Quantitation of cell proliferation,colony formation,and carcinogen induced cytotoxicity of rat tracheal epithelial cells grown in culture on 3T3 feeder layers

Summary A cell culture system is described for the growth of rat tracheal epithelial (RTE) cells at clonal density. The system uses normal, early passage RTE cells grown on feeder layers of lethally irradiated 3T3 cells. The RTE cells have a high colony forming efficiency (5 to 10%) in culture, can be passaged up to 5 times, and are capable of more than 20 cumulative doublings per colony forming cell. The epithelial nature of the cells was confirmed by cell and colony morphology, immunoperoxidase staining of intracellular keratin, and cellular ultrastructural studies. The cytotoxic response of RTE cells to a variety of carcinogens, including a direct acting chemical carcinogen, a physical carcinogen, and a series of polycyclic aromatic hydrocarbons, was quantitated. A linear decrease in the logarithm of survival was observed with increasing doses ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG), γ-irradiation, 7,12-dimethylbenz(a)anthracene, and a diol-epoxide of benzo(a)pyrene. No toxicity was observed after treatment with benzo(a)pyrene or 3-methylcholanthrene over the concentration range examined. In contrast, phorbol ester tumor promoters stimulated cell growth markedly. Based on these and other studies, the RTE cell culture system represents a model system that will be useful for quantitative studies of epithelial cell growth, differentiation, and carcinogenesis.     

Expression and quantification of firefly luciferase under control of Rhizobium meliloti symbiotic promoters

We have tested the use of firefly luciferase for monitoring regulated symbiotic nitrogen fixation gene expression. Broad-host-range plasmids carrying translational fusions of Rhizobium meliloti nifH, fixA and nifA promoters were constructed. Despite low levels of promoter activity the absence of Escherichia coli endogenous luminescence and the high sensitivity of the bioluminescent assay for firefly luciferase allowed rapid screening for functional luciferase expression. Plasmids containing symbiotic promoter-luc fusions were established in R. meliloti. Luciferase activity was detected and measured in both vegetative and symbiotic cells giving comparable results with those obtained by beta-galactosidase assays. In addition, the luciferase assay was quicker, more sensitive and could be carried out with unrestricted cells. Furthermore, bioluminescence was high enough in alfalfa nodules containing nifH—luc fusion to be observed by a dark-adapted eye and photographed.     

Biological N2 Fixation in wetland rice fields: Estimation and contribution to nitrogen balance

