on PC12 cells in suspension or adherent to collagen-coated coverslips . For suspension cells, we obtained 32% recovery with D approximately 2.5 X 10(-9) cm2/s . On adherent cells, we obtained 17% recovery with 6 X 10(-9) cm2/s . Binding of NGF enhanced lateral diffusion of NGFr in A875 cells and in PC12 cells in suspension but did not alter lateral diffusion of NGFr in COS cells or in adherent PC12 cells . NGF had no effect on the diffusing fraction or the distribution of NGFR for any cell line.

Mutat Res, 1990 Feb, 228(2), 211 - 9
Effects of trypsin on X-ray-induced cell killing, chromosome abnormalities and kinetics of DNA repair in mammalian cells; Sprunt EA et al.; When cells are trypsinized before irradiation a potentiation of X-ray damage may occur . This is known as the 'trypsin effect' . Potentiation of X-ray damage on cell killing was seen in V79 Chinese hamster cells but was marginal in Chinese hamster ovary (CHO K1) cells and not evident in murine Ehrlich ascites tumour (EAT) cells . Trypsinization did however increase the number of X-ray-induced chromosomal abnormalities in all 3 lines . To investigate the possibility that trypsin acts by digestion of proteins in chromatin, further experiments were performed to monitor DNA damage and repair . Induction of DNA breaks by X-rays was unaffected by trypsin but trypsinized EAT (suspension) cells repaired single-strand breaks (ssb) less rapidly than controls indicating an inhibitory effect of trypsin on ssb repair . However double-strand break (dsb) repair was unaffected by trypsin . It was also found that the EDTA solution in which the trypsin was dissolved also contributes to the inhibition of dsb repair . The results show that trypsinization can enhance X-ray-induced cell killing, chromosomal damage and DNA repair, the effect varying between cell lines.

Proc Natl Acad Sci U S A, 1990 Feb, 87(4), 1406 - 10
Nuclear proteins bind conserved elements in the abscisic acid-responsive promoter of a rice rab gene; Mundy J et al.; We have previously shown that the expression of a rice gene, rab-16A, is responsive to abscisic acid (ABA) and osmotic stress in plant tissues and cultured suspension cells . We demonstrate here that transcriptional elements between -294 and -52 of this gene are sufficient to confer ABA-dependent expression on the chloramphenicol acetyltransferase reporter gene in rice protoplasts . Sequence motifs within this 242-base-pair region of the rab-16A gene are conserved among the 5' upstream regions of other ABA-responsive genes . Gel retardation and DNAse I experiments show nuclear factor(s) binding to these sequences . This correlative data indicate that these motifs are involved in the transcription of the rab genes and suggest that they may be ABA-responsive-elements (ABREs).

Ultrasound Med Biol, 1990, 16(7), 719 - 24
Protein synthesis stimulated in sonicated sugar beet cells and protoplasts; Joersbo M et al.; Sugar beet suspension cells and protoplasts were exposed to 20 kHz ultrasound and the amount of 35S-methionine incorporated into cellular protein was determined after 2 days of culture . The incorporation of 35S-methionine in cells was enhanced up to 90% after sonication for 220-770 ms at 1.2-3.5 W/cm2 which reduced viability 5-11% and decreased the average size of cell aggregates . In protoplasts, the 35S-methionine incorporation was increased up to 31% after sonication for 220-550 ms at 0.3-1.0 W/cm2 with a concomitant reduction in viability of 10-16% . At higher ultrasonic energies, 35S-methionine incorporation declined proportionally to the reduction in protoplast viability . At concentrations up to 30 mM, cystamine and cysteamine did not affect 35S-methionine incorporation or viability of the sonicated protoplasts . These results show that mild ultrasonic irradiation can stimulate protein synthesis in plant cells and protoplasts significantly.

Chin J Biotechnol, 1990, 6(2), 125 - 9
High frequency plant regeneration from protoplasts of wheat; Sun BL et al.; Calli were initiated from the mature seeds of wheat (Triticum asetivum L.cv . Xuzhou 211), and suspension cultures were established . The protoplasts isolated from suspension cells were cultured in the modified MS medium solidified with 0.8% agarose . Regenerated cells divided and calli formed . Whole plants were regenerated from protoplast-derived calli . Colony formation was promoted when the medium with lower osmotic pressure was added after two weeks culture . The frequency of regenerated plants was increased with lower concentration of sucrose in the differentiation medium . Shoots were induced effectively with high concentration of cytokinins and calliferous shoots were avoided . The frequency of regenerated plants was affected when protoplast-derived calli were transferred onto the differentiation medium in different periods.

Exp Hematol, 1989 Dec, 17(11), 1110 - 5
Hydrocortisone promotes survival and proliferation of granulocyte-macrophage progenitors via monocytes/macrophages; Pasquale D et al.; We have determined the mechanism by which hydrocortisone (Hc) promotes the survival and proliferation of normal human granulocyte-macrophage progenitors (granulocyte-macrophage colony-forming units, CFU-GM) . Peripheral blood mononuclear cells were cultured in a liquid system with 0-10.0 microM Hc over 3 weeks . At 7-day intervals 50% of the culture media along with the cells (suspension cells) present in the media were removed and replaced with fresh media . No CFU-GM or very small numbers of CFU-GM were contained in the suspension cells of the 14- and 21-day-old liquid cultures without Hc; CFU-GM were present and increased with increasing concentrations of Hc . The CFU-GM content in suspension cells of 14- and 21-day-old liquid cultures with 1.0 microM Hc was at least threefold higher compared to liquid cultures without Hc . In a double-layer CFU-GM agar culture system, the suspension cells from liquid cultures with 1.0 microM Hc, but not from liquid cultures without Hc, supported CFU-GM proliferation from normal human bone marrow cells . The CFU-GM proliferation-inducing ability was confined to the monocytes/macrophages (Mo) . CFU-GM colony inhibitory and stimulatory activities were detected in cell-free media recovered from liquid cultures without Hc, but only colony stimulatory activity was detected in the media from cultures with 1.0 microM Hc . These results indicate that greater than or equal to physiological concentrations of Hc (0.1-1.0 microM) are required for the persistence and proliferation of CFU-GM, and the effect of Hc is mediated through the Mo, probably by inhibiting the production of one or more of the CFU-GM colony inhibitory molecules.

Plant Cell, 1989 Dec, 1(12), 1137 - 46
Novel regulation of heat shock genes during carrot somatic embryo development; Zimmerman JL et al.; We have determined that somatic embryos of carrot exhibit a number of interesting and unusual properties when exposed to heat shock at different times in their development . Specifically, we have seen that mid-globular embryos can be arrested irreversibly in their development when heat-shocked, whereas all other stages of embryogenesis, both before and after this stage, are fully capable of normal development after the stress . In investigating the molecular basis of this developmental sensitivity to heat shock, using a cloned heat shock gene encoding a small heat shock protein, we have determined that globular embryos both synthesize and accumulate significantly less heat shock mRNA when compared with embryos of any other stage or to callus suspension cells . In fact, there appears to be no transcriptional induction of heat shock gene expression in response to heat shock during this time period; the gene is expressed at the same relatively low level both before and after heat shock . However, in spite of the low level of heat shock mRNA available, globular embryos synthesize the full complement of heat shock proteins in response to heat treatment . The globular embryos appear to accomplish this by translating the existing heat shock mRNAs at an elevated rate, which compensates for the low level of available mRNA . Once the embryos have progressed beyond the globular stage of development, regulation at the transcriptional level resumes, and the embryos again exhibit normal development after heat shock.

Plant Mol Biol, 1989 Nov, 13(5), 503 - 11
Transient gene expression in electroporated Solanum protoplasts; Jones H et al.; Electroporation was used to evaluate parameters important in transient gene expression in potato protoplasts . The protoplasts were from leaves of wild potato Solanum brevidens, and from leaves, tubers and suspension cells of cultivated Solanum tuberosum cv . Desiree . Reporter enzyme activity, chloramphenicol acetyl transferase (CAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, depended on the field strength and the pulse duration used for electroporation . Using field pulses of 85 ms duration, the optimum field strengths for maximum CAT activity were: S . brevidens mesophyll protoplasts--250 V/cm; Desiree mesophyll protoplasts--225 V/cm; Desiree suspension culture protoplasts--225 V/cm; and Desiree tuber protoplasts--150 V/cm . The optimum field strengths correlated inversely with the size of the protoplasts electroporated; this is consistent with biophysical theory . In time courses, maximum CAT activity (in Desiree mesophyll protoplasts) occurred 36-48 h after electroporation . Examination at optimised conditions of a chimaeric gene consisting of a class II patatin promoter linked to the beta-glucuronidase (gus) gene, showed expression (at DNA concentrations between 0-10 pmol/ml) comparable to the CaMV 35S promoter in both tuber and mesophyll protoplasts . At higher DNA concentrations (20-30 pmol/ml) the patatin promoter directed 4-5 times higher levels of gus expression . Implications and potential contributions towards studying gene expression, in particular of homologous genes in potato, are discussed.

Biochem J, 1989 Jul 15, 261(2), 679 - 82
Reconstitution of intermediate filaments from a higher plant; Hargreaves AJ et al.; Immunological studies have shown that plants contain intermediate-filament antigens, but it is not known whether these proteins are capable in themselves of forming filaments . To address this problem, a detergent-resistant and high-salt-insoluble fraction from carrot (Daucus carota L.) suspension cells was solubilized with 9 M-urea and then subjected to a two-step dialysis procedure, devised for the reconstitution of animal intermediate filaments . This induced the self-assembly of 10 nm filaments and large bundles of filaments . The predominant components of reconstituted material were polypeptides with apparent molecular masses between 58 and 62 kDa . These polypeptides immunoblotted with two monoclonal antibodies known to show broad cross-reactivity with intermediate filaments across the phylogenetic spectrum . This establishes that the antigens are able to self-assemble into intermediate-sized filaments.

