Intracellular Localization of Lunularic Acid and Prelunularic Acid in Suspension Cultured Cells of Marchantia polymorpha

Intracellular localization of lunularic acid and prelunularic acid in suspension cultured cells of Marchantia polymorpha L. was studied. The sum of both compounds was determined as lunularic acid group (LNAs) because of the instability of prelunularic acid to convert into lunularic acid.

Mechanical disruption of the cells followed by differential centrifugation showed that LNAs was associated with the supernatant of 100,000g centrifugation. Protoplasts isolated from the cells were osmotically ruptured and the distribution of LNAs among the organelles was examined by discontinuous density gradient centrifugation of the protoplast contents. Successful isolation of intact chloroplasts, mitochondria and peroxisomes free from cytoplasm indicated that LNAs was not accumulated in these organelles. Flotation techniques resulted in an efficient isolation of pure vacuoles and revealed that LNAs was distributed almost equally in the vacuoles and cytoplasm.

     

In Vitro DNA Synthesis by Chloroplasts Isolated from Marchantia polymorpha L. Cell Suspension Cultures

Lysate of chloroplasts prepared from liverwort Marchantia polymorpha L. cell suspension cultures incorporated [³H]-dTTP into acid insoluble materials when DNA was added exogenously as a template. The incorporation was highly dependent on the addition of template DNA, four deoxynucleoside triphosphates and magnesium ions (maximum incorporation at 5mM). Magnesium ions could be replaced by manganese ions. DNA synthesis inhibitors, N-ethylmaleimide (NEM) and ethidium bromide (EtBr), strongly inhibited the incorporation. Dideoxythymidine triphosphate (ddTTP), an inhibitor of DNA polymerases β and γ, inhibited the incorporation at the concentration of 50 μM (molar ratio of ddTTP/dTTP = 17). On the other hand, the incorporation by the chloroplast lysate was resistant to arabinofuranosyl cytosine triphosphate (araCTP) and aphidicolin as well as the RNA polymerase inhibitors, rifampicin and α-amanitin. The chloroplast lysate highly utilized denatured calf thymus DNA and bacteriophage ?X174 single-stranded DNA as templates when added exogenously, while a synthetic homopolymer, poly(rA)-oligo(dT)₁₂ ~ ₁₈, did not stimulate the incorporation at all. Autoradiographic analysis of DNA synthesized in isolated chloroplasts showed that the chloroplast DNA synthesis took place at several specific sites on the chloroplast DNA from cells of the liverwort, Marchantia polymorpha.     

Effect of Introducing a Lactone Group into Typhasterol and Teasterone to Promote Rice Lamina Inclination

Protein phosphorylation may be required for plant cell response to phytohormones and other extracellular signals. Protein phosphorylation and protein kinase activity in the culm of heading time of rice (Oryza sativa L.) were studied. Before heading, protein kinase activity was increased by Ca²⁺ in the membrane fraction of the panicle and culm. The protein kinases with M_r of 51,900, 49,200, and 45,500 isolated from the membrane fraction of culm increased the protein phosphorylation of M_r and pI of 40,000/7.5 and 40,000/7.6 in the culm extract. The activation of protein kinases, associated with membrane and subsequent protein phosphorylation, thus appears to be involved in the regulation of heading time in rice.     

Identification of Castasterone,Typhasterol and Teasterone from the Pollen of Zea mays

From the pollen of Zea mays, three brassinosteroids, castasterone, typhasterol and teasterone, were identified by GC/MS and/or ¹H NMR. Their concentrations in the pollen were shown by GC/SIM to be about 120 μg/kg fr.wt. (castasterone), 6.6 μg/kg fr.wt. (typhasterol) and 4.1 μg/kg fr.wt. (teasterone). It was also found that the anther contained a fairly large amount of brassinosteroids by a bioassay.     

