Micropropagation of Cowania subintegra and Cowania stansburiana

Both Cowania subintegra Kearney and C. stansburiana Torr. were successfully propagated in vitro. Shoot proliferation occurred from shoot tips of green-house grown C. subintegra using a modified Murashige and Skoog medium supplemented with 4.4 M 6-benzyladenine and 0.5 M indole butyric acid. Excised microshoots (1.5–3.0 cm long) of both species were rooted using a two-step process in which they were cultured for 3 days in a root initiation medium with 2.7 M naphthaleneacetic acid and then transferred to a low nitrogen root elongation medium without auxin. Plantlets were successfully transferred to soilless potting mix.     

Effect of temperature on carbohydrate content,ADP glucose pyrophosphorylase,and ATP- and PPi-dependent phosphofructokinase activity of potato tuber callus tissue

Callus derived from Lemhi Russet and Russet Burbank tuber tissue was incubated at 20°C and 30°C on a high sucrose medium for starch-formation and subjected to simulated storage and reconditioning treatments at 5°C and 25°C after transfer of the callus to a medium without a carbon source. Percent dry weight of callus from both cultivars averaged about 21% after starch formation and 5% after storage and reconditioning treatments. Total sugars were higher in callus incubated on the starch forming treatment. Lemhi Russet callus contained predominantly reducing sugars, while Russet Burbank callus contained mostly non-reducing sugars. Total sugar content was generally lower for both cultivars after the storage and reconditioning treatment in callus after starch formation at 20°C. Starch content was generally higher in Lemhi Russet tissue. After starch formation at 20°C Lemhi Russet had higher starch after the storage and reconditioning treatment than tissue from 30°C, while the opposite trend was found in Russet Burbank tissue. Total protein declined in Russet Burbank tissue during the storage and reconditioning treatment regardless of prior incubation conditions, while this decline only occurred in Lemhi Russet tissue initially incubated at 30°C during the starch formation treatment. In tissue of both cultivars, ATP- and PPi-dependent phosphofructokinase activities were inversely proportional to total sugar concentrations, while in RB callus ADP glucose pyrophosphorylase activities were proportional to starch content.Research Paper 91B1 of the Idaho Agricultural Experiment Station.     

Hominization in the rainforest: The chimpanzee's piece of the puzzle

Many models of human evolution propose that key behavioral innovations were involved in the divergence of the human line from the ape line. An ecological reason, the shift from dense forests to a more open habitat, is suggested as the basis for these innovations. Primate models can be useful to our understanding of how environmental factors can affect such key behaviors. New results on forest chimpanzees demonstrate that for most patterns of behavior considered to favor a savanna model, the environmental influences are in opposition to the expectations. A new evaluation of the environmental influences on human evolution is required. We propose that either patterns of key human behaviors were inaccurately distinguished or that the hominization process started while the common ancestor of the chimpanzee and man was alive.     

Altered free calcium transients in pig kidney cells (LLC-PK1) cultured with penicillin/streptomycin

Summary The effect of a conventional antibiotic (penicillin/streptomycin) mixture on the widely used kidney epithelial cell line, LLC-PK₁, was investigated by measuring growth and intracellular free calcium. Free calcium concentration was the same in cells cultured for 3 to 7 wk with (“plus”) and without (“minus”) antibiotics both at rest and when challenged with high (14 mM) external calcium. When exposed to vasopressin, minus cells exhibited significantly smaller calcium transients than plus cells. A similar difference existed for transients elicited by a calcium ionophore, 4-br-A23187. After longer periods of culture (>20 wk), minus cells grew slower than plus cells but on reaching confluence (minus cells took 1 day longer) the morphologies and viabilities were indistinguishable. The finding that culture with penicillin/streptomycin reversibly modified some properties of LLC-PK₁ cells, at least partly through altered calcium homeostasis, is of importance for workers using this cell model to study drug effects and raises the general possibility of similar effects on other cultured cells.     

