Three-dimensional growth and morphogenesis of mouse submandibular epithelial cells in serum-free primary culture

Mouse submandibular epithelial cells can be grown in primary culture using the collagen gel matrix and a chemically defined medium consisting of insulin, transferrin, cholera toxin, and BSA (or FGF). Sustained cell growth leading to a 5–10-fold increase in cell number was observed in less than 2 weeks. In the presence of these additives, clumps of cells proliferate by extending ‘star-like’ projections into the matrix, resulting in three-dimensional outgrowths. The morphology of these outgrowths can be modulated to form a ‘cyst-like’ appearance by deleting BSA and adding cortisol to the basal medium containing insulin, transferrin, cholera toxin and FGF. In brief, a serum-free medium for sustained growth has been devised and a simple manipulation of supplements can modulate the three-dimensional colony morphology in the collagen gel matrix. Finally, the resulting outgrowths can produce epidermal growth factor (EGF) in response to dihydrotestosterone.     

Iron-molybdenum cofactor from nitrogenase. Modified extraction methods as probes for composition

Five modifications of the preparative procedure for isolating iron-molybdenum cofactor (FeMoco) from the molybdenum-iron (MoFe) protein of Azotobacter vinelandii nitrogenase have been developed. This variety of isolation methods has established that no single component of the original isolation protocol, i.e. Tris, Cl-, citrate, HPO4(2-), N,N-dimethylformamide, and N-methylformamide, is essential for the effective isolation and/or structural stability of FeMoco, although any of them may act as ligands to FeMoco when present. The acid-bse status (effective pH) of the extracting solvent is a key adjustable parameter in the isolation procedure. The new procedures produced FeMoco with yields, metal analysis, charge, EPR spectrum, and specific activity (after reconstituting crude extracts from A. vinelandii UW45 mutant cells) essentially identical with FeMoco isolated by the original procedure. After purification, FeMoco apparently contains molybdenum, iron, and sulfide in a 1:7:4 ratio with N-methylformamide as a ligand but no amino acid residues, common sugars, coenzyme A, or lipoic acid. Reaction with o-phenanthroline allows quantitation of both adventitious and FeMoco-associated iron. Correlations of total activity after UW45 reconstitution with molybdenum, total iron, and o-phenanthroline-resistant iron contents show that only the last gives a consistent relationship of 35 +/- 5 nmol of C2H4/min/ng atom of Fe. Both o-phenanthroline and EDTA interact with FeMoco to abolish its EPR signal in reactions reversible by additions of Fe2+ or Zn2+, respectively. These and related reactions point against the presence of an endogenous organic component in FeMoco and toward the presence of exogenous ligands and imply a relatively labile coordination sphere whose nature may be determinable by a systematic investigation.     

The dominant fish fauna in the North Sea and its determination

The 224 species of fish reported from the North Sea are regarded as being composed of elements of three faunas—Boreal, Lusitanian and Atlantic, and they can be grouped accordingly on both a number of species and a biomass basis. The estimation of biomass of more than 10 important species is based on stock estimates obtained from population data. For 65 non-standard species the estimate is based on a comparison of catch rates for 'standard'and non-standard species from groundfish survey data. This involves assumption about the relative catchability of different sets of species. For the other species the biomass are computed in different ways. An analysis of the dominant fish fauna in the North Sea is attempted. The fish fauna in the area is analysed in two ways: by the conventional total number of species from each zoogeographic area, and by the biomass of the representatives of each fauna. It is concluded that for establishing the dominant faunal element of fish, biomass is a better index than is the number of species. The dominant faunal element of fish in the North Sea is Boreal.     

Evidence for Ascending and Descending Intraspinal as Well as Primary Sensory Somatostatin Projections in the Rat Spinal Cord

Somatostatin distribution was measured quantitatively in the rat spinal cord by radioimmunoassay. Rostro-caudally, somatostatin content was about 50% higher in lumbar-sacral cord than in cervical or thoracic levels. The dorso-ventral distribution is more uneven: somatostatin is highest in the dorsal horn, where the peptide is 15 times as concentrated as it is in the ventral white matter, the region of lowest concentration. However, measurable amounts of the peptide were found in all regions studied. Dorsal root ganglionectomy decreased somatostatin levels in the dorsal cord, supporting the previously proposed role for this peptide as a primary sensory neurotransmitter or modulator; but somatostatin content also was decreased both rostral and caudal to spinal transection, indicating the presence of ascending and descending somatostatin pathways within the spinal cord. Brain levels did not change. Met-enkephalin and substance P were also measured after the above surgical manipulations. Met-enkephalin content was not altered and substance P content was lowered significantly only after ganglionectomy. Although this study confirms the primary sensory neuron as the origin of a part of spinal cord somatostatin, it further indicates the presence of ascending and descending somatostatin pathways within the rat spinal cord.     

