Translational alterations in maize leaves responding to pathogen infection, paraquat treatment, or heat shock : polysome dissociation and accumulation of a 57 kilodalton protein

Translational alterations occur in maize (Zea mays L.) leaves stressed by pathogen infection or herbicide paraquat treatment. These translational changes include: (a) dissociation of large polysomes to small polysomes, monosomes, and subunits; (b) a decreased rate of total protein synthesis; and (c) a reduced synthesis of several proteins by polysomes in vitro. The polysome dissociation was neither due to an extraction artifact nor to degradation of RNA by RNase. The protein patterns of polysomes isolated from leaves inoculated with Bipolaris maydis at 6 to 48 hours showed an increase in the intensity of a 57 kilodalton protein. When inoculated with less virulent pathogens, such as B. zeicola, Exserohilum turcicum, or Colletotrichum graminicola, the protein was accumulated in polysomes of leaves at 24 to 48 hours after inoculation. The 57 kilodalton protein was also accumulated in polysomes of maize leaves responding to heat shock or herbicide paraquat treatments. The purified 57 kilodalton protein reassociated with polysomes isolated from healthy leaves and inhibited polysomal translation in vitro. Since the 57 kilodalton protein is rapidly accumulated in maize polysomes in response to various biological and environmental stresses and may affect protein synthesis, it may be involved in translational regulation of maize leaves during stress response.     

Characterization of the H Translocating Adenosine Triphosphatase and Pyrophosphatase of Vacuolar Membranes Isolated by Means of a Perfusion Technique from Chara corallina

Sealed tonoplast vesicles were isolated from single cells of Chara corallina with the aid of an intracellular perfusion technique in combination with a 3/10% Percoll two step gradient centrifugation. The isolated tonoplast fraction was free from plasmalemma and chloroplasts, and showed no activities of cytochrome c oxidase, and latent IDPase, but had about 10% of the NADH-cytochrome c reductase activity. The vesicles had both ATPase and PPase activities, which could be stimulated in the presence of 10 micromolar gramicidin by 170 and 130%, respectively, demonstrating the existence of sealed vesicles. Furthermore, ATP- and PPi-dependent H⁺ pumping through the membrane into the vesicles was shown. Both ATPase and PPase had pH optima around pH 8.5. At the physiological pH, 7.3, they still had more than 80% of their maximal activities. Ammonium molybdate, azide, and vanadate had no or little effect on the activities of both enzymes or their associated H⁺ pumping activities. N,N′-dicyclohexylcarbodiimide inhibited the ATPase strongly (I₅₀ = 20 micromolar) but the PPase only weakly. The ATPase was also more sensitive to N-ethylmaleimide than the PPase. 4,4′-Stilbenedisulfonic acid affected both enzyme activities and their associated H⁺ pumping activities. This is in contrast to the H⁺-PPase of higher plants which is 4,4′-stilbenedisulfonic acid insensitive.     

CO(2) Fixation Rate and RuBisCO Content Increase in the Halotolerant Cyanobacterium, Aphanothece halophytica, Grown in High Salinities

The growth of the halotolerant cyanobacterium Aphanothece halophytica, previously adapted to 0.5 molar NaCl, was optimal when NaCl concentration in culture medium was in the range 0.5 to 1.0 molar. The growth was delayed at either too low or too high salinities with lag time of ca. 0.5 day in 0.25 molar NaCl and ca. 2 days in 2 molar NaCl under the experimental conditions. However, the growth rates at the logarithmic phase were similar in the culture media containing NaCl in the range 0.25 to 2.0 molar. The capacity of photosynthetic CO₂ fixation increased 3.7-fold in the cells at the logarithmic phase as NaCl concentration in the culture medium increased from 0.25 to 2.0 molar. The protein level of ribulose 1,5-bisphosphate carboxylase/oxygenase was also found to increase with increasing salinity using both an immunoblotting method and protein A-gold immunoelectron microscopy. These results indicate that high photosynthetic capacity and high ribulose 1,5-bisphosphate carboxylase/oxygenase content may entail an important role in betaine synthesis and adaptation of the A. halophytica cells to high NaCl level.     

Approach to maceration mechanism in enzymatic pulping of bast fibers by alkalophilic pectinolytic enzymes produced by Erwinia species

Tissue maceration was generally elucidated by the action of endo-polygalacturonase and endo-pectate or -pectin lyase (endo-PAL or -PNL). In a process of screening of Erwinia and Pseudomonas strains for enzymatic pulping of pectocellulosic bast fibers, it was found that their PAL productivity was not completely related with defibration activity, i.e., the fact that an E.chrysanthemi strain showed high PAL productivity but possessed rather low defibration activity. Moreover, defibration activity was parallel to the amount of neutral sugars released during pulping. Based on these fact, the maceration or enzymatic pulping of basts was estimated to proceed not only by cleavage of interfiber bonding cause by PAL action but also another factors. Among three possibilities proposed on the maceration mechanism of basts, it was elucidated by a concerted action of PAL and PNL with an aid of xylanase. In addition, a quantitative determinative method of maceration activity toward basts was also presented.     

