Characterization of starch produced by suspension cell cultures of Indica rice (Oryza sativa L.)

Suspension cultures of rice (Oryza sativa L.), initiated from seed, produced significant amounts of starch. Starch accumulated in the cultured cells throughout the growth phase and reached a maximum of 7% of the cell dry weight at stationary phase. Starch was present in compound granules which were birefringent under polarized light. Suspension culture starch had a higher amylose content and a lower gelatinization temperature than rice grain starch. Additionally, starch branching enzyme, an enzyme involved in starch biosynthesis, was characterized by anion exchange chromatography in culture cells and endosperm. Culture cells had at least one major form of starch branching enzyme which differed from the multiple enzyme forms present in endosperm.     

Characterization of aggregate size in Taxus suspension cell culture

Plant cells grow as aggregates in suspension culture, but little is known about the dynamics of aggregation, and no routine methodology exists to measure aggregate size. In this study, we evaluate several different methods to characterize aggregate size in Taxus suspension cultures, in which aggregate diameters range from 50 to 2,000 μm, including filtration and image analysis, and develop a novel method using a specially equipped Coulter counter system. We demonstrate the suitability of this technology to measure plant cell culture aggregates, and show that it can be reliably used to measure total biomass accumulation compared to standard methods such as dry weight. Furthermore, we demonstrate that all three methods can be used to measure an aggregate size distribution, but that the Coulter counter is more reliable and much faster, and also provides far better resolution. While absolute measurements of aggregate size differ based on the three evaluation techniques, we show that linear correlations are sufficient to account for these differences (R ² > 0.99). We then demonstrate the utility of the novel Coulter counter methodology by monitoring the dynamics of a batch process and find that the mean aggregate size increases by 55% during the exponential growth phase, but decreases during stationary phase. The results indicate that the Coulter counter method can be routinely used for advanced process characterization, particularly to study the relationship between aggregate size and secondary metabolite production, as well as a source of reliable experimental data for modeling aggregation dynamics in plant cell culture.     

Characterization of DNA lesions produced by HgCl2 in cell culture systems

HgCl2 is extremely cytotoxic to Chinese hamster ovary (CHO) cells in culture since a 1-h exposure to a 75- microM concentration of this compound reduced cell plating efficiency to 0 and cell growth was completely inhibited at 7.5 microM . The level of HgCl2 toxicity depended upon the culture incubation medium and has previously been shown to be inversely proportional to the extracellular concentration of metal chelating amino acids such as cysteine. Thus, HgCl2 toxicity in a minimal salts/glucose maintenance medium was about 10-fold greater than the toxicity in McCoy's culture medium. The HgCl2 toxicity in the latter medium was 3-fold greater than that in alpha-MEM which contains more of the metal chelating amino acids. When cells were exposed to HgCl2 there was a rapid and pronounced induction of single strand breaks in the DNA at time intervals and concentrations that paralleled the cellular toxicity. The DNA damage was shown to be true single strand breaks and not alkaline sensitive sites or double strand breaks by a variety of techniques. Consistent with the toxicity of HgCl2, the DNA damage under an equivalent exposure situation was more pronounced in the salts/glucose than in the McCoy's medium and more striking in the latter medium than in alpha-MEM. Most of the single strand breaks occurred within 1 h of exposure to the metal. We believe that the DNA damage caused by HgCl2 leads to cell death because the DNA single strand breaks are not readily repaired. DNA repair activity measured by CsCl density gradient techniques was elevated above the untreated levels at HgCl2 concentrations that produced little measurable binding of the metal to DNA or few single strand breaks assessed by the alkaline elution procedure. DNA repair activity decreased at HgCl2 concentrations that produced measurable DNA binding and single strand breaks. These irreversible interactions of HgCl2 with DNA may be responsible for its cytotoxic action in cells.     