J Virol, 1989 Jul, 63(7), 2921 - 8
Semliki Forest virus capsid protein acts as a pleiotropic regulator of host cellular protein synthesis; Elgizoli M et al.; The Semliki Forest virus capsid (C) protein was introduced into various target cells by electroporation-, liposome-, and erythrocyte-ghost-mediated delivery . Data are presented which show that the incorporated C protein is biologically active and, at low concentrations (10(3) to 10(4) molecules per cell), markedly induces host cellular protein synthesis (average value, up to 90%) . On the other hand, high concentrations (10(5) to 10(6) molecules per cell) led to a significant inhibition (average value, up to 60%) . The cellular response to C protein was found to be identical in P3X63Ag8 suspension cells, CV-1 cells, and GpBind4 cells . Following electroporation-mediated delivery of C-protein molecules, both induction and repression of cellular protein synthesis were immediate, whereas with liposome-mediated delivery these events were delayed by about 1 h . Maximum stimulation and repression occurred between 0 and 1 h after delivery of C protein and decreased thereafter to reach control values at about 4 h . The analysis of the proteins synthesized suggests that low amounts of microinjected C protein are responsible for the induction of classes with specific Mrs, whereas high amounts lead to an inhibition of overall protein synthesis.

J Cell Biochem, 1989 Jul, 40(3), 331 - 40
Evidence of an auxin-mediated phosphoinositide turnover and an inositol (1,4,5)trisphosphate effect on isolated membranes of Daucus carota L; Zbell BA et al.; Microsomal membranes from carrot suspension cells were phosphorylated in vitro with {gamma-32P}ATP . In the presence of submicromolar concentrations of the natural auxin indoleacetic acid (IAA), a rapid, but transient decrease of the {32P} label could be detected in the phospholipid extracts of the membranes . The phytohormone effect was not the result of an inhibition of the lipid phosphorylation reactions, but was caused by a simultaneous release of water-soluble compounds, which, according to their chromatographic properties, were assumed to contain inositol polyphosphates . Although the {32P}-labeled lipids, as well as the inositol polyphosphates, were not identified unequivocally by chemical analysis, these findings point to an auxin-mediated control of a phosphoinositidase C-like reaction similar to the hormone-stimulated phosphoinositide response in animals . Exogenously applied inositol (1,4,5)trisphosphate {(1,4,5)IP3} was found to release 45Ca2+ from preloaded membrane vesicles of carrot cells . Both the detection of the auxin-stimulated phosphoinositide response and the (1,4,5)IP3-mediated Ca2+ release on isolated cell membranes offer new experimental approaches for the identification of the putative auxin receptor and its signal transduction pathway.

Eur J Cell Biol, 1989 Jun, 49(1), 189 - 95
Organization of the vimentin system and its spatial relationship to the microtubule complex during the division of mammalian cells growing attached and in suspension; Paulin-Levasseur M et al.; We have used immunofluorescence staining with antibodies that detect vimentin, tubulin and the centrioles to compare the distributions of these respective antigens during the division of several suspension and attached cultured cells . Our observations demonstrate that 1) from distinct interphase organizations in suspension and attached cells, the vimentin system consistently rearranges with the onset of mitosis into a filamentous cage-like structure enclosing the spindle, 2) during cytokinesis, the polar centrosomes relocalize near the midbody in suspension cells while they remain at the pole opposite to it in attached cells, and 3) the vimentin cage is disintegrated and aggregated on each side of the midbody during cytokinesis in lymphoid cells but may be retained in other suspension cells.

Int J Radiat Biol, 1989 Mar, 55(3), 423 - 33
Interaction of hyperthermia and radiation in tolerant and nontolerant HeLa S3 cells: role of DNA polymerase inactivation; Kampinga HH et al.; The activities of DNA polymerase alpha and beta were measured in tolerant and nontolerant HeLa S3 suspension cells . The heat-inactivation of the enzymes and their recovery when cells were incubated at 37 degrees C after the heat challenge was compared to the synergistic action of heat and radiation and its disappearance at the level of cell survival . Thermotolerant cells were radiosensitized by heat similarly to nontolerant cells, but the sensitization decreased more rapidly in the tolerant cells when time at 37 degrees C was allowed between the two treatments . For polymerase activities the extent of inactivation, as well as the kinetics of recovery, were similar in tolerant and nontolerant cells . The results show that the activities of DNA polymerase alpha and beta do not always correlate with the extent of heat radiosensitization . It is concluded that heat inactivation of these enzymes may not be taken as a general cause for the synergistic effect of hyperthermia and radiation . As an alternative mechanism, changes in nuclear protein binding due to cellular heating are suggested, since these correlate well with effects observed for radiosensitization under different experimental conditions, including the use of thermotolerant cells.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1989 Feb, 22(1), 1 - 20
Serological analysis and biochemical characterization of monoclonal antibodies defining antigens of human hepatocellular carcinoma; Chang KJ et al.; A panel of seven murine monoclonal antibodies reactive with human hepatocellular carcinoma (HCC) cell line, SK- HEP-1, resulted in the definition of four distinct antigen systems, designated HB4, HB5, HB1 and HJ2 . HB4 antigen was found to be expressed specifically on HCC cell lines and fresh HCC specimens but not on normal liver . Immunoprecipitation tests suggest that the HB4 epitope may be a heat-stable carbohydrate determinant on a high molecular mass molecule . HB5 antigen was found to have less-restricted expression on a panel of normal adult tissues and on melanoma, astrocytoma, sarcoma, neuroblastoma and epithelial cancer cell lines . In fetal and adult liver, HB5 antigen localized to bile canaliculi and ducts . Under reducing conditions, three mAbs detected a Mr 140,000 glycoprotein using lysates of {125-I}, {3-H}-glucosamine and {35-S}-methionine labeled SK-HEP-1 cells . Under non-reducing conditions an additional component of greater than Mr 200,000 was also detected . HB1 antigen was found on almost all monolayer cell lines and not on most cultured suspension cells . This antigen was also detected on cultured HCC cells inoculated into nu/nu mice . Immunoprecipitation experiments revealed that the HB1 antigen is a bimolecular complex with an Mr 170,000 alpha chain and Mr 130,000 beta chain under non-reducing conditions, and three subunits of Mr 140,000, Mr 30,000 and Mr 130,000 under reducing conditions . Two antibodies reacted with epitopes on the alpha chain . HJ2 antigenic determinant is a heat-stable component which could not be immunoprecipitated . This most widely expressed antigen was found in secreted form in many of the cells and tissues examined . These antibodies introduce new antigens which may serve as useful markers for the diagnosis, classification and investigation of HCC and other liver diseases.

Biochem Biophys Res Commun, 1989 Jan 31, 158(2), 576 - 83
Multi-copy introduction and high-level expression of interleukin-3 genes by retroviral vector superinfection; Tsuchiya T et al.; We constructed a retroviral expression vector carrying multiple cloning sites . This vector was found to express efficiently the cloned gene . Using this vector and a helper virus-free system, a murine interleukin-3 (mlL-3) high-producing cell line was established by multiple cycles of infection with recombinant retroviruses carrying mlL-3 cDNA . The infected cells produced a considerable amount of mlL-3 and the concentration of mlL-3 in culture media increased as a function of the frequency of infection . High levels of mlL-3 cDNA, mRNA and protein in this cell line were confirmed by Southern, Northern and biological assays, respectively . These results suggest that artificial gene amplification is possible in a helper-free retroviral system . This should be applicable to efficient expression of bioactive molecules in a wide variety of mammalian cells including suspension cells.

Nature, 1989 Jan 26, 337(6205), 387 - 8
Cationic liposome-mediated transfection; Felgner PL et al.; A lipid named DOTMA has been created that forms unilamellar liposomes which complex with DNA and RNA for the transfection of mammalian cells, including suspension cells and hybridomas.

Ann N Y Acad Sci, 1989, 567, 104 - 21
SV40 T-antigen as a dual oncogene: structure and function of the plasma membrane-associated population; Butel JS et al.; SV40 T-antigen (T-ag) is localized in both the nucleus (nT-ag) and plasma membrane (pmT-ag) of cells and provides multiple functions necessary for cell transformation . The pmT-ag population is structurally very similar to the nT-ag . Transport to the cell surface is by an unknown mechanism that does not involve the secretory pathway . The disposition of T-ag in the membrane exposes both the amino and the carboxyl terminus on the exterior of the cell . Nuclear-transport-defective mutants of T-ag can transform established cells in culture, but not primary cells, suggesting that non-nuclear forms of T-ag may mediate some transformation-related process(es) . A non-cytolytic protein extraction technique utilizing 1-butanol solubilized from SV40-transformed cells a multimeric complex composed of pmT-ag and at least five cellular proteins ranging in size from 35,000 (35K) to 60K M . Both amino- and carboxylterminal T-ag-specific monoclonal antibodies co-precipitated T-ag and the 35-60K Mr proteins, but antibodies against the internal portion of T-ag precipitated only uncomplexed T-ag . The growth state of the cells markedly influenced the expression of the T-ag-containing surface complexes; more complexes were recovered from actively dividing cells than from confluent cell cultures, and suspension cells yielded more complexes than cells on a substratum . The complex exhibited a highly dynamic association with the cell membrane, as demonstrated by pulse-chase analysis . The characteristics of growth-dependent expression and rapid turnover rate suggest a functional role for the membrane complex . The identities of the cellular proteins in the complex with pmT-ag are unknown, although one member (56K) is recognized by p53-specific monoclonal antibodies.

EMBO J, 1988 Aug, 7(8), 2279 - 86
Abscisic acid and water-stress induce the expression of a novel rice gene; Mundy J et al.; We have identified a novel rice gene, called RAB 21, which is induced when plants are subject to water-stress . This gene encodes a basic, glycine-rich protein (mol . wt 16,529) which has a duplicated domain structure . Immunoblots probed with antibodies raised against beta-galactosidase/RAB 21 fusion protein detect RAB 21 protein only in cytosolic cell fractions . RAB 21 mRNA and protein accumulate in rice embryos, leaves, roots and callus-derived suspension cells upon treatment with NaCl (200 mM) and/or the plant hormone abscisic acid (10 microM ABA) . The effects of NaCl and ABA are not cumulative, suggesting that these two inducers share a common response pathway . Induction of RAB 21 mRNA accumulation by ABA is rapid (less than 15 min in suspension cells) and does not require protein synthesis, indicating that preformed nuclear and/or cytosolic factors mediate the response to this hormone . We have characterized the RAB 21 gene by determining the complete nucleotide sequence of a nearly full-length cDNA and corresponding genomic copy, and by mapping the start site of its major transcript . The proximal promoter region contains various GC-rich repeats.

