Induction of accumulation and degradation of the 18.4-kDa oleosin in a triacylglycerol-storing cell culture of anise (Pimpinella anisum L.)

Two closely related anise cell-culture lines, Pa15 and Pa19, differ considerably in growth rate, potential to form somatic embryoids, triacylglycerol (TAG) storage and pattern of lipid-body proteins. Line Pa15 grows very fast (doubling rate: 3 d), mainly as single cells, exhibits a low potential for somatic embryogenesis and its TAG content is relatively low (5–20 mg TAG per g dry weight). In contrast, the line Pa19 shows lower growth rates (doubling rate: 8 d), tends to form clusters of somatic cells, has a higher TAG content (100–150 mg TAG per g dry weight), and somatic embryoids are easily induced. Under defined culture conditions, the TAG content of the line Pa19 can be increased to approximately 70% of that of ripe anise seeds (150 and 220 mg TAG per g dry weight, respectively). Polyclonal antibodies prepared against the most abundant protein (relative molecular mass 18.4 kDa) from the lipid-body fraction of anise seeds (Radetzky et al. 1993, Planta 191, 166–172) react also with a 18.4-kDa protein from the lipid-body fraction of cells of the Pa19 culture. In contrast, only fairly low levels of the 18.4-kDa oleosin were detected in Pal5. Limited sucrose supply in the medium resulted in TAG degradation and the concomitant decrease in the amount of immunodetectible 18.4-kDa protein in the Pa19 cell culture. Treatment with sorbitol, or abscisic acid and sorbitol in combination, enhanced TAG contents and also the amount of immunostained 18.4-kDa protein in the cell culture Pa19, whereas no effect was found on either TAG content or 18.4-kDa protein in the cell-culture line Pa15. The 18.4-kDa protein can be classified as an oleosin, a proposal which is supported by the similarity in molecular mass compared with other known oleosins, its occurrence in the lipid-body fraction and the fact that its amount correlates with the TAG content. The results of this study indicate that the Pa19 cell culture provides a valid model system for investigations of lipid storage and mobilization in higher-plant cells.Abbreviations ABA cis-abscisic acid - TAG triacylglycerol(s) - 2,4-D 2,4-dichlorophenoxyacetic acid The authors thank Christiane Bernshausen for kind technical assistance.     

Isolation of Typical Marine Bacteria by Dilution Culture: Growth, Maintenance, and Characteristics of Isolates under Laboratory Conditions

Marine bacteria in Resurrection Bay near Seward, Alaska, and in the central North Sea off the Dutch coast were cultured in filtered autoclaved seawater following dilution to extinction. The populations present before dilution varied from 0.11 × 10⁹ to 1.07 × 10⁹ cells per liter. The mean cell volume varied between 0.042 and 0.074 μm³, and the mean apparent DNA content of the cells ranged from 2.5 to 4.7 fg of DNA per cell. All three parameters were determined by high-resolution flow cytometry. All 37 strains that were obtained from very high dilutions of Resurrection Bay and North Sea samples represented facultatively oligotrophic bacteria. However, 15 of these isolates were eventually obtained from dilution cultures that could initially be cultured only on very low-nutrient media and that could initially not form visible colonies on any of the agar media tested, indicating that these cultures contained obligately oligotrophic bacteria. It was concluded that the cells in these 15 dilution cultures had adapted to growth under laboratory conditions after several months of nutrient deprivation prior to isolation. From the North Sea experiment, it was concluded that the contribution of facultative oligotrophs and eutrophs to the total population was less than 1% and that while more than half of the population behaved as obligately oligotrophic bacteria upon first cultivation in the dilution culture media, around 50% could not be cultured at all. During one of the Resurrection Bay experiments, 53% of the dilution cultures obtained from samples diluted more than 2.5 × 10⁵ times consisted of such obligate oligotrophs. These cultures invariably harbored a small rod-shaped bacterium with a mean cell volume of 0.05 to 0.06 μm³ and an apparent DNA content of 1 to 1.5 fg per cell. This cell type had the dimensions of ultramicrobacteria. Isolates of these ultramicrobacterial cultures that were eventually obtained on relatively high-nutrient agar plates were, with respect to cell volume and apparent DNA content, identical to the cells in the initially obligately oligotrophic bacterial dilution culture. Determination of kinetic parameters from one of these small rod-shaped strains revealed a high specific affinity for the uptake of mixed amino acids (a°_A, 1,860 liters/g of cells per h), but not for glucose or alanine as the sole source of carbon and energy (a°_A, ± 200 liters/g of cells per h). The ultramicrobial strains obtained are potentially a very important part of picoplankton biomass in the areas investigated.     