Sulfite-induced lipid peroxidation in chloroplasts as determined by ethane production

Ethane formation, as a measure of lipid peroxidation, was studied in spinach (Spinacia oleracea L.) chloroplasts exposed to sulfite. Ethane formation required sulfite and light, and occurred with concomitant oxidation of sulfite to sulfate. In the dark, both ethane formation and sulfite oxidation were inhibited. Ethane formation was stimulated by ferric or ferrous ions and inhibited by ethylenediamine tetraacetate. The photosynthetic electron transport modulators, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and phenazine methosulfate, inhibited both sulfite oxidation and ethane formation. Methyl viologen greatly stimulated ethane formation, but had little effect on sulfite oxidation. Methyl viologen, in the absence of sulfite, caused only a small amount of ethane formation in comparison to that produced with sulfite alone. Sulfite oxidation and ethane formation were effectively inhibited by the radical scavengers, 1,2-dihydroxybenzene-3,5-disulfonic acid and ascorbate. Ethanol, a hydroxyl radical scavenger, inhibited ethane formation only to a small degree; formate, which converts hydroxyl radical to superoxide radical, caused a small stimulation in both sulfite oxidation and ethane formation. Superoxide dismutase inhibited ethane formation by 50% when added at a concentration equivalent to that of the endogenous activity. Singlet oxygen did not appear to play a role in ethane formation, inasmuch as the singlet oxygen scavengers, sodium azide and 1,4-diazobicyclo-[2,2,2]-octane, were not inhibitory. These data are consistent with the view that O₂ is reduced by the photosynthetic electron transport system to superoxide anion, which in turn initiates the free radical oxidation of sulfite, and the free radicals produced during sulfite oxidation were responsible for the peroxidation of membrane lipids, resulting in the formation of ethane.     

Dark metabolism of carbon monoxide in lettuce leaf discs

In the dark, leaf tissue of crisphead lettuce (Lactuca sativa L.) metabolized ¹⁴CO to ¹⁴CO₂ and acid-stable products. Tissue incubated at 2.5°C for 3.5 hours and 48 hours converted about 1% and 17%, respectively, of the applied ¹⁴CO to ¹⁴CO₂, and incorporated about 0.04% and 0.6% of the ¹⁴C in acid-stable products. Examination of soluble acid-stable products from ¹⁴CO and ¹⁴CO₂-treated leaf tissue revealed that the labeling patterns of both treatments were identical during the 3.5-hour and the 48-hour incubation periods. Malate, citrate, and aspartate together comprised 70% or more of the soluble radioactivity from both treatments. Incorporation of radioactivity from CO into soluble acid-stable products during a 3-hour incubation period at 2.5°C was inhibited 90% by adding 3% nonradioactive CO₂. These results indicate that in head lettuce in the dark ¹⁴CO is metabolized primarily to ¹⁴CO₂ which is the precursor of acid-stable products. In leaf discs at 2.5°C, the apparent K_m for CO oxidation to CO₂ was 5.3 microliters per liter and the V_max was 9.7 nanoliters per gram per hour. The mitochondrial fraction of the leaf homogenate was the most active fraction to oxidize CO to CO₂, and this activity was heat-labile and cyanide-sensitive.     

Inhibition of ethylene synthesis in tomato plants subjected to anaerobic root stress

Enhanced ethylene production and leaf epinasty are characteristic responses of tomato (Lycopersicon esculentum Mill.) to waterlogging. It has been proposed (Bradford, Yang 1980 Plant Physiol 65: 322-326) that this results from the synthesis of the immediate precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), in the waterlogged roots, its export in the transpiration stream to the shoot, and its rapid conversion to ethylene. Inhibitors of the ethylene biosynthetic pathway are available for further testing of this ACC transport hypothesis: aminooxyacetic acid (AOA) or aminoethoxyvinylglycine (AVG) block the synthesis of ACC, whereas CO²⁺ prevents its conversion to ethylene. AOA and AVG, supplied in the nutrient solution, were found to inhibit the synthesis and export of ACC from anaerobic roots, whereas Co²⁺ had no effect, as predicted from their respective sites of action. Transport of the inhibitors to the shoot was demonstrated by their ability to block wound ethylene synthesis in excised petioles. All three inhibitors reduced petiolar ethylene production and epinasty in anaerobically stressed tomato plants. With AOA and AVG, this was due to the prevention of ACC import from the roots as well as inhibition of ACC synthesis in the petioles. With Co²⁺, conversion of both root- and petiole-synthesized ACC to ethylene was blocked. Collectively, these data support the hypothesis that the export of ACC from low O₂ roots to the shoot is an important factor in the ethylene physiology of waterlogged tomato plants.     