Exp Cell Res, 1988 Jun, 176(2), 309 - 18
Wet cleaving of cells: a method to introduce macromolecules into the cytoplasm . Application for immunolocalization of cytosol-exposed antigens; Brands R et al.; A new method, called "wet cleaving," has been introduced to allow direct exposure of cytoplasm to externally supplied macromolecules by mechanical rupture of the plasma membrane . Monolayers of adherent cells or poly-L-lysine-attached suspension cells are overlayed with nitrocellulose sheets . By subsequent removal of the sheets, cells are cleaved, thereby exposing the cytoplasm . The method allows bulk quantities of cells to be cleaved in an efficient manner . Cleavage, although imposing some mechanical stress on the cells, leaves most if not all organelles morphologically intact, as shown by electron microscopy . Mechanically ruptured cells are well suited for use in immunocytochemical studies, as is demonstrated with the immunofluorescence localization of vinculin in chicken embryo fibroblasts.

Clin Exp Metastasis, 1988 Mar-Apr, 6(2), 141 - 52
Development of lymphosarcoma lines with high metastatic ability to lymph nodes and visceral organs in BALB/c mice; Tsuruo T et al.; A metastatic tumor population was isolated in BALB/c mice during routine s.c . passage of the colon 26 adenocarcinoma . The tumor metastasized to lymph nodes, liver, spleen, ovary and kidney . A primary culture established from the s.c . growing tumor was composed of both adherent and nonadherent cells . These two cell types were successfully separated from the primary culture and designated CMS (suspension cells) and CMA (adherent cells) . The CMS and CMA cell lines are morphologically distinct in culture; however both formed similar histopathologic tumors when inoculated s.c . Furthermore, both tumor lines showed identical metastatic patterns in BALB/c mice with involvement of lymph node, liver, spleen, ovary and kidney . CMS and CMA expressed T-antigen as revealed by FITC-labeled-anti-Thy 1.2 antibody . Chromosome analysis and morphologic studies by light and electron microscopy indicated that the present metastatic lines have no relationship with the colon 26 adenocarcinoma and seem to be non-thymic T-cell lymphosarcomas which developed spontaneously in BALB/c mice.

Tsitologiia, 1987 Sep, 29(9), 1007 - 11
{Characteristics of the structural organization of the cytoskeleton in suspension and monolayer sublines of mouse fibroblasts depending upon the type of growth in culture}; Bobrova IF et al.; In attached cells, the main part of filaments is localized in submembrane area, whereas suspension fibroblasts are situated deeper in the cytoplasm . Another difference involves the form of filament gatherings . In the former these are bundles stretched parallel to the cell surface, whereas in the latter these gatherings are in the form of loose balls near the nucleus . According to their diameter and type of cell localization the latter filaments are similar to intermediate ones . In suspension cells with caps formed by ligand treatment, gatherings of fibrillar elements are observed within the cytoplasm but these filaments are shorter and looser, being located in disorder . In addition, the increase in the number of electron dense granules of unknown nature is observed between these filaments . The above observations suggest that the surface processes may be associated with the inner parts of cytoskeleton.

J Cell Biol, 1987 Jul, 105(1), 387 - 95
An actin network is present in the cytoplasm throughout the cell cycle of carrot cells and associates with the dividing nucleus; Traas JA et al.; We have studied the F-actin network in cycling suspension culture cells of carrot (Daucus carota L.) using rhodaminyl lysine phallotoxin (RLP) . In addition to conventional fixation with formaldehyde, we have used two different nonfixation methods before adding RLP: extracting cells in a stabilizing buffer; inducing transient pores in the plasma membrane with pulses of direct current (electroporation) . These alternative methods for introducing RLP revealed additional features of the actin network not seen in aldehyde-fixed cells . The three-dimensional organization of this network in nonflattened cells was demonstrated by projecting stereopairs derived from through-focal series of computer-enhanced images . F-actin is present in interphase cells in four interconnected configurations: a meshwork surrounding the nucleus; thick cables in transvacuolar strands and deep in the cytoplasm; a finer network of bundles within the cortical cytoplasm; even finer filaments that run in ordered transverse array around the cell periphery . The actin network is organized differently during division but it does not disappear as do the cortical microtubules . RLP stains a central filamentous cortical band as the chromatin begins to condense (preprophase); it stains the mitotic spindle (as recently shown by Seagull et al . {Seagull, R . W., M . Falconer, and C . A . Weerdenburg, 1987, J . Cell Biol., 104:995-1004} for aldehyde fixed suspension cells) and the cytokinetic apparatus (as shown by Clayton, L., and C . W . Lloyd, 1985, Exp . Cell Res., 156:231-238) . However, it is now shown that an additional network of F-actin persists in the cytoplasm throughout division associating in turn with the preprophase band, the mitotic spindle, and the cytokinetic phragmoplast.

Blood, 1987 Apr, 69(4), 1021 - 5
Erythropoietic repopulating ability of stem cells from long-term marrow culture; Harrison DE et al.; Hemopoietic precursors are heterogeneous with respect to their capacity for self-renewal and long-term repopulating ability . Bone marrow cultures produce a variety of precursors over many weeks, including CFU-S; however, it is important to determine whether these populations retain the functional ability shown by fresh marrow . The most primitive precursor or stem cells have the most long-term repopulating ability . We here describe direct measurements of this ability in cells from marrow cultures by using competitive repopulation assays . Cultured adherent cells repeatedly showed less capacity than fresh marrow cells to repopulate erythropoiesis in irradiated recipients, whereas cultured suspension cells consistently had less capacity than adherent cells . Concentrations of macroscopic CFU-S measured at nine or 12 days were similar in cultured adherent and suspension cells and generally lower than those in fresh marrow . In every experiment, the long-term repopulating ability of the marrow cells used was substantially reduced after transfer into tissue culture . Thus, primitive stem cells may not proliferate in such cultures despite extensive production of CFU-S and more differentiated cell types.

Cell Tissue Kinet, 1987 Jan, 20(1), 89 - 98
Maintenance of granulopoiesis in long-term bone marrow cultures from W/Wv mice and effects of lipopolysaccharide on granulopoiesis in culture; Kirikae T et al.; An attempt was made to establish long-term cultures of marrow cells from genetically anaemic W/Wv mice . Two batches of horse sera were used . One batch of horse serum (HS-lot A) supported long-term maintenance (up to 20 weeks) of granulopoiesis in vitro . The number of suspension cells in W/Wv marrow culture was maintained at the same level as that in the control +/+ culture, but the number of granulocyte-macrophage progenitor cells (GM-CFC) and the ratio of immature to mature granulocytes were at a lower level than those in +/+ culture . These data suggest that haemopoietic progenitors in W/Wv cultures maintain a higher level of differentiation, and hence an increased self-renewal than those in +/+ cultures . Another batch of horse serum (HS-lot B) was less effective in the maintenance of the cultures, and the cultures deteriorated within 10 weeks . Addition of bacterial lipopolysaccharide (LPS) induced increased granulopoiesis in +/+ cultures, whereas such treatment resulted in the depletion of suspension cells in W/Wv cultures . The results suggest that haemopoietic cells of W/Wv mouse cannot cope with the strong stimulus for differentiation that occurs after the administration of LPS, although the cells can continue a moderately increased self-renewal and differentiation, as indicated by the results in the culture with HS-lot A.

Radiat Res, 1986 Jul, 107(1), 115 - 24
DNA conformation of Chinese hamster V79 cells and sensitivity to ionizing radiation; Olive PL et al.; Chinese hamster V79 cells grown for 20 h in suspension culture form small clusters of cells (spheroids) which are more resistant to killing by ionizing radiation than V79 cells grown as monolayers . This resistance appears to be due to the greater capacity of cells grown in contact to repair radiation damage . Attempts to relate this "contact effect" to differences in DNA susceptibility or DNA repair capacity have provided conflicting results . Two techniques, alkaline sucrose gradient sedimentation and alkaline elution, show no difference in the amounts of radiation-induced DNA single-strand breakage or its repair between suspension or monolayer cells . However, using the alkali-unwinding assay, the rate of DNA unwinding is much slower for suspension cells than for monolayer cells . Interestingly, a decrease in salt concentration or in pH of the unwinding solution eliminates these differences in DNA unwinding kinetics . A fourth assay, sedimentation of nucleoids on neutral sucrose gradients, also shows a significant decrease in radiation damage produced in suspension compared to monolayer cultures . It is believed that this assay measures differences in DNA conformation (supercoiling) as well as differences in DNA strand breakage . We conclude from these four assays that the same number of DNA strand breaks/Gy is produced in monolayer and spheroid cells . However, changes in DNA conformation or packaging occur when cells are grown as spheroids, and these changes are responsible for reducing DNA damage by ionizing radiation.

Nucleic Acids Res, 1986 Jun 25, 14(12), 4953 - 68
The structure of the maize ribosomal DNA spacer region; McMullen MD et al.; A combination of a single stranded plasmid vector and ordered deletions were used to determine the complete nucleotide sequence of a rDNA spacer region isolated from the DNA of maize Black Mexican Sweet suspension cells . The sequence reveals the presence of ten "200 base" subrepeats within the maize rDNA spacer region . By S1 protection experiments we have tentatively determined that the start of maize rRNA transcription is 144 bases 3' of the end of the last spacer subrepeat . We propose that the spacer subrepeats may have an important role in regulating maize rRNA transcription.

Radiat Res, 1986 Mar, 105(3), 405 - 12
Changes in bleb formation following hyperthermia treatment of Chinese hamster ovary cells; Kapiszewska M et al.; Chinese hamster ovary cells in suspension cultures were heated for various times at 41.5, 43.5, and 45.5 degrees C, and quantitative determinations of microblebbing and macroblebbing of the cell membrane were performed for cells maintained at 4, 25, and 37 degrees C after hyperthermia . The percentage of cells with blebs following heating at 45.5 degrees C was dependent upon the duration of heating with increases from 40% for 5 min to 90% for 30 min . Cells exposed to lower temperatures exhibited less blebbing which was not quantifiable . The changes in bleb formation following 45.5 degrees C were dependent upon the posthyperthermia temperature: a slight decrease of macroblebbing at 25 degrees C, a decrease to 50% by 2 h at 37 degrees C, and a sharp decrease of macroblebbing to less than 10% by 1 h at 4 degrees C . Microblebbing increased slightly at 37 degrees C . When cells were transferred rapidly from the 4 degrees C posthyperthermia incubation to 37 degrees C, the bleb formation percentages returned rapidly to the higher levels which existed before posthyperthermia incubation at the lower temperatures . Gamma irradiation of 20 and 50 Gy produced only a small increase in microblebbing at longer periods (5 to 6 h) but no increase in macroblebbing . The survival of cells heated for 20 min at 45.5 degrees C was decreased 40% for suspension cells maintained at 4 degrees C for 2 to 3 h before incubation at 37 degrees C for colony formation compared to cells immediately incubated at 37 degrees C after heating . The survival of cells maintained at 25 degrees C after heating was not altered in comparison.