Biochemical Characterization of Casein Kinases II (CK-II) from Cultured Cells of the Liverwort, Marchantia polymorpha

Monomeric and oligomeric forms of CK-II have been purified froma 1.0 M KC1 extract of the liverwort, Marchantia polymorpha,by means of heparin-agarose column chromatography and gel filtrationon Superose 6HR (HPLC). It was found that (i) a monomeric kinase(approximately 38 kDa) is the main form of CK-II in the cells;and (ii) the enzymatic properties of oligomeric kinase (approximately140 kDa), which cross-reacts with anti-serum against DrosophilaCK-IIß, are similar to those of CK-II (₂ß₂)in various animal cells. (Received November 10, 1992; Accepted February 18, 1993)     

Assessment of In Vivo and In Vitro Genotoxicity of Glibenclamide in Eukaryotic Cells

Glibenclamide is an oral hypoglycemic drug commonly prescribed for the treatment of type 2 diabetes mellitus, whose anti-tumor activity has been recently described in several human cancer cells. The mutagenic potential of such an antidiabetic drug and its recombinogenic activity in eukaryotic cells were evaluated, the latter for the first time. The mutagenic potential of glibenclamide in therapeutically plasma (0.6 μM) and higher concentrations (10 μM, 100 μM, 240 μM and 480 μM) was assessed by the in vitro mammalian cell micronucleus test in human lymphocytes. Since the loss of heterozygosity arising from allelic recombination is an important biologically significant consequence of oxidative damage, the glibenclamide recombinogenic activity at 1 μM, 10 μM and 100 μM concentrations was evaluated by the in vivo homozygotization assay. Glibenclamide failed to alter the frequency of micronuclei between 0.6 μM and 480 μM concentrations and the cytokinesis block proliferation index between 0.6 μM and 240 μM concentrations. On the other hand, glibenclamide changed the cell-proliferation kinetics when used at 480 μM. In the homozygotization assay, the homozygotization indices for the analyzed markers were lower than 2.0 and demonstrated the lack of recombinogenic activity of glibenclamide. Data in the current study demonstrate that glibenclamide, in current experimental conditions, is devoid of significant genotoxic effects. This fact encourages further investigations on the use of this antidiabetic agent as a chemotherapeutic drug.     

地钱、拳卷地钱和粗裂地钱的紫外谱线组法鉴别

本文建立了紫外谱线组法鉴别地钱、拳卷地钱和粗裂地钱的方法。通过这三种药材的石油醚、氯仿、乙醇和水提取液的紫外谱线比较,发现地钱、拳卷地钱和粗裂地钱的紫外谱线图、最大吸收峰数目及峰位值具有明显差异。该法简单、准确,可用来鉴别地钱、拳卷地钱和粗裂地钱的原药材。     

Thymoquinone Inhibits Murine Leukemia WEHI-3 Cells In Vivo and In Vitro

BackgroundThymoquinone is an active ingredient isolated from Nigella sativa (Black Seed). This study aimed to evaluate the in vitro and in vivo anti-leukemic effects of thymoquinone on WEHI-3 cells.ConclusionThymoquinone promoted natural killer cell activities. This compound showed high toxicity against WEHI-3 cell line which was confirmed by an increase of the early apoptosis, followed by up-regulation of the anti-apoptotic protein, Bcl2, and down-regulation of the apoptotic protein, Bax. On the other hand, high reduction of the spleen and liver weight, and significant histopathology study of spleen and liver confirmed that thymoquinone inhibited WEHI-3 growth in the BALB/c mice. Results from this study highlight the potential of thymoquinone to be developed as an anti-leukemic agent.     

In vitro Propagation,Dedifferentiation and redifferentiation of Marchantia polymorpha L.

The gemma and gametophyte of Marchantia #olymorpha were propagated in vitro. Dedifferentiation and redifferentiation as well as the media used and cultural conditions reguired were described. Since the differentiation of bryophytes was very difficult, it was necessary to culture the tissue through initiation of partial dedifferentiation on MS agar medium supplemented with 1 mg/1 2,4-D and 3% sucrose, and then subsequently the tissue was transplanted onto 1/2 Knop agar medium with addition of 4–8 mg/1 2,4-D, 0.25–0.5 mg/1 BA and Fe salt of MS medium. The formed calli were visual but still contained rhizoid, in this stage. The small calli finally were subcultured in white agar medium supplemented with mixture of pyruvic acid, citric acid and fumaric acid (5 mmol/1); 1 mg/1 2,4-D and 4% glucose. They could be differentiated thoroughly into normal tissue. The time of the total process for differentiation requied as long as 10 months. The redifferentiation and regeneration of thalli were far easier than those of higher plants even if they were transplanted onto MS phytohormone-free medium.     