This paper 1) reviews improvements and new approaches in methodologies for estimating biological N₂ fixation (BNF) in wetland soils, 2) summarizes earlier quantitative estimates and recent data, and 3) discusses the contribution of BNF to N balance in wetland-rice culture.Measuring acetylene reducing activity (ARA) is still the most popular method for assessing BNF in rice fields. Recent studies confirm that ARA measurements present a number of problems that may render quantitative extrapolations questionable. On the other hand, few comparative measures show ARA's potential as a quantitative estimate. Methods for measuring photodependent and associative ARA in field studies have been standardized, and major progress has been made in sampling procedures. Standardized ARA measurements have shown significant differences in associative N₂ fixation among rice varieties.The ¹⁵N dilution method is suitable for measuring the percentage of N derived from the atmosphere (% Ndfa) in legumes and rice. In particular, the ¹⁵N dilution technique, using available soil N as control, appears to be a promising method for screening rice varieties for ability to utilize biologically fixed N. Attempts to adapt the ¹⁵N dilution method to aquatic N₂ fixers (Azolla and blue-green algae [BGA]) encountered difficulties due to the rapid change in ¹⁵N enrichment of the water.Differences in natural ¹⁵N abundance have been used to show differences among plant organs and species or varieties in rice and Azolla, and to estimate Ndfa by Azolla, but the method appears to be semi-quantitative.Recent pot experiments using stabilized ¹⁵N-labelled soil or balances in pots covered with black cloth indicate a contribution of 10–30 kg N ha^-1 crop^-1 by heterotrophic BNF in flooded planted soil with no or little N fertilizer used.Associative BNF extrapolated from ARA and ¹⁵N incorporation range from 1 to 7 kg N ha^-1 crop^-1. Straw application increases heterotrophic and photodependent BNF. Pot experiments show N gains of 2–4 mg N g^-1 straw added at 10 tons ha^-1.N₂ fixation by BGA has been almost exclusively estimated by ARA and biomass measurements. Estimates by ARA range from a few to 80 kg N ha^-1 crop^-1 (average 27 kg). Recent extensive measurements show extrapolated values of about 20 kg N ha^-1 crop^-1 in no-N plots, 8 kg in plots with broadcast urea, and 12 kg in plots with deep-placed urea.Most information on N₂ fixed by Azolla and legume green manure comes from N accumulation measurements and determination of % Ndfa. Recent trials in an international network show standing crops of Azolla averaging 30–40 kg N ha^-1 and the accumulation of 50–90 kg N ha^-1 for two crops of Azolla grown before and after transplanting rice. Estimates of % Ndfa in Azolla by ¹⁵N dilution and delta ¹⁵N methods range from 51 to 99%. Assuming 50–80% Ndfa in legume green manures, one crop can provide 50–100 kg N ha^-1 in 50 days. Few balance studies in microplots or pots report extrapolated N gains of 150–250 kg N ha^-1 crop^-1.N balances in long-term fertility experiments range from 19 to 98 kg N ha^-1 crop^-1 (average 50 kg N) in fields with no N fertilizer applied. The problems encountered with ARA and ¹⁵N methods have revived interest in N balance studies in pots. Balances are usually highest in flooded planted pots exposed to light and receiving no N fertilizer; extrapolated values range from 16 to 70 kg N ha^-1 crop^-1 (average 38 kg N). A compilation of balance experiments with rice soil shows an average balance of about 30 kg N ha^-1 crop^-1 in soils where no inorganic fertilizer N was applied.Biological N₂ fixation by individual systems can be estimated more or less accurately, but total BNF in a rice field has not yet been estimated by measuring simultaneously the activities of the various components in situ. As a result, it is not clear if the activities of the different N₂-fixing systems are independent or related. A method to estimate in situ the contribution of N₂ fixed to rice nutrition is still not available. Dynamics of BNF during the crop cycle is known for indigenous agents but the pattern of fixed N availability to rice is known only for a few green manure crops.     

Genetic control and expression of the major ejaculatory bulb protein (PEB-me) inDrosophila melanogaster

PEB-me is a predominant protein of matureDrosophila melanogaster ejaculatory bulbs. It is resolved into four or five closely spaced subfractions (apparent molecular weight 35–39 kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four electrophoretic variants of PEB-me differing in apparent molecular weight by 200–800 daltons were found. These appear to be controlled by four alleles of a gene (peb) located by recombination and deletion mapping to the 60F1-2 region of chromosome 2. A minor ejaculatory bulb protein of ca. 80 kD (hPEB) was found to be immunochemically related to PEB and possibly encoded bypeb. PEB is not detected by immunoblotting techniques in virgin females, in male tissues other than the ejaculatory bulb, or during developmental stages preceding the formation of this organ. The results of transplantations of genital imaginal discs and of immature ejaculatory bulbs between two strains having different PEB alleles suggest that the ejaculatory bulb is the site of PEB synthesis. In flies mutant fortra, tra-2, dsx, orix, tissue specificity of PEB localization is retained and the protein is found whenever the ejaculatory bulb is formed, regardless of the chromosomal sex of the fly. The protein is transferred into the female genital duct during mating, where it can be detected for up to 12 hr. Possible functions of PEB inDrosophila reproduction are discussed.     

