Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit

We have used a gene transfer system to investigate the relationship between expression of the rat Na,K-ATPase alpha 1 subunit gene and ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATPase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat alpha 1 subunit gene is directly responsible for ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.     

Artificial incubation of California condor Gymnogyps californianus eggs removed from the wild

The currrent California condor (Gymnogyps californianus) recovery plan entails increasing the reproductive rate via replacement-clutch manipulation of eggs. During the period from 1983 to 1985, 15 eggs were removed from wild nesting pairs for artificial incubation. The eggs were incubated at a dry bulb temperature of 36.4°C in modified forced-air Lyon Electric incubators. The incubation humidity was adjusted for individual eggs based on weight loss data (water = weight), 25.6–30.0°C wet bulb (41.0–63.0% Relative Humidity (RH)). The chicks were hatched initially under forced-air conditions of 36.1°C dry bulb, 31.1–01.7°C wet bulb (70.0–73.0% RH). In 1984, hatching parameters were changed to still-air conditions, 36.1°C dry bulb (top of the egg), 35.0°C dry bulb (bottom of the egg), 31.1–31.7°C wet bulb (70.0-73.0% RH). Tactile and auditory stimulation was utilized during the pip-to-hatch interval. From among 15 eggs collected, 13 hatched, and 12 condor chicks were raised successfully (hatchability: 86.7%; survivability: 92.3%).     

3-Mercaptopropionic acid administration increases the affinity of [H]Quinuclidinyl benzilate binding to membranes of the striatum and cerebellum

It is known that quinuclidinyl benzilate (QNB) binds specifically and with high affinity to the cholinergic muscarinic receptor and that behaves as a potent antagonist of this receptor.

We have analysed -[³H]QNB binding to rat CNS membranes after the administration of the convulsant 3-mercaptopropionic acid (MP) (150 mg·kg⁻¹, i.p.). The studies were done in rats killed at two stages: during and after seizures. No changes in [³H]QNB binding to hippocampus and cerebral cortex membranes were found. [³H]QNB binding increased about 40 and 80% in striatum and cerebellum membranes, respectively. The changes were observed both in seizure and postseizures states. The study was extended to the assay of [³H]QNB binding kinetic constants in the anatomical areas modified by the convulsant. The analysis of the saturation curves indicated an increase in the binding affinity but no change in the number of binding sites. Hill number values were near the unit suggesting a non-cooperative interaction between the ligand and the receptor, and the labelling of a homogeneous population of receptor sites.

The results suggest the participation of some cholinergic pathways in the development and maintenance of MP-induced seizures.     

The principal islet of the coho salmon (Oncorhyncus kisutch) contains the BB isoenzyme of creatine kinase

The importance of creatine kinase (E.C. 2.7.3.2) in endocrine tissues has been generally overlooked. Using a specific radiometric assay, we have demonstrated the existence of CK in the Brockmann body (principal islet) of the Coho salmon. We have purified this protein from insular tissue and concurrently purified CK from brain and muscle of the salmon. Purification characteristics, immunological cross-reactivity, and N-terminal sequence analysis have demonstrated that the predominant cytosolic CK from the Brockmann body is indistinguishable from the BB (brain) isoenzyme. Immunocytochemical studies indicated that the enzyme resides in the endocrine parenchyma. Phosphocreatine may serve as a reservoir of energy in the islet and augment its capacity to secrete hormones. The induction of CK-BB in the islet by other hormones could influence the secretion of insular hormones. Interorgan flux of the substrate creatine may be an undescribed mechanism of physiological regulation.     

Immature maize spikelets develop and produce pollen in culture

Immature maize spikelets have been successfully grown in vitro. Culture conditions were refined to maximize development of normal pollen grains. Kinetin was not required for normal development, in contrast to the absolute requirement for this plant growth regulator for in vitro tassel development. Development occured in all stages sampled, from premeiosis to postvacuolation, and there was no lag in progression through the various stages of development as compared to greenhouse-grown material. Cultured spikelets produced pollen that appeared morphologically normal, accumulated starch and had the normal two sperm nuclei and single vegetative nucleus.     