Influence of Metronidazole, CO, CO2, and Methanogens on the Fermentative Metabolism of the Anaerobic Fungus Neocallimastix sp. Strain L2

The effects of metronidazole, CO, methanogens, and CO₂ on the fermentation of glucose by the anaerobic fungus Neocallimastix sp. strain L2 were investigated. Both metronidazole and CO caused a shift in the fermentation products from predominantly H₂, acetate, and formate to lactate as the major product and caused a lower glucose consumption rate and cell protein yield. An increased lactate dehydrogenase activity and a decreased hydrogenase activity were observed in cells grown under both culture conditions. In metronidazole-grown cells, the amount of hydrogenase protein was decreased compared with the amount in cells grown in the absence of metronidazole. When Neocallimastix sp. strain L2 was cocultured with the methanogenic bacterium Methanobrevibacter smithii, the fermentation pattern changed in the opposite direction: H₂ and acetate production increased at the expense of the electron sink products lactate, succinate, and ethanol. A concomitant decrease in the enzyme activities leading to these electron sink products was observed, as well as an increase in the glucose consumption rate and cell protein yield, compared with those of pure cultures of the fungus. Low levels of CO₂ in the gas phase resulted in increased H₂ and lactate formation and decreased production of formate, acetate, succinate, and ethanol, a decreased glucose consumption rate and cell protein yield, and a decrease in most of the hydrogenosomal enzyme activities. None of the tested culture conditions resulted in changed quantities of hydrogenosomal proteins. The results indicate that manipulation of the pattern of fermentation in Neocallimastix sp. strain L2 results in changes in enzyme activities but not in the proliferation or disappearance of hydrogenosomes.     

Immuno- and lectin histochemistry of epithelial subtypes and their changes in a radiation-induced lung fibrosis model of the mini pig

Cell types of lung epithelia of mini pigs have been studied using a panel of monoclonal and polyclonal antibodies against cytokeratins (CKs) and vimentin and three lectins before and after radiation-induced fibrosis. In normal tissues, CK18 specific antibodies reacted above all with type II alveolar epithelial cells, while CK7 and pan CK-specific antibodies stained the whole alveolar epithelium. In bronchial epithelial cells, CKs 7, 8, 18 and focally CKs 4 and 13 as well as vimentin were found. Cell specificity of the CK pattern was confirmed by double label immunofluorescence using type II cell-specific Maclura pomifera (MPA) lectin, type I cell specific Lycopersicon esculentum (LEA) lectin and capillary endothelium-binding Dolichos biflorus (DBA) lectin. In experimental pulmonary fibrosis, enhanced coexpression of CK and vimentin was observed in bronchial epithelium. Subtypes of alveolar epithelial cells were no longer easily distinguishable. CK18 was found to be expressed in the entire alveolar epithelium. The gradual loss of the normal alveolar epithelial marker, as seen by the binding of MPA to type I-like cells, of LEA to type II-like cells and the partial loss of MPA-binding to type II cells, was paralleled by the appearance of CK4, typical for squamous epithelia, and the occurrence of DBA-binding in epithelial cells. Implications of these results for general concepts of intermediate filament protein expression and lectin binding in the fibrotic process are discussed.     

Changes in cytokeratin,vimentin and desmoplakin distribution during the repair of irradiation-induced lung injury in adult rats

Virchows Archiv B Cell Pathology - The expression of cytokeratins, desmoplakin and vimentin has been studied immunohistochemically in the rat lung injured by x-irradiation using 14 well...     

Two N 5, N 10-methylenetetrahydromethanopterin dehydrogenases in the extreme thermophile Methanopyrus kandleri: characterization of the coenzyme F420-dependent enzyme

It was recently reported that the extreme thermophile Methanopyrus kandleri contains only a H₂-forming N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase which uses protons as electron acceptor. We describe here the presence in this Archaeon of a second N ⁵,N ¹⁰-methylenetetrahydromethanopterin dehydrogenase which is coenzyme F₄₂₀-dependent. This enzyme was purified and characterized. The enzyme was colourless, had an apparent molecular mass of 300 kDa, an isoelectric point of 3.7±0.2 and was composed of only one type of subunit of apparent molecular mass of 36 kDa. The enzyme activity increased to an optimum with increasing salt concentrations. Optimal salt concentrations were e.g. 2 M (NH₄)₂SO₄, 2 M Na₂HPO₄, 1.5 M K₂HPO₄, and 2 M NaCl. In the absence of salts the enzyme exhibited almost no activity. The salts affected mainly the V _max rather than the K _m of the enzyme. The catalytic mechanism of the dehydrogenase was determined to be of the ternary complex type, in agreement with the finding that the enzyme lacked a chromophoric prosthetic group. In the presence of M (NH₄)₂SO₄ the V _max was 4000 U/mg (k _cat=2400 s^-1) and the K _m for N ⁵,N ¹⁰-methylenetetrahydromethanopterin and for coenzyme F₄₂₀ were 80 M and 20 M, respectively. The enzyme was relatively heat-stable and lost no activity when incubated anaerobically in 50 mM K₂HPO₄ at 90°C for one hour. The N-terminal amino acid sequence was found to be similar to that of the F₄₂₀-dependent N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Archaeoglobus fulgidus.Abbreviations H₄MPT tetrahydromethanopterin - F₄₂₀ coenzyme F₄₂₀ - CH₂=H₄MPT N ⁵,N ¹⁰-methylenetrahydromethanopterin - CHH₄MPT⁺ N ⁵,N ¹⁰-methenyltetrahydromethanopterin - methylene-H₄MPT dehydrogenase N ⁵,N ¹⁰-methylenetetrahydromethanopterin dehydrogenase - Mops N-morpholinopropane sulfonic acid - Tricine N-[Tris(hydroxymethyl)-methyl]glycine - 1 U = 1 mol/min