Kinetics of growth and sugar consumption in yeasts

An overview is presented of the steady- and transient state kinetics of growth and formation of metabolic byproducts in yeasts.Saccharomyces cerevisiae is strongly inclined to perform alcoholic fermentation. Even under fully aerobic conditions, ethanol is produced by this yeast when sugars are present in excess. This so-called Crabtree effect probably results from a multiplicity of factors, including the mode of sugar transport and the regulation of enzyme activities involved in respiration and alcoholic fermentation. The Crabtree effect inS. cerevisiae is not caused by an intrinsic inability to adjust its respiratory activity to high glycolytic fluxes. Under certain cultivation conditions, for example during growth in the presence of weak organic acids, very high respiration rates can be achieved by this yeast.S. cerevisiae is an exceptional yeast since, in contrast to most other species that are able to perform alcoholic fermentation, it can grow under strictly anaerobic conditions.Non-Saccharomyces yeasts require a growth-limiting supply of oxygen (i.e. oxygen-limited growth conditions) to trigger alcoholic fermentation. However, complete absence of oxygen results in cessation of growth and therefore, ultimately, of alcoholic fermentation. Since it is very difficult to reproducibly achieve the right oxygen dosage in large-scale fermentations, non-Saccharomyces yeasts are therefore not suitable for large-scale alcoholic fermentation of sugar-containing waste streams. In these yeasts, alcoholic fermentation is also dependent on the type of sugar. For example, the facultatively fermentative yeastCandida utilis does not ferment maltose, not even under oxygen-limited growth conditions, although this disaccharide supports rapid oxidative growth.     

Molecular integrity of monoclonal antibodies produced by hybridoma cells in batch culture and in continuous-flow culture with integrated product recovery

The molecular integrity of monoclonal antibodies (MCAB) produced by murine hybridoma cell line TB/C3 was studied in batch and continuous-flow cultures. In batch culture, one band of MCAB was detected initially by Western blotting of sodium dodecyl sulfate (SDS)-polyacrylamide gels run under unreduced conditions, but heterogenous MCAB bands appeared as the culture aged. The latter were due to the degradation of MCAB by proteases active at the neutral pH of the culture. The deleterious effect of proteases was minimized in the continuous-flow cultures which were integrated for product recovery. The MCAB of high quality was purified over 26 days from a culture grown at a dilution rate of 0.025 h(-1) (experiment 1). However, at a lower dilution rate of 0.015 h(-1) (experiment 2), the integrity of MCAB was compromised after the initial 13 days of culture. This was shown to be due to the variation in the carbohydrate content of MCAB produced, as judged by the increased sialylation of heavy chains and the varied reactivity of MCAB with lectins (Maackia amurensis agglutinin, Galanthus nivalis agglutinin, and Datura stramonium agglutinin) as the age of the culture increased. The concentration of the purified MCAB samples by enzyme-linked immunosorbent assay (ELISA) (used normally) was usually higher than that estimated by absorbance at 280 nm. Best correlation between the two methods (ELISA-280 nm ratio of 1.02-1.25) was obtained with experiment 1 samples. This ratio increased in experiment 2 and batch culture samples as the heterogeneity of MCAB produced increased, being 1.03-2.94 and 2.53-4.62, respectively. Therefore, ELISA overestimated MCAB concentration when the molecular integrity of the latter was compromised. The ELISA-A(280) nm ratio might hence provide a useful indicator for assessing the quality of MCAB produced. Comparison of SDS-polyacrylamide gels stained with Coomassie Brilliant Blue R and silver showed that the former correlated better with the MCAB activity stain, whereas the silver stained both the protein- and carbohydrate-rich components. Comparison of the patterns produced with these two stains might therefore offer another parameter to monitor the overall integrity of MCAB produced. Finally, the data presented have important implications on the validity of using long-term and intensive cultures for generating MCAB because such cultures would be subjected to the additive effects reported for batch and continuous modes of growth. (c) 1993 John Wiley & Sons, Inc.     