Stimulation of ethylene production in citrus leaf discs by mannitol

Wound ethylene formation induced in leaf tissue of citrus (Citrus sinensis [L.] Osbeck cv. “Washington Navel”) by excision was significantly stimulated by mannitol after a lag period of about 6 hours. The extent of stimulation was dependent upon the concentration of mannitol (10 to 100 millimolar). This increased ethylene production was not simply due to osmotic effect or water stress as other osmoticums tested failed to exert such an effect. The stimulatory effect of mannitol resulted from both the enhancement of 1-aminocyclopropane-1-carboxylic acid (ACC) formation and the conversion of ACC to ethylene. The effect on the latter step was particularly pronounced in aged discs. The use of labeled mannitol showed that it was taken up by the leaf discs, utilized for respiration, and metabolized to sucrose, but no radioactivity was detected in the ethylene.     

Relationship between excision repair and the cytotoxic and mutagenic effect of the ‘anti’ 7,8-diol-9,10-epoxide of benzo[a]pyrene in human cells

The cytotoxic and mutagenic effect of (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti BPDE) in normally excision diploid human cells treated just prior to onset of S was compared with that of cells allowed ～ 16 h for excision repair before onset of S and with that observed in excision-deficient serodema pigmentosum (SP12BE) cells. The cells were synchronized by release from density inhibition of cell replication. DNA synthesis began ～ 22 h after the cells were plated at lower density (i.e., 1.4 × 10⁴ cells/cm²). The frequency of thioguanine-resistant mutants induced in normal cells treated just prior to onset of S was ～ 12- to 16-fold higher than that observed in cells treated in early G₁ or treated in G₀ (confluence) and then plated at lower density. The frequency approximated that expected for XP12BE cells from extrapolation of data obtained at lower doses. The frequency of mutants measured in normal cells treated in exponential growth was also much higher than that in the cells treated in early G₁ or in G₀, No such difference could be seen in XP12BE cells treated in exponential growth or in G₀. In contrast to the mutagenicity data in the normal cells, there was no significant difference in the slope of the survival curve of normal cells treated at various times prior to S phase at low densities. However, normal cells treated even at the onset of S exhibited survival equal to XP12BE cells give a 4- to 5-fold lower dose. The data support the hypothesis that DNA synthesis is the cellular event which converts unexcised DNA lesions into mutations. However, they indicate that S is not the event primarily responsible for translating DNA damage into cell death. Accompanying studies on the rate of excision of anti BPDE adducts from the normal cells during the period priot to S support the conclusions.     

The effect of plant-hormone pretreatments on ethylene production and synthesis of 1-aminocyclopropane-1-carboxylic acid in water-stressed wheat leaves

Excised wheat (Triticum aestivum L.) leaves, when subjected to drought stress, increased ethylene production as a result of an increased synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) and an increased activity of the ethyleneforming enzyme (EFE), which catalyzes the conversion of ACC to ethylene. The rise in EFE activity was maximal within 2 h after the stress period, while rehydration to relieve water stress reduced EFE activity within 3 h to levels similar to those in nonstressed tissue. Pretreatment of the leaves with benzyladenine or indole-3-acetic acid prior to water stress caused further increase in ethylene production and in endogenous ACC level. Conversely, pretreatment of wheat leaves with abscisic acid reduced ethylene production to levels produced by nonstressed leaves; this reduction in ethylene production was accompanied by a decrease in ACC content. However, none of these hormone pretreatments significantly affected the EFE level in stressed or nonstressed leaves. These data indicate that the plant hormones participate in regulation of water-stress ethylene production primarily by modulating the level of ACC.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - BA N⁶-benzyladenine - EFE ethylene-forming enzyme - IAA indole-3-acetic acid