Embryogenesis and oögenesis in alate virginoparae,gynoparae, and oviparae of the aphid Myzus persicae, in relation to photoperiod

The study reports on the effects of prenatal and/or postnatal exposures to short-night or long-night conditions, and of crowding, on embryogenesis and oögenesis in alate virginoparae, gynoparae, and oviparae of a holocyclic strain of the green peach aphid, Myzus persicae, from Yakima, Washington State.In alate virginoparae raised at a density of 10–20 per radish seedling in a short-night regime (8 hr darkness per diem), 3–4 embryos occurred in each of their 10 ovarioles, when the aphids attained adulthood. More than 30 larvae were deposited by most of these alatae. However, in young adult gynoparae, raised at these densities in a long-night regime (15 hr darkness per diem), only one viable embryo (a presumptive ovipara) occurred per ovariole. The follicle containing this embryo was followed by 1–2 abnormal follicles in each ovariole, and the number of larvae deposited by a gynopara was generally less than 10. In young adult oviparae similarly raised under a long-night regime, only one egg typically occurred in each of their 10 ovarioles, and the eggs deposited by an ovipara (only after it had mated) generally numbered less than 10. Alate virginoparae and gynoparae contained an additional embryo in some of their ovarioles when these morphs were raised at a lower density (1–5 per plant).Presumptive gynoparae partially developed the reproductive features of alate virginoparae when transferred to a short-night regime at birth; the converse was true when presumptive alate virginoparae were transferred to a long-night regime early in larval life. Oviparae maintained in short nights from before birth developed the appearance of apterous virginoparae but still produced eggs rather than embryos. However, their oögenesis was enhanced and eggs (10–20) were deposited by them without prior mating. Under all regimes tested, oviparae were always deposited early in the larviposition sequence of their alate mothers, and the number of oviparae deposited never exceeded 15.The possible involvement of juvenile hormone in the regulation of these events and the ecological significance of the results are discussed.     

Population Dynamics of Meloidogyne incognita on Corn Grown in Soil in Fested with Arthrobotrys conoides

Microplot and greenhouse experiments were conducted to evaluate the effects of soil incorporation of the nematophagous fungus Arthrobotrys conoides and green alfalfa mulch on the population dynamics of Meloidogyne incognita on corn. Reproduction of M. incognita and the incidence of root galling were reduced by the addition of A. conoides and/or green alfalfa in all tests. Numbers of juveniles were reduced by as much as 84%, and eggs were fewest in early to mid-season soil samples from microplots. Yields increased in treatments with A. conoides and/or green alfalfa in greenhouse tests and in the microplot tests in 1979. No interaction was found between the fungus and green alfalfa in the reduction of the nematode population.     

MORPHOLOGY AND DISTRIBUTION OF NUCLEI DURING DEVELOPMENT IN CYMOPOLIA BARBATA (CHLOROPHYCOPHYTA,DASYCLADALES)1

The morphology and distribution of a variety of types of nucleus in the apex, in young and mature gametangia, and in older regions of the Cymopolia cell were studied by light and electron microscopy. Spherical and convoluted nuclei 2–7μm in diameter were observed in apical regions of the vegetative siphon. Nuclei 4 μm in diameter were present in the young primary laterals and in developing gametangia. A single characteristic nucleus migrates from the siphon into the primary lateral of the second basipetal whorl. It is further transported into one of several possible secondary laterals and determines the development of gametangia which become multinucleate in the fourth or fifth whorls. Nuclei are characterized by size, shape, nucleolar morphology, nucleoplasmic inclusions and the ultrastructure of the perinuclear cytoplasm. Although no “primary nucleus” characteristic of the uninucleate genera, Acetabularia and Batophora was observed, some nuclei of Cymopolia have features in common with secondary nuclei of these genera.     

Effect of sulphite on adenine nucleotides of the green alga Trebouxia

The amounts of AMP, ADP and ATP were determined after sulfite incubation in photo-organotrophically cultivated cells of the green alga Trebouxia. D