EMBO J, 1985 Dec 1, 4(12), 3123 - 30
Immuno-isolation of a plasma membrane fraction from the Fao cell; Devaney E et al.; A plasma membrane was immuno-isolated from a post-nuclear supernatant of a cultured rat hepatocyte, the Fao cell, using a cellulose immuno-adsorbent and antibodies raised against a variety of endogenous antigens of hepatocytes: 5'-nucleotidase, a plasma membrane fraction and the whole Fao cell . The antibodies which recognize antigens on the cell surface were selected from the total serum by first binding the antiserum to suspension cells . Alternatively, the plasma membrane and Fao antisera were affinity purified on a column prepared from a Triton X-114 extract of a plasma membrane fraction . The immuno-isolation was most efficient when carried out with either the plasma membrane or the Fao anti-serum . When alkaline phosphodiesterase I or 5'-nucleotidase was used as the plasma membrane marker, 40-60% of the plasma membrane of the post-nuclear supernatant was isolated representing a maximum 34-fold increase in the specific activity of the enzymes in the bound material . Using the NaB-{3H}4-labelled glycoproteins of the plasma membrane or the IgG bound to the plasma membrane as alternative markers, an 80% isolate of the plasma membrane of the post-nuclear supernatant was achieved, resulting in an estimated 40-fold purification . The non-specific binding was low despite the use of a post-nuclear supernatant as the input fraction . The characterization of the bound materials suggested that the whole plasma membrane was immuno-isolated and not a particular domain.

Virology, 1985 Oct 15, 146(1), 111 - 9
Characterization and assembly of poliovirus-related 45 S particles; Rombaut B et al.; Using detergents, 45 S particles could be extracted from poliovirus-infected Vero and human embryonic kidney monolayer cells, but not from HeLa suspension cells . They were composed of the capsid proteins VP0, VP1, and VP3, devoid of RNA, and extremely sensitive to heat or to a slightly alkaline pH . The 45 S particles possessed neutralization epitopes of the N1 and N2 classes, as well as a VP3-linked epitope . The N2 epitopes were lost upon denaturation . In the presence of cell extracts 45 S particles were, like 14 S subunits, assembled to 71 S empty capsids expressing the N1 and N2 epitopes.

J Clin Invest, 1985 Jun, 75(6), 2085 - 90
Extracellular matrix promotes the growth and differentiation of murine hematopoietic cells in vitro; Campbell A et al.; We have developed a long-term culture system in which murine marrow cells are cultured on a complex extracellular matrix (ECM) that is derived from marrow and extracted with guanidine hydrochloride and dithiothreitol . Marrow cultures were established with fresh murine marrow cells and recharged at 2 wk (week 0) . Phase microscopy showed a dramatically increased adherent cell layer development on ECM compared with controls within a week after recharge . By electron microscopy, this adherent layer was composed of numerous reticular cells apparently attached to the ECM which extended cytoplasmic projections to the surrounding hematopoietic cells . Adherent cellularity on ECM-coated dishes increased to 30 times the control values by week 2 . Cumulative suspension cells on ECM dishes were eight times controls . ECM influenced both hematopoietic progenitor cell proliferation and differentiation . Adherent colony-forming unit-granulocyte/macrophage and colony-forming unit-megakaryocyte were greater than 30 and 15 times the control values, respectively, by week 2 (P less than or equal to 0.05) . There were more mature granulocytic and megakaryocytic cells in ECM-coated dishes than in controls at all time points . This new culture system directly demonstrates that ECM is an important component of the hematopoietic microenvironment.

Mol Cell Biol, 1985 May, 5(5), 1188 - 90
Gene transfer method for transient gene expression, stable transformation, and cotransformation of suspension cell cultures; Gopal TV; A new method was developed to study transient gene expression, stable transformation, and cotransformation in suspension cells, such as mouse myeloma and erythroleukemia cells . This method involves attachment of cells to a concanavalin A-coated tissue culture dish, treatment of cells with DEAE-dextran to adsorb plasmid DNA to the attached cells, and finally treatment with a 40% solution of polyethylene glycol to facilitate the uptake of DNA by the cells . Plasmids pSV2cat and pSV2neo were used as markers to optimize the conditions for transient gene expression and stable transformation, respectively, of mouse myeloma and erythroleukemia cells . This method was successfully used to obtain cotransformants of mouse myeloma cells.

J Cell Biol, 1985 May, 100(5), 1793 - 8
Monoclonal antibody to intermediate filament antigen cross-reacts with higher plant cells; Dawson PJ et al.; The monoclonal antibody (anti-IFA) raised (Pruss et al., 1981, Cell 27:419-428) against an intermediate filament antigen, which is widespread throughout phylogeny, has been shown here to cross-react with higher plants . On immunoblotting, anti-IFA cross-reacted with proteins in homogenates of carrot suspension cells and of meristematic cells from onion root tips . A 50-kD cross-reactive protein was enriched in a fraction that consisted of detergent-insoluble bundles of 7-nm fibrils from carrot protoplasts (Powell et al., 1982, J . Cell Sci . 56:319-335) . By use of indirect immunofluorescence, anti-IFA stained formaldehyde-fixed onion meristematic cells and carrot protoplasts in patterns approximating those obtained with monoclonal anti-tubulins . That anti-IFA was not recognizing plant tubulins was established by use of immunoblots of two-dimensional gels on which the proteins that comprised isolated fibrillar bundles and taxol-purified carrot tubulins had been separated . The two groups of proteins had different positional coordinates: anti-IFA recognized the fibrillar bundle proteins, and anti-tubulins recognized plant microtubule proteins with no cross-reaction to the heterologous proteins . Likewise, formaldehyde-fixed taxol microtubules from carrot cells could be stained with anti-tubulin but not with anti-IFA . It is concluded that an epitope common to intermediate filaments from animals co-distributes with microtubules in higher plant cells.

Blood, 1985 Mar, 65(3), 736 - 43
Regulation of erythropoiesis in long-term hamster marrow cultures: role of bone marrow-adherent cells; Eastment CE et al.; The initial establishment of hamster long-term bone marrow (LTBM) cultures requires formation of an adherent stromal layer, but continued long-term proliferation of these cultures is best accomplished by removal of the suspension cells from the adherent layer and subsequent incubation in liquid suspension culture . Continued maintenance of bone marrow cells in the presence of the adherent layer for more than four to six weeks leads to a decline and eventual disappearance of erythroid and multipotent colony-forming cells . Addition of erythropoietin to LTBM suspension cultures produces mature, hemoglobinized erythrocytes . Incubation of the same cells plus erythropoietin in the presence of autologous parental adherent layers markedly inhibits both terminal erythroid differentiation and the number of detectable erythroid burst-forming units (BFU-Es) . This erythroid inhibition occurs primarily within the first 24 hours with little or no effect on CFU-GEMMs or granulocyte-macrophage colony-forming units (GM-CFUs) . However, continued incubation for seven days produces a reduction in all parameters . Removal of suspension cells from the adherent layer and restimulation with erythropoietin allows regeneration of erythropoiesis . Pretreatment of suspension cells with erythropoietin for 96 hours before exposure to the adherent culture only slightly inhibits erythropoiesis, suggesting that more mature erythroid progenitors are unaffected . Conditioned medium from the marrow adherent layer (ALCM) produces similar erythroid inhibitory effects in LTBM cultures with as little as two hours of incubation . The inhibition is actively produced by the adherent cells, since cycloheximide abolishes its production, while indomethacin has no apparent effect . Adherent marrow stromal cells may regulate hemopoiesis through negative as well as positive humoral signals, and they are particularly effective in erythroid regulation.

Eur J Biochem, 1985 Jan 15, 146(2), 323 - 9
Ether glycerolipids: novel substrates for studying specificity of enzymes involved in glycerolipid biosynthesis in higher plants; Weber N et al.; Ether glycerolipids, predominantly alkylacylglycerols and alkylacylglycerophosphocholines, are synthesized in photomixotrophic rape (Brassica napus) suspension cells from various exogenous monoalkylglycerols . The stereospecific distribution of acyl moieties was studied in these ether glycerolipids with regard to chain-length and degree of unsaturation of alkyl moieties and compared with the distribution of acyl moieties in the corresponding endogenous acyl glycerolipids . The results show the following: (1) Alkylacylglycerophosphocholines replaced up to one-half of the corresponding physiological membrane lipids, i.e . diacylglycerophosphocholines, without changing the total amount of cholineglycerophospholipids as compared to untreated cells . (2) The composition of acyl moieties in total lipids of rape cells was practically unaltered by fatty acids derived via oxidative cleavage from the various alkyl moieties of either glycerolipids . (3) In 1-O-alkyl-2-acylglycerols derived from exogenous alkylglycerols and in endogenous 1,2-diacylglycerols compositions of acyl moieties were found to be different indicating that different pathways were operative in the biosynthesis of these two neutral glycerolipids . (4) Enzymes involved in synthesizing molecular species of 1-O-alkyl-2-acylglycerophosphocholines or 2-O-alkyl-1-acylglycerophosphocholines as well as 1,2-diacylglycerophosphocholines showed similar specificities with regard to chain-length and degree of unsaturation of both alkyl and corresponding acyl moieties . Thus, ether glycerolipids formed by plant cells from exogenous alkylglycerols are suitable metabolites for studying the specificity of enzymes involved in the biosynthesis of glyerolipids.