Characterization of rice transformed via an Agrobacterium-mediated inflorescence approach

Rice inflorescences were inoculated with Agrobacterium tumefaciens strain LBA4404 carrying plasmid pJD4 with application of vacuum infiltration. After co-cultivation, callus was initiated and subjected to hygromycin selection, and plants were regenerated from resistant callus lines. Based on the total number of co-cultivated inflorescences bearing flowers 1 to 3 mm in length, the average frequency for recovering independent transgenic rice plants was at least 30%. Seeds from selfed R0 plants were harvested within 6 months after initiation of the experiments. Genomic DNA blot analysis showed that genes in the T-DNA of the binary plasmid were stably integrated into the rice genome, typically at low copy number. In most, but not all, cases the transgene was transmitted to R1 progeny at a frequency characteristic for Mendelian inheritance of a single dominant trait. For selfed progeny of one two-locus insertion line, reactivation of GUS expression was observed for a single copy locus that segregated from a silenced multicopy locus. For this line and some additional plants, fluorescence in situ hybridization was used to visualize the chromosomal location of the transgene insert.     

Reduced protease activity in transformed rice cell suspension cultures expressing a proteinase inhibitor

In this study, we synthesized a synthetic serine proteinase inhibitor II gene (sPI-II) that harbored the chymotrypsin and trypsin inhibitor domains of the PI-II gene from Nicotiana alata. In an effort to reduce protease activity in a rice cell suspension culture, we first synthesized sPI-II using overlap PCR and then introduced the gene into a rice calli (Oryza sativa L. cv. Dongin) by particle bombardment-mediated transformation. The sPI-II gene was under the control of a rice alpha-amylase 3D promoter induced by sugar starvation. To verify the integration and expression of the sPI-II gene in the transformed rice cells, we employed genomic DNA PCR amplification and Northern blot analysis, respectively. The relative protease activity of the transformed cell suspension culture was reduced to approximately 23% when compared to the non-transformed culture. This indicates that a transformed suspension culture system expressing a proteinase inhibitor, may be a useful tool to protect against recombinant protein losses resulting from extracellular proteases.     

Expression of human growth hormone in transgenic rice cell suspension culture

Human growth hormone (hGH), a pituitary-derived polypeptide, evidences a wide range of biological functions, including protein synthesis, cell proliferation, and metabolism. A synthetic hGH gene (shGH) has been synthesized on the basis of plant-optimized codon usage via an overlap PCR strategy and located in a plant expression vector under the control of the rice amylase 3D (Ramy3D) promoter, which is induced by sugar starvation. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Donjin) via particle bombardment transformation methods. The integration of the shGH gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification and shGH expression in transgenic rice suspension cells was confirmed via Northern blot analysis. The shGH protein was detected in the transgenic rice cell suspension culture medium following induction with sugar starvation, using Western blot analysis. The quantity of shGH that accumulated in the transgenic rice cell suspension medium was 57 mg/l. The shGH accumulated in the transgenic rice cell suspension culture medium evidenced a biological activity similar to that of Escherichia coli-derived recombinant hGH. These results indicate that the shGH was generated and accumulated in the transgenic rice cell suspension culture medium, and manifested biological activity.     

Cloning of murine transformed cell lines in suspension culture with efficiencies near 100%

Addition of 3 × 10⁶ thymus cells from either syngeneic, allogeneic or xenogeneic animals increases the cloning efficiencies of murine thymomas (EL-4, WC-2), B-lymphomas (McPC 1748, 38C-13), Abelson-virus transformed cell lines (F and K), mastocytomas (P815), myelomas (AbPC22, X63-AG8, 5563, MOPC 104 E, RFC 5, W 3469) and hybrids of myelomas and normal B-lymphocytes (Sp-1), all adapted to tissue culture, to near 100%. Thymus cells also increase the efficiencies of growth initiation in primary in vitro cultures of myeloma tumor cells (S117) transplanted in vivo, and of cells fused between the azaguanine-resistant X63-AG8 myeloma cell line and normal, LPS-stimulated B-lymphocyte blasts.     