High cell density perfusion culture of hybridoma cells recycling high molecular weight components

We have developed a high cell density and high product concentration culture system recycling high molecular weight components. The production of monoclonal antibodies in high concentration was performed by this culture system with mouse human hybridoma H2 and V6 cells in serum-free defined media.The concentration of IgG after 48 days culture of H2 cells in ITES-eRDF reached 2 mg/ml and the purity of IgG in culture fluid was 61%. In addition, high molecular weight components in serum-free media, such as transferrin or BSA, could be reduced to 5% of the original concentration.     

Egg yolk lipoprotein,a new supplement for the growth of mammalian cells in serum-free medium

Egg yolk lipoprotein promoted growth of a wide variety of mammalian cell lines, including plasma-cytomas and epithelial cell lines, in serum-free medium. The lipoprotein was active for cell growth when used with insulin, transferrin, ethanolamine and selenite. The most active lipoprotein fraction (YLP-pI7.5) was purified to give a single peak by chromatofocusing and gel filtration, and was homogeneous on a 0.35% agarose gel electrophoretogram. The lipoprotein was characterised as a very low density lipoprotein with a protein content of only 1.3%. This lipoprotein had an optimal concentration of 300 g/ml (4 g protein/ml). It was easily separable from proteinous molecules secreted into the serum-free medium by the cells, since it floated on the surface of the medium after addition of ammonium sulfate, to precipitate protein, and centrifugation. An associated structure of lipid and protein seemed to be still necessary for the lipoprotein to exhibit a growth promoting activity.     

Purification and characterization of lymphocytic clonal growth factor (LCGF) derived from human-human hybridoma SH-76 cells

Human-human hybridoma SH-76 cells were found to produce a factor that supported the growth of lymphocytic cells at low densities. The factor was purified from serum-free conditioned medium of the hybridoma cells by a successive application of ammonium sulfate precipitation, DEAE-Toyopearl, TSK G3000 SW and DEAE-5PW column chromatograph. The purified factor was a 72K single protein. The factor showed marked growth stimulating effect on lymphocytic cell lines, but had no effect on the growth of human adhesive cancer cell lines. Thus, the factor is a lymphocytic clonal growth factor (LCGF), as found previously in human plasma (Miyata, 1988). The LCGF of SH-76 cells could be produced in growth factor-free RPMI medium and purified easily from the conditioned medium. The factor is inactivated by heating at over 80°C, but is much more stable than the LCGF in human plasma.     

The usefulness of CHAPS as a non-cytotoxic stabilizing agent in purification of growth factors

Among several detergents, a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), was found to be least cytotoxic for cultured mammalian cells. CHAPS improved the activity recovery and elution profile of crude and purified fibroblast growth factors (FGFs) during chromatographies. Diluted preparations of FGFs were stabilized by CHAPS against the loss during storage. Amino acid sequence analysis was not disturbed by CHAPS. CHAPS was removable by reversed-phase high-performance liquid chromatography. These results indicate that CHAPS is useful as a non-cytotoxic stabilizing agent in purification of various kinds of bioactive polypeptides.Abbreviations -MEM Alpha Modification of Eagle's Minimal essential medium - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - CHAPSO 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate - CS Calf Serum - EGF Epidermal Growth Factor - FGF Fibroblast Growth Factor - HPLC High-Performance Liquid Chromatography - NGF Nerve Growth Factor - NOG 1-O-n-octyl--D-glucopyranoside - NP-40 Nonidet P-40 - PBS Phosphate-Buffered Saline - SB 12 3-(dodecylmethylammonio)-1-propane sulfonate - SDS Sodium Dodecyl Sulfate - TGF- and Transforming Growth Factor type and      

QALYs, age and fairness

... We can therefore conclude that either we should go for equality; and in that case QALYs are unfair because they haven't got enough of an ageist bias. Or we should accept consequentialism; and in that case QALYs have just the right sort of ageist bias. No plausible case can, however, be made for the claim that QALYs have an unfair bias against old people. Other things being equal we ought when distributing resources essential for survival favour the young. This ethical claim can be supported both by reference to equality (the life-time-view) and by reference to consequentialism (and the premises that resources generally will be more useful when given to young people).     

Transport regulation of recombinant gene expression in E. coli and B. subtilis

Expression kinetics of the lactose (lac) operon in Escherichia coli are reviewed for both wild-type and recombinant cell cultures under chemostatic conditions. A unified model which involves regulation of active inducer (lactose) transport, promoter-operator regulated expression of the lac operon, glucose-mediated inducer exclusion, and catabolite repression is summarized and supporting data is shown to verify its accuracy. The synthesis of alpha-amylase with a recombinant form of Bacillus subtilis is also reviewed to point out generic features in transport regulation, the lac operon model providing a point of departure. While there are many similarities in the influence of transport on both regulating models, there are also important differences. In a chemostat system, the synthesis of alpha-amylase is nongrowth associated, while beta-galactosidase is a growth-associated enzyme. Nevertheless, transport regulation is an important feature in both instances.