Dev Biol Stand, 1985, 60, 179 - 83
Parvovirus serologically related to the minute virus of mice (MVM) as contaminant of BHK 21 cl . 13 suspension cells; Zoletto R; From BHK 21 cl . 13 suspensions cells we have isolated a virus serologically related to the Minute Virus of Mice (MVM); the same virus has been isolated from the same cells in U.K . in 1980 . Besides isolating this virus from large scale cultures, we have also isolated it from many of the sublines stored in nitrogen we received at different times from different laboratories . At this time it is difficult to ascertain if the contamination originated from the serum or from other components of the medium; furthermore we do not know if many of the cells of our stock became contaminated in our laboratory or if they were already contaminated.

Cell Biol Int Rep, 1985 Jan, 9(1), 5 - 12
Some biochemical properties of higher plant tubulins; Mizuno K et al.; Tubulin was isolated by a combination of affinity (ethyl N-phenylcarbamate-Sepharose) and ion exchange (DEAE-Sephacel) chromatography from mung bean and cultured carrot suspension cells . SDS-PAGE (Blose 1981) of mung bean tubulin has shown it to consist of two major subunits (MBT1 and MBT2) and a minor subunit (MBT3) . Tubulin isolated from carrot cells was resolved into only two bands on SDS-PAGE (slow moving subunit was named CT1) . However, the faster moving subunit on SDS-PAGE was resolved into two bands (CT2 and CT3) on SDS-4M urea-PAGE . On SDS-4M urea-PAGE, CT1 migrated faster than CT2, CT3 . By contrast in SDS-4M urea-PAGE, mung bean tubulin remains unresolved . Mammalian tubulin could be resolved into alpha and beta-subunits in both electrophoretic systems . Monoclonal antibodies to mammalian alpha and beta-tubulin subunits (MCA-T alpha and MCA-T beta, respectively) and Western blot analysis clearly demonstrated a cross-reactivity of MCA-T alpha with MBT2, MBT3, CT2 and CT3, while MCA-T beta showed cross-reactivity with MBT1 and CT1 . Although MBT2, MBT3, CT2 and CT3 are immunologically related to the alpha-subunit of mammalian tubulin, their migration on SDS-PAGE was reversed with respect to MBT1 or CT1, which were immunologically related to the beta-subunit of mammalian tubulin . Peptide mapping patterns also supported above the results.

Cancer Surv, 1985, 4(2), 399 - 419
Phenotypic and genotypic analysis of Hodgkin's disease derived cell lines: histopathological and clinical implications; Diehl V et al.; Five Hodgkin's disease (HD) derived cell lines were established in vitro in our laboratory in the last seven years . Morphological, cytochemical, immunological and cytogenetic marker analysis demonstrated that the in vitro cells represent genotypically and phenotypically the in vivo Hodgkin (H) and Sternberg-Reed (SR) cells in biopsy specimens . The cultured cells resemble haematolymphoid cells at different stages of maturation . Four of the five continue to grow in vitro as suspension cells after more than 50 months . Four more in vitro HD-derived lines were described recently by several authors . A summary of the various marker characteristics of these in vitro lines is given as a synopsis of the phenotypic marker spectrum and is discussed in comparison with our own cell lines . There is a striking similarity between two of the newly established lines (CO, HDLM-2) and our lines whereas the two other in vitro established cultures seem to resemble cell species further along the line of maturation to B lymphocytes (DEV) and monocytes (SU-HD-1) . Gene rearrangement experiments undertaken with the L428, L540, L591 and the CO cell line show that the L428 and 591 cells have undergone gene rearrangement, the L428 being compatible with the genotypic state of a pre-B cell; the L591 cells, similarly rearranged furthermore demonstrated functional light chain rearrangement, compatible with B-cell development . By cytogenetic analysis chromosome 7 was found to be affected in all our described lines . This chromosome appears to be particularly unstable and vulnerable in patients with HD, since all tested cell lines revealed multiple abnormalities of this chromosome, a finding which is in accordance with observations made by other investigators in HD-biopsy cells . Since similar structural changes or loss of chromosome 7 is a characteristic event in cases of secondary acute non-lymphocytic leukaemia, it is speculated that this form of secondary neoplasia could resemble the blast crisis, as observed in chronic myeloid leukaemia.

Dev Biol Stand, 1985, 60, 163 - 70
The use of BHK suspension cells for the commercial production of foot and mouth disease vaccines over a twenty year period; Radlett PJ et al.; The Wellcome Foundation Ltd first used BHK suspension cells for the commercial production of foot and mouth disease vaccine in 1964 . Since that time both the scale of operation and the volume of production has increased dramatically and in 1983 the Wellcome Group produced almost 350 million monovalent equivalent doses of vaccine from just over 2 million litres of antigen . Experience gained during this period has shown that with careful attention to plant design and operation this large industrial scale tissue culture process can be managed reliably and efficiently . Data will be presented to show the level of production and success rate achieved in our Foot and Mouth Vaccine production units together with an analysis of antigen yields and the comparatively few failures experienced.

J Cell Biol, 1984 Sep, 99(3), 788 - 95
Tyrosyl kinases acquired from anchorage-independent cells by a membrane-enveloped virus; Clinton GM et al.; Tyrosyl kinase activity in vesicular stomatitis virus (VSV) acquired from host cells that differ in morphology was investigated . VSV grown in baby hamster kidney (BHK) cells with rounded morphology and a high efficiency of colony formation in soft agar (Rous sarcoma virus {RSV}-transformed and suspension BHK cells) was compared with VSV grown in BHK cells with a flattened morphology and lower efficiency of colony formation in soft agar (RSV-infected revertant and control BHK cells) . Tyrosyl kinase activity measured with the substrates angiotensin II peptide or casein was found at 7-10-fold higher levels in virus released from the anchorage-independent BHK cells . Most of the VSV-associated tyrosyl kinases acquired from the RSV-transformed BHK cells reacted with antiserum to pp60src, whereas the activity acquired from the suspension BHK cells was unaffected by anti-src serum . The overall levels of tyrosyl kinase in subcellular fractions of the host BHK cells were also measured . Like the VSV released from them, the RSV-transformed cell extracts contained high levels . The suspension cells, however, contained the same low levels of tyrosyl kinase as was found in the control BHK cell extracts . Therefore, tyrosyl kinase was concentrated and acquired by VSV from the anchorage-independent suspension BHK cells . VSV-associated protein kinases acquired from other cell types followed a similar pattern . Tyrosyl kinase levels were high in VSV released from suspension cultures (Chinese hamster ovary and HeLa) and from virally transformed cells (Kirsten murine sarcoma virus-transformed rat kidney cells) and low in VSV released from an anchorage-dependent primary cell culture (chick embryo fibroblasts).

Eur J Biochem, 1984 Jan 2, 138(1), 93 - 9
Role of the ATPase of sugar-cane vacuoles in energization of the tonoplast; Thom M et al.; Vacuoles of sugar-cane suspension cells contained a tonoplast-bound ATPase which was exclusively located on the cytoplasmic side of the vacuole . Vanadate and diethylstilbestrol had little effect on the vacuolar ATPase . ATP was the optimum substrate for the tonoplast ATPase, but there was also evidence for tonoplast-bound GDP-hydrolyzing and GTP-hydrolyzing enzymes which can interfere with the ATPase assay . Other phosphate anhydrides and esters were not hydrolyzed . The addition of MgATP polarized the tonoplast from about 0 mV to an interior-positive value of about +20 mV; MgADP and MgGTP had much less effect; MgGDP and ATP (in the absence of magnesium) had no effect on the membrane potential . The polarization of the tonoplast was insensitive to valinomycin, nigericin, and inhibitors of plasmalemma ATPase, but was strongly reduced by the uncoupler carbonylcyanide m-chlorophenylhydrazone . These data are interpreted as evidence for the action of tonoplast-bound ATPase as a pump which translocates protons into the vacuoles . The activity of the ATPase was highly specific for MgATP2-; the other important ionic states of ATP:ATP4-, HATP3-, MgHATP-, and Mg2ATP neither stimulated nor inhibited . The same was true for Mg2+ . Since the protons were not brought to the catalytic site by protonation of the substrate, the tonoplast-ATPase may pick up the proton for translocation from the cytoplasm . The saturation kinetics for MgATP2- hydrolysis were biphasic, the higher affinity ATPase with Km value of 0.7 mM seems to be the physiologically relevant activity.

J Biol Stand, 1983 Jul, 11(3), 145 - 55
The improvement and standardization of the simplified process for the production of foot and mouth disease virus from BHK suspension cells; Whiteside JP et al.; Improvements have been made to the methodology for the production of foot and mouth disease (FMD) virus from BHK 21 clone 13 suspension cells by the simplified process . Data derived from some 600 individual 8-1 cultures covering all seven types of FMD virus has been analysed . The production of virus was shown to (1) have no direct relationship to cell passage level and (2) to be inversely related to the cell multiplication factor observed during the cell growth cycle immediately prior to infection.

Biochim Biophys Acta, 1983 Feb 16, 762(1), 86 - 93
Isolation and characterization of phosphoprotein pp 105 from simian virus 40-transformed mouse fibroblasts; Pfeifle J et al.; Investigation of the cellular distribution of a 105 kDa phosphoprotein (pp 105) in transformed mouse fibroblasts, showed that only a minor amount was located on the surface of logarithmically grown suspension cells . More than 90% of total pp 105 was contained in the cytosolic fraction representing about 0.2% of total cytosolic proteins . Surface and cytosolic pp 105 had identical phosphopeptide patterns . Cytosolic pp 105 was highly purified by ammonium sulfate precipitation followed by three chromatographic steps and gel electrophoresis . The purified pp 105 was capable of weak autophosphorylation . In the stationary growth phase of suspension cells, the amount of pp 105 detectable by endogenous phosphorylation was only 10-15% of that observed during logarithmic growth . pp 105 was also detected in normal mouse tissue and its distribution determined.