The exudation and characterization of iron-solubilizing compounds in a rice cell suspension culture

Some compounds capable of solubilizing ferric chloride as theiron source were released into the growth medium from culturedrice cells in the latter half of the growth cycle. This exudationof iron-solubilizing compounds was repressed by an additionof ammonium ion, which, of five cations, was taken up most rapidly.These iron-solubilizing compounds from cultured rice cells wereidentified by chromatography to be principally malic acid andsome citric acid. In the medium containing almost insolubleferric chloride as the iron source, the growth rate of the culturedcells decreased when cells were inoculated at a low populationdensity. This iron deficiency at the low initial populationwas rectified by an addition of a small amount of conditionedmedium or malic acid. (Received May 22, 1980; )     

The exudation and characterization of iron-solubilizing compounds in a rice cell suspension culture

Some compounds capable of solubilizing ferric chloride as theiron source were released into the growth medium from culturedrice cells in the latter half of the growth cycle. This exudationof iron-solubilizing compounds was repressed by an additionof ammonium ion, which, of five cations, was taken up most rapidly.These iron-solubilizing compounds from cultured rice cells wereidentified by chromatography to be principally malic acid andsome citric acid. In the medium containing almost insolubleferric chloride as the iron source, the growth rate of the culturedcells decreased when cells were inoculated at a low populationdensity. This iron deficiency at the low initial populationwas rectified by an addition of a small amount of conditionedmedium or malic acid. (Received May 22, 1980; )     

Characterization of extracellular matrix-associated glycosaminoglycans produced by untransformed and transformed bovine corneal endothelial cells in culture

A cloned bovine corneal endothelial cell line was transformed in vitro by simian virus 40, and the subendothelial extracellular matrix-associated sulfated glycosaminoglycans synthesized by the cells were isolated and compared with their untransformed counterpart. The transformed endothelial cells grew at faster rates to higher stationary cell densities in the absence of fibroblast growth factor than did the untransformed cells. On a per-cell basis, the transformed cells produced slightly lower amounts of sulfated glycosaminoglycans. The rate of production of sulfated glycosaminoglycans in extracellular matrix increased during seven days of culture. At confluency the extracellular matrix-associated sulfated glycosaminoglycans synthesized by the untransformed endothelial cells consisted of about 80% heparan sulfate and about 20% chondroitin sulfate. Extracellular matrix-associated sulfated glycosaminoglycans of transformed endothelial cells were composed of about 70% heparan sulfate and about 30% chondroitin sulfate plus dermatan sulfate. High-speed gel permeation chromatography profiles on Fractogel TSK HW-55(S) of matrix-associated heparan sulfate from untransformed and transformed endothelial cells were very similar, and gave single peaks (Kav = 0.19). Apparent Mr estimated from the eluting position of the peaks were approximately 47000. Heparan sulfate from both untransformed and transformed endothelial cells was degraded by incubation with a metastatic B16 melanoma cell lysate containing heparanase (heparan-sulfate-specific endo-beta-glucuronidase). The eluting position of the heparan sulfate degradation products on gel permeation column were similar (Kav = 0.43). Size analysis and anion-exchange chromatography of the degradation products after nitrous acid deamination at low pH indicated that the degree of N-sulfation of heparan sulfate was similar in untransformed and transformed endothelial cells. The results indicated that transformation of endothelial cells only slightly changes the molecular nature of subendothelial matrix-associated sulfated glycosaminoglycans.     

Production of a self-activating CBM-factor X fusion protein in a stable transformed Sf9 insect cell line using high cell density perfusion culture

Factor Xa is a serine protease, whose high selectivity can be used to cleave protein tags from recombinant proteins. A fusion protein comprised of a self-activating form of factor X linked to a cellulose-binding module, saCBMFX, was produced in a stable transformed Sf9 insect cell line. The activity of the insect cell produced saCBMFX was higher than the equivalent mammalian cell produced material. A 1.5 l batch fermentation reached a maximum cell concentration of 1.6 × 10⁷ cells ml⁻¹ and a final saCBMFX concentration of 4 mg l⁻¹. The production of saCBMFX by this cell line was also analyzed in a 1.5 l perfusion system using an ultrasonic filter as a cell-retention device for flow rates up to 3.5 l day⁻¹. The cell-retention efficiency of an air backflush mode of acoustic filter operation was greater than 95% and eliminated the need to pump the relatively shear sensitive insect cells. In the perfusion system over 4 × 10⁷ Sf9 cells ml⁻¹ were obtained with a viability greater than 80%. With a doubling of viable cell concentration from 1.5 to 3 × 10⁷ cells ml⁻¹ the saCBMFX production rate was doubled to 6 mg l⁻¹ day⁻¹. The saCBMFX volumetric productivity of the perfusion system was higher than the batch fermentations (0.6 mg l⁻¹ day⁻¹) by an order of magnitude.     