Cell retention-chemostat studies of hybridoma cells-analysis of hybridoma growth and metabolism in continuous suspension culture in serum-free medium

The steady-state metabolic parameters for a hybridoma cell line have been determined in continuous suspension-perfusion culture over a wide range of perfusion rates and cell bleed rates. Significant increases in viable cell concentrations and volumetric productivities were achieved at high perfusion rates and low cell bleed rates. At the low growth rates examined in this study, cellular metabolism shifted to become more oxidative, and as a result, the fraction of consumed substrate converted to inhibitory metabolic by-products was reduced. Specific antibody productivity was found to be non-growth associated. (c) 1993 John Wiley & Sons, Inc.     

Cell wall synthesis during growth and maturation of Nitella internodal cells

An improved ¹³C-density-labeling method was used to study cell wall synthesis in rapidly expanding, slowly expanding and recently mature internodes of Nitella translucens var axillaris (A.Br.) R.D.W. As cells matured, the rate of wall synthesis slowed and the deposition of cellulose microfibrils changed from a predominantly transverse direction in the primary wall of rapidly expanding internodes to a helicoidal array in the secondary wall of mature internodes. The secondary wall was characterized by relatively higher rates of cellulose synthesis and lower rates of pectin synthesis than the primary wall. The synthesis of xyloglucan also decreased markedly at the transition to secondary wall synthesis, while the synthesis of mannose-rich hemicellulose increased. Even though structural differences were striking between the primary and secondary walls of Nitella, compositional differences between the two types of wall were quantitative rather than qualitative. The authors appreciate the assistance of Martin Yousef with the electron microscopy.     

Rapid changes in amplification and methylation pattern of genomic DNA in cultured carrot root explants (Daucus carota L.)

Summary Rapid genomic DNA variation due to methylation and copy number alteration was observed in carrot root explants 6 h after inoculation and during a 36-h period of exponential callus growth. De novo methylation and amplification of restricted BspNI fragments of low molecular weight occurred before cell cycle activation and should, therefore, be independent of progression through the S-phase of the cell cycle. Growth regulators seemed to influence the amplification pattern indirectly by regulating cell division activity. In exponentially growing callus tissue the copy number of most of the repetitive fragments was dramatically reduced. It is presumed that this reduction in the copy number of repetitive fragments is characteristic of rejuvenilization. 3-Indole-acetic-acid (IAA) and inositol in the medium increased the degree of unspecific genomic DNA methylation in growing rhizogenic carrot callus tissue in the absence of kinetin, which inhibits root induction at that stage. A possible relation to the induction of rhizogenesis is considered. The observed reduction in number of repetitive restriction fragments and the increase in DNA methylation are gross changes covering the total genome. The results are discussed in relation to the controversy concerning the general biological significance of the methylation and amplification of DNA sequences.     

EFFECTS OF VASOACTIVE INTESTINAL PEPTIDE ON ACTIVITIES OF SUCCINIC DEHYDROGENASE AND ALKALINE PHOSPHATASE IN RAT HEPATOMA CELLS

Using cytochemical method,microspectrophotometry and image analysis,effects of va-soactive intestinal peptide(VIP)on activities of succinic dehydrogenase(SDH)and alkalinephosphatase(ALP)in rat hepatoma cells were studied in vitro.The results showed that thehepatoma cell expressed potent positive reactions of SDH and ALP,the positive positionswere located at the cell membranes and/or cytoplasm.Having been treated with VIP,ALPdecreased obviously in activity(P<0. 01,compared with hepatoma cells untreated by VIP).The sites of ALP activty were chiefly located at the cell membranes,particularly at the cell-cell contacts.Cultured rat hepatoma cells had intensive SDH activity in their cytoplasm.Compared with untreated eclls,there was no marked difference in the intensity of SDH activ-ity in VIP-treated hepatoma cells(P>0.05).