Arch Virol, 1983, 77(2-4), 97 - 108
The attachment of the foot-and-mouth disease virus Asia I Iran 1/73 to BHK suspension cells does not require virus specific cell receptors; Spier RE et al.; Experiments are described which show that a member of the picornaviridae (FMD virus Asia I Iran 1/73) attaches to BHK suspension cells in a manner which precludes a requirement for virus specific receptors on the cell plasma membrane . While it may be possible to demonstrate the apparent saturation of the cell surface with multiple doses of virus, an increase of the concentration of the dosing suspension results in more virus attachment . Indeed, it was found that with the amounts of virus which were made available it was not possible to saturate the ability of the cell to take up virus particles . This, coupled with the demonstration that the uptake of virus followed the pattern of uptake of gas molecules on to a solid surface (Freundlich adsorption isotherm), drew us to the conclusion that, in contrast to other reported systems with similar viruses and cells, the uptake of the FMD virus we used to BHK suspension cells did not require virus specific cell receptor sites.

Blood, 1982 Jul, 60(1), 130 - 5
In vitro proliferation of hematopoietic stem cells in the absence of an adherent monolayer; Eastment C et al.; Experiments on long-term murine bone marrow cultures indicate that the production and maintenance of the hematopoietic stem cell is dependent on the establishment of an adherent monolayer and a secondary repopulation of the culture with fresh marrow . In contrast, we have found that bone marrow cultures derived from the Syrian hamster do not require a repopulation step and produce stem cells that proliferate and differentiate for more than 12 wk in the absence of an adherent layer . Stem cells were grown in Fisher's medium (pH 7.0-7.2) containing 20% horse serum in a fully humidified atmosphere of 5% CO2 in air at 37 degrees C . Cultures were fed twice weekly by removal of half of the medium and supernatant cells and replacement with an equal volume of fresh medium . No hormones or exogenous growth factors were required for the maintenance of myeloid cells, monocytes, and megakaryocytes in either the adherent or suspension cells cultures.

Br J Radiol, 1982 May, 55(653), 362 - 5
Ultrasonically induced nuclear aberrations in an in vitro multicellular tumour system; Sacks PG et al.; Chinese hamster V-79 cells, attached to an acoustically transparent membrane and EMT6/Ro mouse mammary sarcoma cells as small multicellular spheroids or in suspension, were exposed to therapeutic intensities of 1 MHz ultrasound (0-10 W/cm2) and examined for the production of atypical nuclear or chromosomal aberrations . In the monolayer configuration no abnormalities occurred . In multicell spheroids or suspension cells 3 classes of abnormalities were found: (1) pyknotic/irregular nuclei; (2) lysed nuclei with extruding nucleoplasm; and (3) agglomerations which have an irregular shape and "fused" morphology similar to that which has been reported for plant tissues (Cataldo et al, 1973).

Tsitologiia, 1982 May, 24(5), 586 - 91
{Characteristics of the distribution in a 2-phase polymer system of L cells at different stages of culture growth}; Petrov IuI et al.; By the method of counter-current distribution in two-phase polymer system Dextran-500/poly (ethylene)glycol-6000, surface properties of monolayer and suspension sublines of L cells were investigated in relation to the stage of culture growth . The cultivation conditions of both the cell types were identical . It has been shown that with the growth of cell population, the number of cells with lower partition coefficient increases in both the sublines, mostly in cells of monolayer subline . These data suggest more essential changes of surface properties of cells growing in monolayer . It is supposed that this can be associated with the inability of suspension cells to form adhesion contacts.

Tsitologiia, 1982 Jan, 24(1), 35 - 40
{Circahoral fluctuations in protein release from hepatocytes in a monolayer culture}; Novikova TE et al.; An account is presented on morphological and functional properties in primary monolayer culture . Advantages of this model, as compared to the suspension cells, for studying various aspects of the liver vital activity are described . Dynamics of protein secretion by hepatocytes during a 2-hour period was investigated by different methods . The data obtained point to cyclic changes of the rate of protein secretion in the liver cells with circahoralian periodicity, which is typical of the changes in the rate of protein synthesis we revealed earlier for the same cells.

Biochim Biophys Acta, 1981 Sep 29, 670(2), 274 - 84
Cell adhesion-dependent differences in endogenous protein phosphorylation on the surface of various cell lines; Pfeifle J et al.; Endogenous phosphorylation of intact cells was studied with four mouse, hamster and human cell lines using {gamma-32P}ATP and {gamma-32P}GTP as exogenous substrates . With all four cell lines distinct differences in the phosphoprotein patterns could be demonstrated for cells grown in suspension culture compared to cells grown in monolayers . Two major, apparently ubiquitous phosphoproteins with molecular weights of 135 000 (128 000 in HeLa cells) and 105 000, representing up to 60% of total phosphorylation, were phosphorylated only in cells grown in suspension . These phosphoproteins and the kinase(s) were located on the surface of the suspension cells . Evidence showed that phosphorylation was apparently not a true endogenous reaction, that rather it occurred by cell-cell collision, showing exponentially increasing 32P incorporation with increasing cell population density . Phosphorylation of pp135 and pp105 was established with ATP as well as with GTP and was not dependent on cyclic nucleotides cyclic AMP, cyclic GMP and cyclic CMP . The substrate-attached cells of all four cell lines have protein kinases on the cell surface . The lack of pp135 and pp105 phosphorylation may be due to the fact that these phosphoproteins are not expressed at all on the surface of substrate-attached cells or that these phosphoproteins are already fully phosphorylated.

Eur J Biochem, 1981 Jun 1, 116(3), 527 - 33
Mechanism of uptake of L-arginine by sugar-cane cells; Komor E et al.; Suspension cells of sugar cane were used as a model system for cells of higher plants to study the mechanism of L-arginine uptake . The uptake system is specific for the L-arginine molecule in the fully ionized state, i.e . delta-guanidino group and alpha-amino group positively charged and carboxyl group negatively charged . This was concluded because the Km value for uptake increased strongly for: (a) L-arginine analogues which lack the charged carboxyl group (L-arginine methyl ester, agmatin); (b) L-arginine analogues, which lack the charged alpha-amino group (L-arginine acid, gamma-guanidinobutyric acid); (c) L-arginine analogues, which lack the charged delta-guanidino group or gamma-guanidinoxy group (L-citrulline, L-canavanine at neutral and alkaline pH-values) . The importance of the positive charge of the delta-guanidino group or gamma-guanidinoxy group was further documented by Km values for L-arginine and L-canavanine at different pH values . Only at pH values where the gamma-guanidinoxy group is protonated, was there an effective uptake of L-canavanine and effective competition of L-canavanine with L-arginine . The length of the L-arginine molecule was less important: slightly larger (L-homoarginine) or shorter analogues (L-lysine) were taken up rather well . A spatial rearrangement at the alpha-carbon (D-ariginine) was, however, not tolerated . The uptake of L-arginine proceeds by electrogenic uniport, there is no evidence for symport or antiport of another molecule (though L-canavanine uptake at neutral pH value causes a transient alkalinization of the suspension medium) . Charge equilibration is brought about by efflux of protons and potassium ions.

Infect Immun, 1981 Mar, 31(3), 1054 - 61
Lysis of herpes simplex virus-infected cells early in the infectious cycle by human antiviral antibody and complement; Cromeans TL et al.; Chang liver (CL) cells and human embryonic lung fibroblasts (MRC-5) were infected with type 1 herpes simplex virus (HSV), ant the time postinfection at which these cells became susceptible to lysis by antiviral antibody and complement of human origin was determined in a 51Cr release assay . Using a 1:2 dilution of fresh HSV antibody-positive human serum, we initially detected specific lysis by 4 h postinfection in HSV-infected CL cells and by 3 h postinfection in HSV-infected MRC-5 cells in suspension . MRC-5 cells were more completely lysed than CL cells . Protein inhibition studies with cycloheximide showed that all of the HSV-infected CL cells and most (83%) of the HSV-infected MRC-5 cells injured early in the infectious cycle were attacked because of newly synthesized viral surface antigens rather than because of adherent input virus . Suspension cells early in the infectious cycle were less completely lysed and required higher concentrations of both antiviral antibody and complement for lysis than cells that were in the later stages of infection (18 h postinfection) . Guinea pig serum was inferior to human serum as a complement source for lysis of early infectious cycle cells . Lysis early in the infectious cycle was directly proportional to the multiplicity of infection and inversely proportional to the cell concentration . Infected cells in monolayers were lysed less readily and about 1 to 2 h later in the infectious cycle than infected cells in suspension . This difference was pronounced for CL cells, but modest for MRC-5 cells . These studies demonstrate that, despite previously held notions, HSV-infected tissue culture cells can be lysed by antiviral antibody and complement early in the infectious cycle before the initial production of progeny virus particles . The demonstration of lysis was highly dependent on experimental conditions, however, including cell type, suspension versus monolayer culture, cell density, and concentration of antibody and complement as well as the source of complement.

Dev Biol Stand, 1981, 50, 145 - 54
Uptake of amino acids and glucose by BHK 21 clone 13 suspension cells during cell growth; Arathoon WR et al.; The uptake of amino acids and glucose by BHK 21 clone 13 suspension cells grown in Modified Eagles Medium was monitored . The Growth Yields for sixteen amino acids and glucose were determined . Four "non essential" amino acids were utilised . Glutamine, glutamate, leucine and asparagine were used in greatest quantity by the cells which also utilised virtually all the cystine in the medium . The implications of these results are discussed.

J Cell Sci, 1980 Oct, 45, 257 - 68
The mode of action of 2,4-D in counteracting the elongation of carrot cells grown in culture; Lloyd CW et al.; The growth regulator 2,4-D (2,4-dichlorophenoxyacetic acid) has been used to investigate the inter-relationship between cell elongation and cell division in carrot suspension cells . Maintained in 1 mg/l . 2,4-D, dividing populations of cells remain spheroidal and in clusters . But when subcultured into lower levels or zero, 2,4-D they increasingly elongate at the expense of division . Over the range of 0 to 1.0 mg/l, 2,4-D, elongation and division are therefore inversely related . However, by suppressing the mitogenic effect with FUdR it can be shown that cells do elongate in 1 mg/l . 2,4-D--a concentration which otherwise produces dividing, spheroidal cells . This indicates that mitogenc levels of 2,4-D do not perturb structures which support cellular elongation . This conclusion is confirmed by immuno- and electron-microscopy which show that development of elaborate arrays of cytoplasmic microtubules is unaffected by 1 mg/l . 2,4-D when FUdR is present . It is concluded that over the time periods under study here, 2,4-D regulates cell size (and shape) by stimulating growing cells to enter the division cycle and not by inhibiting elongation per se.