High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection

Endochitinases (E.C. 3.2.14, chitinase) are believed to be important in the biochemical defense of plants against chitin-containing fungal pathogens. We introduced a gene for class I (basic) tobacco chitinase regulated by Cauliflower Mosaic Virus 35S-RNA expression signals into Nicotiana sylvestris. The gene was expressed to give mature, enzymatically active chitinase targeted to the intracellular compartment of leaves. Most transformants accumulated extremely high levels of chitinase-up to 120-fold that of non-transformed plants in comparable tissues. Unexpectedly, some transformants exhibited chitinase levels lower than in non-transformed plants suggesting that the transgene inhibited expression of the homologous host gene. Progeny tests indicate this effect is not permanent. High levels of chitinase in transformants did not substantially increase resistance to the chitin-containing fungus Cercospora nicotiana, which causes Frog Eye disease. Therefore class I chitinase does not appear to be the limiting factor in the defense reaction to this pathogen.     

Identification and sequence of a Na+-linked gene from the marine bacterium Alteromonas haloplanktis which functionally complements the dagA gene of Escherichia coli

A 4.0 kb fragment from a plasmid genomic DNA library of the marine bacterium Alteromonas haloplanktis ATCC 19855 was found in the presence of Na+ to complement the dagA gene of Escherichia coli. We have completely sequenced this fragment and the position of the Na(+)-linked D-alanine glycine permease gene (dagA) on the fragment has been determined by complementation. The predicted carrier protein consists of 542 amino acid residues (M(r) 58,955). Its hydropathy profile suggests it is composed of eight transmembrane segments with a long hydrophilic region between segments six and seven. Significant similarity has been found between this Na(+)-linked permease and the Na+/proline permeases of E. coli and Salmonella typhimurium and the human and rabbit intestinal Na+/glucose cotransporters.     

The role of malic acid in the metabolism of Schizosaccharomyces pombe: substrate consumption and cell growth

Summary The effect of initial concentrations of malate varying from 0 to 28.6 g/l was studied. The acid was found to be inhibitory for growth of Schizosaccharomyces pombe but not for its deacidification activity. Malate was never integrated into biomass but partly transformed into ethanol if the aeration rate was weak (oxygen limitation). In the absence of glucose, resting cells of S. pombe were able to degrade malic acid if their concentration was sufficient, but their viability gradually decreased. However, for 0.15 g/l of growing cells (inoculum) 6 g/l of glucose was necessary to consume 8 g/l of malate. When the medium did not contain sugar no growth was observed despite the partial consumption of malate, showing that the acid was neither a carbon source nor an energy source. Offprint requests to: P. Strehaiano     

Inactivation of recombinant plasmid DNA from a human erythropoietin-producing mouse cell line grown on a large scale

Summary Experiments were carried out to assess the survival of recombinant plasmid DNA during large-scale production of recombinant human erythropoietin (rhuEPO) in a fermentation pilot plant. The analyses revealed DNA-degrading activities in the fermentation broth and in the waste-water, leading to rapid destruction of plasmid DNA added to medium or waste-water. The capability of the plasmid-DNA-spiked samples to transform competent bacteria was drastically reduced. The DNA-degrading activity in the waste-waters could be blocked by addition of EDTA or by boiling, indicating the presence of DNA-degrading enzymes (DNases). No plasmid-specific DNA sequences were detected in waste-water samples by in-vitro amplification with Taqpolymerase. Genomic DNA preparations of cell debris collected from waste-water samples only contained degraded plasmid DNA. Furthermore, it was shown that intact plasmid DNA could be degraded to fragments of less than 1000 bp by incubation at 121°C for 20 min, leading to a decrease in the plasmid-specific transforming capacity by a factor of 10³ per minute. Thus, DNA from the rhuEPO production pilot plant was efficiently inactivated at three different levels: (i) in the fermentation medium (DNase), (ii) in the waste-water container (DNase), and (iii) by heat inactivation for 20 min at 120°C. These results indicate that the probability of delivery of recombinant DNA into the environment is extremely low in such biotechnological production processes. Offprint requests to: M. R. Fibi