Effects of culture media on hCTLA4Ig production and protein expression patterns in transgenic rice cell suspension cultures

The effects of culture media on the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) and intracellular protein expression patterns were investigated in transgenic rice cell suspension cultures. Using comparative proteomic analysis, changes in the intracellular proteome in different culture media were identified. Culture media were found to be an important factor for the production of the recombinant target protein in this expression system, which was under the control of the rice α-amylase 3D (RAmy3D) promoter. In terms of hCTLA4Ig production, the N6 medium produced a 3.7-fold higher level of protein than the AA medium. In addition, the N6 medium provided better protein stability and cell viability. In the intracellular proteome analysis, we identified eight proteomes that were differentially expressed. These results could provide valuable information for the improvement of cell growth and target protein production.     

Tumor targeting of humanized fragment antibody secreted from transgenic rice cell suspension culture

The tumor-associated glycoprotein 72 (TAG 72) has been shown to be expressed in the majority of human adenocarcinomas. In an effort to develop a technique for the safe and inexpensive production of large quantities of anti-TAG 72 humanized antibody fragments (hzAb) as a future source of clinical-grade proteins, we developed a transgenic rice cell suspension culture system. The in vivo assembly and secretion of hzAb were achieved in a transgenic rice cell culture under the control of the rice alpha amylase 3D (RAmy 3D) expression system, and the biological activities of plant-derived hzAb were determined to be quite similar to those of animal-derived antibody. Purified hzAb was shown to bind to the recombinant antigen, TAG 72, and to bind specifically to human LS 174T colon adenocarcinoma cells expressing the TAG 72 antigen, and this binding occurred to the same extent as was seen with animal-derived antibody. Plant-derived hzAb proved as effective as animal-derived antibody in targeting tumors of xenotransplanted LS 174T cells in nude mice. The results of this study indicate that the hzAb derived from plant cell suspension cultures may have great potential for pharmaceutical applications in the development of future cancer therapeutic and diagnostic protocols.     

Characterization of rat liver cells transformed in culture bydl-ethionine

Summary A rat liver-derived epithelial cell line transformed withdl-ethionine and the corresponding control cell line were characterized according to morphological and cytochemical criteria to establish their origin from liver epithelium and to identify cellular changes due to transformation bydl-ethionine. The presence of intermediate junctions confirms the epithelial nature; glycogen accumulation and glucose-6-phosphatase activity confirm the hepatic origin of the cells. Persistent alterations resulting from ethionine transformation were variations in cell shape and size, focal multilayered growth, an increase in the nucleolar: nuclear ratio, and a reduction in the number of cells displaying a primary cilium. Hyperplasia of the inner nuclear membrane, elongation and branching of mitochondria, and a reduction in the length and frequency of cell junctions were also characteristic of the transformed cells.     

Establishment of rapidly proliferating rice cell suspension culture and its characterization by fluorescence-activated cell sorting analysis

A rapidly growing and fine-textured cell line, NB2P, was established from Japonica rice cultivar Nipponbare and characterized in this study. Addition of casein enzymatic hydrolysate (2 g/L) and pectinase (0.005%) to the suspension medium resulted in a 2-fold-increased rate of cell growth and reduced aggregation. Remarkably, the medium and conditions described here resulted in growth leading to a 9-fold increase in fresh weight 7 d after subculture. High-quality, well-dispersed nuclei were obtained from this NB2P cell culture. Fluorescence-activated cell sorting (FACS) analysis of the isolated nuclei showed a clear separation of each cell cycle phase in both small- and large-scale preparations. On the basis of representative data from the nuclei fraction in the G1 phase, purity of the sorted and recovered nuclei was higher than 98%. The studies described here demonstrate that NB2P culture can be a powerful tool for studying many important plant processes, including DNA replication and cell cycle-related pathways.     

Isolation of nuclei from rice tissue grown in suspension culture

Summary A simple method is described for the isolation of intact nucleic from rice tissue (Oryza sativa L.) grown in suspension culture. The procedure involves incubation of the tissue for 4 h with cellulase and pectinase prior to disruption of the cells. The yield of nucleic is approximately 40% (DNA basis) and the preparations are capable of synthesizing RNA in vitro. The method may be valuable to biochemically oriented research requiring plant nuclei.