J Biol Chem, 1980 May 10, 255(9), 3871 - 7
Neuroblastoma differentiation involves both the disappearance of old and the appearance of new poly(A)+ messenger RNA sequences in polyribosomes; Grouse LD et al.; The cholinergic mouse neuroblastoma cell line NS20Y was adapted to undifferentiated growth in suspension culture . When suspension cells were transferred to surface culture and treated with dibutyryl cyclic AMP, the cells underwent differentiation as assessed by biochemical, morphological, and physiological criteria . Differentiated NS20Y cells in co-culture with mouse muscle cells had the capacity to form functional neuromuscular junctions with the muscle cells . The sequence complexities of the poly(A)-containing messenger RNA (poly(A)+ mRNA) of the differentiated, process-forming cells (P-cells) and undifferentiated cells in suspension culture (S-cells) were measured by analysis of the kinetics of hybridization of the mRNAs with their complementary DNAs (cDNAs) . There were less than 100 high abundance and approximately 8000 low abundance poly(A)+ mRNAs in both differentiation states . Heterologous hybridization reactions and recycling of the cDNA probes revealed that 9.7% and 6.8% of the messages in P- and S-cells, respectively, were specific to those differentiation states . The P-cell-specific sequences included approximately 3 high abundance and 320 low abundance poly(A)+ mRNAs . The S-cell-specific sequences included approximately 3 high abundance and 250 low abundance poly(A)+ mRNAs . We conclude that the increment in NS20Y differentiation results in both the disappearance of old, and the appearance of new mRNAs in polyribosomes.

J Cell Physiol, 1980 May, 103(2), 239 - 46
Limitation of macrophage production in long-term marrow cultures containing prostaglandin E; Williams N et al.; Addition of prostaglandin E2 (PGE2) significantly altered the cellular composition of murine long-term bone marrow cultures . After 4--5 weeks of culture, increased cellularity in the suspension phase was observed in all cultures containing prostaglandin . These suspension cells contained markedly higher proportions of differentiated neutrophils than did cells cultured in the absence of PGE2 . Granulocyte-macrophage progenitor cell levels in the suspension layer were increased 3--20 fold after five weeks in prostaglandin-containing cultures compared with control cultures . Fewer cells comprised the adherent layer in cultures containing prostaglandin . The number of macrophages in this layer was reduced 3--8 fold in these cultures compared with control cultures, while the number of granulocytes was increased 2--3 fold . The progenitor cells biased toward macrophage development were selectively inhibited in the cultures with PGE2 . There was no significant effect of PGE2 on pluripotent stem cell levels or on the longevity of the cultures . It is concluded that excessive monopoiesis in bone marrow may be limited by PGE2 without influencing either stem cell maintenance or the development of other marrow-derived cell types.

J Cell Sci, 1980 Apr, 42, 401 - 15
Plasma membrane ultrastructure during plant protoplast plasmolysis, isolation and wall regeneration: a freeze-fracture study; Wilkinson MJ et al.; The freeze-fracture morphology of the plasma membrane of cells and isolated protoplasts of plant callus suspensions has been investigated . Plasmolysis of suspension cells leads to the formation of 2 types of hexagonal arrays of intramembrane particles situated on the inner fracture face (PF) . These arrays are interpreted as proteins that have 'crystallized' in the plane of the membrane as the area of surrounding lipid bilayer is reduced during protoplast retraction from the cell wall . Time-course studies have revealed no positive relationship between the distribution of hexagonal arrays and the occurrence of microfibrils regenerated around isolated protoplasts during periods of culture . No evidence for the specialized transport functions attributed to hexagonal arrays of plant cells by previous workers has been found.

J Cell Physiol, 1980 Mar, 102(3), 287 - 95
The effect of mouse lung granulocyte-macrophage colony-stimulating factor and other colony-stimulating activities on the proliferation and differentiation of murine bone marrow cells in long-term cultures; Williams N et al.; The roles of colony-stimulating factors in long-term bone marrow cultures were studied and compared . After single additions of high concentrations of unpurified colony-stimulating activities to the cultures, rapid deterioration of the cultures was observed . This appears to result from contaminants in the stimulatory preparations . Cultures to which one purified colony-stimulating factor (csf) from endotoxin mouse lung-conditioned medium was added did not run down ten weeks after addition and were found to be the same as the controls . The deterioration of the cultures to which unpurified stimulators were added could not be accounted for by accelerated granulopoiesis leading to subsequent exhaustion of the cultures . The inability of purified CSF to affect the cellularity of the suspension cells did not result from instability or masking of the activity in the cultures, nor did CSF preferentially stimulate the cells within the adherent layer . The suspension cells responded to purified CSF after separation from the adherent cells . The data suggest that if CSFs are marrow stimulators, their effects in turn may be stringently regulated within the marrow.

Proc Natl Acad Sci U S A, 1980 Feb, 77(2), 1044 - 8
A role for cyclic AMP in expression of developmentally regulated genes in Dictyostelium discoideum; Landfear SM et al.; Starved cells of Dictyostelium discoideum begin to synthesize a new class of developmentally regulated proteins at about 13 hr of the 24-hr developmental program, concomitant with the formation of tips on the tight cell aggregates {Alton, T . H . & Lodish, H . F . (1977) Dev . Biol . 60, 180--206} . Continued synthesis of these proteins is normally dependent upon the integrity of the multicellular aggregates, because cells that have been disaggregated at 13 hr and shaken in suspension for 5 hr do not make these proteins . We show here that addition of 20 microM cyclic AMP to suspension cultures of disaggregated 13-hr cells caused synthesis of most of these late proteins to be maintained . Translation in an in vitro wheat germ system of total cellular RNA isolated from these cyclic AMP-stimulated suspension cells, or from normal aggregates, generated several proteins that were not encoded by the RNA isolated from equivalent suspension cells which had not been treated with cyclic AMP or from preaggregation cells . We conclude that cyclic AMP has a direct role in maintaining the synthesis of aggregation-dependent Dictyostelium proteins and in maintaining the level of the corresponding mRNAs.

Dev Biol Stand, 1980, 46, 159 - 65
Advantages of a microprocessor-monitored and controlled continuous culture of BHK suspension cells; Spier RE; A continuous culture of BHK suspension cells was monitored by a micro-processor . The potential of this system is discussed as well as the results obtained to date on the growth of BHK suspension cells and the susceptibility of those cells to FMDV.

Blood, 1979 Oct, 54(4), 775 - 93
Prolonged hematopoiesis in a primate bone marrow culture system: characteristics of stem cell production and the hematopoietic microenvironment; Moore MA et al.; Maintenance of myelopoiesis and pluripotential stem cell production for prolonged periods in vitro hitherto has been limited to mouse bone marrow culture . In an effort to adapt the system for use in higher species, particularly in human and non-human primates, studies were undertaken using the prosimian species, Tupaia glis (tree shrew) . In a number of experiments the duration of sustained normal hematopoiesis observed in cultures of this species, following a single inoculum of 5 X 10(6)--10(7) bone marrow cells, with or without addition of fresh allogeneic bone marrow exceeded 1 yr . Analysis of suspension cells obtained by weekly demidepopulation of such cultures revealed production of CFU-C, differentiating neutrophils, and basophils at high levels . Direct comparison with murine cultures indicated that in both species a complex series of cellular interactions takes place within an adherent environment of marrow-derived endothelial cells, macrophages, and fat-containing cells . Certain functional and ultrastructural features served to distinguish murine from Tupaia marrow cultures, and the prolonged duration of in vitro hematopoiesis in the latter species could be attributed to a regenerative capacity possessed by its adherent hematopoietic microenvironment . The availability of this primate marrow culture system should facilitate studies of hematopoiesis, viral leukemogenesis, and transplantation biology, which have more direct relevance to man than that provided by the existing murine system.

Cancer Res, 1978 Sep, 38(9), 2827 - 35
Scanning electron microscopic observation of two retinoblastoma cell lines; McFall RC et al.; Two continuous retinoblastoma cell lines were observed by scanning electron microscopy . Both cell lines spontaneously grow as a suspension of round cells in clusters, chains, and unique ring (rosette) formations . Scanning electron microscopy of suspension cells reveals some variation in the number and frequency of surface adornments such as blebs, lamellipodia, and microvilli . Although the cell lines are nonadherent to substratum and therefore assume a spherical form, highly villous cells are not characteristic of the entire cell populations . When WERI-Rb1 and Y79 are seeded onto a polyornithine-treated substrate, attachment and growth as adherent cultures are evident . With selective attachment on a positively charged substrate, we observe alteration of membrane architecture with the extension of cytoplasm and filopidia . In addition, WERI-Rb1 cell-to-substratum adhesion induces morphological changes suggestive of neuronal cell differentiation.

Hum Genet, 1978 Jun 27, 42(3), 319 - 22
Time and cell systems as variables in fusion experiments with polyethylene glycol; Anders GJ et al.; Cell hybridization was done between a monolayer of B14-150 Chinese hamster cells and a suspension of either mouse leukemia cells or normal human lymphocytes . Cell contact was obtained by centrifugation of the suspension cells onto the monolayer cells in a culture plate . Cell fusion was done by means of polyethylene glycol (PEG) . The optimum time for PEG exposure as well as the yield of hybrid cells differed markedly with the different combinations.

Biochem Genet, 1977 Oct, 15(9-10), 1001 - 4
A comparison of cAMP phosphodiesterases in normal, malignant, and somatic cell hybrids; Ayad SR et al.; Hybrids (PCM) between a malignant mouse lymphoma suspension cell line (P388F-36) and a normal Chinese hamster fibroblastic cell line (Ch23) have already been isolated in this laboratory . Investigations were carried out on the cAMP phospodiesterases of the parents and two of these hybrids--PCM2 and PCM3 . PCM3 shows a rather unusual growth characteristic in that a considerable proportion of the cells exist at any one time either in suspension or only loosely attached to the substratum, the remaining cell population existing in a monolayer form . It was found that each cell line exhibited multiple forms of the enzyme with varying affinities for cAMP . Both parents, although different, contained high-, low-, and extra-high apparent Km forms of the enzyme . The hybrids exhibited characteristics of both parental systems but were different from each other . Neither hybrid exhibited a high-Km enzyme, but both exhibited two low-Km forms . There was also a slight variation between monolayer and suspension cells of PCM3 hybrid . An attempt has been made to explain these phenomena with respect to hybridization and the growth characteristics of the cells.

Eur J Biochem, 1977 Apr 1, 74(2), 405 - 12
Poly(A)-containing RNA in neuroblastoma: immature and differentiated cells in culture; Croizat B et al.; We have analysed the poly(A)-containing RNA from neuroblastoma cells at two different developmental states: either as round, immature neuroblasts grown in suspension, or as differentiated cells exhibiting the morphological properties of mature neurons, when attached to a culture dish . Suspension-grown and monolayer cells were pulse-labelled with tritiated uridine . The profile of cytoplasmic poly(A)-containing RNA from suspension cells is highly heterogeneous with peaks ranging from 16-30 S . The profile obtained from differentiated cells appears somewhat distinct from the previous one . This is evidenced by a relative decrease in the 26-S peak and a virtual disappearance of the 16-S component . In order to compare the 'steady-state' patterns of poly(A)-containing RNA in these two developmental stages, polysomal RNA was prepared from unlabelled cells . Following sucrose gradient sedimentation, each fraction was hybridized to {3H}poly(U) . Examination of the two RNA hybridization profiles reveals striking similarities suggesting that 'steady-state' messenger populations include, on the average, the same subspecies . The 16-S fraction, which was not observed after the pulse-labelling of the monolayer culture, is detected here by hybridization to {3H}poly(U) when using polysomal poly(A)-containing RNA from monolayer cells as substrate . These results suggest that terminal differentiation of neuroblastoma cells is not accompanied by major alterations of the transcription program and is paralleled by a marked stabilization of the 16-S species.

Lipids, 1977 Jan, 12(1), 120 - 4
Uptake and metabolism of fatty acids by soybean suspension cells; Stumpf PK et al.; Soybean suspension cultures very rapidly take up C16 and C18 fatty acids by a nonspecific, nonenzymic binding of exogeneously added fatty acids to cell walls and by a subsequent transfer into the cell where they are rapidly incorporated into triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines . 14C-Palmitic and 14C-stearic acids follow this sequence but are not desaturated, wherease 14C-oleic and 14C-linoleic acids are transferred more rapidly than the saturated fatty acids and are then further modified . All the data fit a sequence of events by which free oleic acid is first activated to a CoA thioester, and then desaturated to linoleyl-CoA; both thioesters are then transferred to triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine.

J Biol Chem, 1976 Nov 10, 251(21), 6747 - 56
Physical properties of membranes isolated from tissue culture cells with altered phospholipid composition; Schroeder F et al.; A choline-requiring strain of mouse fibroblast cells (LM cells) was cultured in suspension with choline, N,N'-dimethylethanolamine, N-monomethylethanolamine, or ethanolamine . These choline analogues were incorporated into membrane phospholipids as phosphatidyl-N,N'-dimethylethanolamine, phosphatidyl-N-monomethylethanolamine, and phosphatidylethanolamine . Plasma membranes, microsomes, mitochondria, and their respective lipids were isolated and the characteristic temperatures were determined by using two types of fluorescent probes: (a) beta-parinaric acid, a naturally occurring molecule, and (b) 8-anilino-1-naphthalene sulfonic acid, a synthetic organic fluorophore . A computer-centered spectrofluorimeter capable of simultaneous measurement of absorbance, absorbance-corrected fluorescence, and relative fluorescence efficiency was utilized for on-line measurement of all fluorescence parameters . Plots of absorbance corrected fluorescence or of relative fluorescence efficiency versus temperature revealed the same five characteristic temperatures with both types of probe . These characteristic temperatures were independent of the phospholipid composition of the LM suspension cell membranes or their extracted lipids . Plasma membranes, microsomes, and mitochondria containing analogue phospholipids had similar (+/- 1 degree) characteristic temperatures . The presence of analogue phopholipids altered the binding characteristics of beta-parinaric acid with plasma membranes and plasma membrane lipids of LM suspension cells . The equilibrium dissociation constant of plasma membranes and plasma membrane lipids was decreased 2- and 5-fold, respectively, when the cells had been supplemented with ethanolamine . The minimum number of phospholipid molecules per probe binding site was approximately constant in the intact plasma membrane but increased (2-fold) in the isolated plasma membrane lipids . The presence of analogue phospholipids also altered the interaction of 8-anilino-1-naphthalene sulfonic acid with LM cell membranes . The equilibrium dissociation constant of this probe interacting with mitochondrial lipid was decreased 40% by ethanolamine supplementation . The fluorescent properties of both probes were sensitive to the degree of methylation of the polar head group . The absolute values of absorbance-corrected fluorescence and relative fluorescence efficiency were different for each type of membrane from LM cells even with the same analogue supplement . Thus, it appears that LM cells maintain the characteristic temperatures which are a measure of the physical properties of their membranes, despite large alterations of the phospholipid polar head group composition.

J Biol Chem, 1976 Aug 25, 251(16), 5015 - 26
Isolation and characterization of subcellular membranes with altered phospholipid composition from cultured fibroblasts; Schroeder F et al.; Plasma membranes, microsomes, and mitochondria were isolated from mouse fibroblast (LM) suspension cells by modification of several established procedures . Choline analogues such as N,N'-dimethylethanolamine, N-monomethylethanolamine, or ethanolamine were incorporated in vivo into phospholipids of all three cell fractions studied, but to varying degrees depending on the type of analogue used . The in vivo incorporation of these bases into membrane phospholipids produced no significant effect on the activities of seven membrane-bound enzymes: (Na+, K+)-ATPase, 5'-nucleotidase (plasma membranes); TPNH-cytochrome c reductase, glucose-6-phosphatase, inosine diphosphatase (microsomes); and succinate cytochrome c reductase (mitochondria) . The incorporation of base analogues into phospholipids was accompanied by several compensatory mechanisms . (a) The quantity of both phosphatidylcholine and phosphatidylethanolamine decreased up to 75% and 50% respectively in 3 days . (b) The molar ratio of desmosterol/phospholipid in the plasma membranes of LM cells grown in suspension culture in the presence of choline analogues decreased from 0.65 to 0.45 . (c) The percentage of lysophosphatidylcholine increased over 2-fold in the phospholipid of all subcellular fractions studied . The quantity of lysophosphatidylcholine was directly proportional to the number of methyl groups on the nitrogen atom of the base analogue supplemented to the cells . This was a specific effect since the quantity of lysophosphatidylethanolamine, the other major lysophospholipid, remained unchanged . (d) The ratio of zwitterionic phospholipids to acidic phospholipids remained relatively constant in all isolated membrane fractions regardless of analogue supplementation . Neither increase in the degree of unsaturation nor shortening of fatty acid chain length was noted in response to analogue supplementation.

Biofizika, 1976 Jul-Aug, 21(4), 661 - 4
{Biphasic nature of the light-dependent transport of C14-alanine into Halobacterium holobium cells}; Plakunova VG; Biphase character of photoinduced pH change in H . halobium R1 suspension cells is shown: in the first illumination period pH rises and only afterwards it decreases . pH increase is accompanied by the introduction of C14-alanine into the cells, while the decrease by its yield up to the level lower than the "dark" one ("negative" photoeffect) . The bi-phase character of transport processes seems to be explained by the reversible character of light-dependent bacteriorodopsin proton pump in H . halobium R1 cells.

Tsitologiia, 1976 Mar, 18(3), 336 - 41
{Characteristics of different sublines of VNK-21 cells}; Kudriavtseva GA et al.; Comparative cytological, histological, histochemical and partially karyological studies were carried out on 8 batches of BHK-21 cell kept at different conditons . It was found that cells adapted to suspension culture differed from monolayer culture cells both morphologically and cytochemically . Suspension cells transmitted into monolayer culture displayed smaller size, changeable form, increased glycogen accumulation, decreased enzymatic activity (acid phosphatase, succinate dehydrogenase), their karyotypes tending to hypodiploidy.

Dev Biol Stand, 1976, 35, 73 - 8
Reproducibility of yields of foot--and--mouth disease virus from BHK monolayer and suspension cells; Spier RE; The production of FMD virus from BHK 21 C13 monolayer and suspension cells was examined under standardized conditions and in different systems . The yields of virus from suspension cells did not significantly exceed the yield from monolayer cells, whereas the monolayer cell was capable of producing virus from some strains of FMD virus which would not grow in suspension cells . When different production systems were examined, the scale of operation did not significantly influence the yield of virus from suspension cells, while the plastic spiral film propagator produced significantly less virus than the other monolayer systems examined . The use of unit monolayer systems on the large scale is discussed.

Dev Biol Stand, 1976, 35, 61 - 6
Studies on the susceptibility to foot--and--mouth disease virus of BHK cell cultures derived from various sources; Clarke JB et al.; Cultures of BHK monolayer and suspension cells obtained from a number of laboratories and a group of cloned sub-lines derived from suspension cells were examined for their susceptibility to three FMD virus strains . It was found that the various cultures were sensitive to the test virus strains to differing degrees . It was shown possible to obtain a clone with high susceptibility to Asia 1 Iran 1/73 virus from a parent culture which had a low susceptibility to that virus . The implication of these findings is discussed.

Biochemistry, 1975 Aug 26, 14(17), 3816 - 25
Surface polypeptides of the cultured Chinese hamster ovary cell; Juliano RL et al.; The organization of the plasma membrane of logarithmically growing Chinese hamster ovary (CHO) suspension cells has been probed using surface label techniques in conjunction with subcellular fractionation and sodium dodecyl sulfate gel electrophoresis . Five components of apparent molecular weights 137,000, 121,000, 97,000, 67,000, and 57,000 have been shown to be exposed at the outer surface of the cell . These components fully meet the criteria of being (a) reactive with two or more surface label reagents, (b) enriched in a purified plasma membrane fraction, and (c) sensitive to proteolytic digestion of intact cells . Three other components of molecular weights 200,000, 44,000 and 30,000 are also reactive with certain surface label reagents, but fail to meet other criteria for cell surface components . Two polypeptides of molecular weights 180,000 and 37,000 are substantially enriched in the plasma membrane fraction, but are unreactive with surface label reagents . The organization of the CHO cell membrane and the applicability of surface label techniques to cultured cell systems are discussed.

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