Protoplasts of Marchantia polymorpha and its Development

For the first time protoplasts from normal differentiated thalliof male and female Marchantia polymorpha were prepared by aone step cell wall digestion with 2% Driselase. Cell wall formationonly takes place in light with a carbohydrate source. Furtherconditions for growth and differentiation were studied. Growthalways starts with a primary callus. This can be maintainedby a higher osmolarity of the medium. After reduction of theosmolarity, differentiation appears in three forms: gemmae formationat the callus surface, production of limited growing "cauloids"and transition to an indefinite growing thallus with one orseveral apical regions. (Received December 21, 1987; Accepted February 12, 1988)     

In Vivo and In Vitro Expression of Gangliosides in Chick Retina Müller Cells

The expression of gangliosides of the lactosylceramide (LC) and of the gangliotetraosylceramide (GTC) series on the surface of cells from the chick neural retina was investigated by double-color indirect immunofluorescence. GD3 was assumed to be representative of LC and was detected using a specific monoclonal antibody. GM1 was assumed to be representative of GTC and was detected using the binding of cholera toxin followed by the binding of cholera toxin antibodies. The expression of polysialosylated GTC (polysialosyl-GTC) was detected using the cholera toxin-cholera toxin antibody experimental approach, after conversion of polysialosyl-GTC to GM1 by treatment of the cells with neuraminidase. In retinas from 6-day-old embryos (R6), most cells (approximately 80%) expressed GD3 but not GTC. After culturing for 7 days, (R6+7), the expression of GTC was found confined to neuron-like cells; flat cells derived from Müller cells expressed GD3 but were negative for GTC expression. On the other hand, postmitotic Müller cells obtained from 13-day-old embryo (R13) or 1-day-old hatched chick retina (RP1) expressed GD3, GM1, and polysialosyl-GTC but were unable to maintain the expression of these GTCs when kept in culture for several days. According to these results, retinal cells can be defined on the basis of their ganglioside expression as follows: (a) retinoblasts, by the expression of GD3; (b) postmitotic neuronal cells, by the expression of GTC; and (c) postmitotic Müller cells, by the expression of GD3 and GTC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Directed In Vitro Myogenesis of Human Embryonic Stem Cells and Their In Vivo Engraftment

Development of human embryonic stem cell (hESC)-based therapy requires derivation of in vitro expandable cell populations that can readily differentiate to specified cell types and engraft upon transplantation. Here, we report that hESCs can differentiate into skeletal muscle cells without genetic manipulation. This is achieved through the isolation of cells expressing a mesodermal marker, platelet-derived growth factor receptor-α (PDGFRA), following embryoid body (EB) formation. The ESC-derived cells differentiated into myoblasts in vitro as evident by upregulation of various myogenic genes, irrespective of the presence of serum in the medium. This result is further corroborated by the presence of sarcomeric myosin and desmin, markers for terminally differentiated cells. When transplanted in vivo, these pre-myogenically committed cells were viable in tibialis anterior muscles 14 days post-implantation. These hESC-derived cells, which readily undergo myogenic differentiation in culture medium containing serum, could be a viable cell source for skeletal muscle repair and tissue engineering to ameliorate various muscle wasting diseases.     

Pseudocompact-Type Growth and Conversion of Growth Types of Strains of Staphylococcus aureus In Vitro and In Vivo

Some strains of Staphylococcus aureus grew as compact colonies in Brain Heart Infusion-serum-soft agar but as diffuse colonies in a modified Staphylococcus 110-serum-soft agar. These strains were designated "pseudocompact." Strains showing compact-type colonial morphology in both media were designated "compact," whereas strains showing diffuse-type growth in both media were designated "diffuse." It was observed that the most recently isolated strains of S. aureus were of the pseudocompact type, whereas most stock culture strains tested were of the compact type. Using cultures recently isolated from clinical material, it was shown that pseudocompact strains convert to compact-type growth after prolonged incubation. Interconversion of compact, diffuse, and pseudocompact growth forms could be induced in vitro by appropriate cultural conditions, and conversion of growth type was also observed in vivo. Femoral abscesses produced in mice by four different compact-type strains showed conversion to diffuse or pseudocompact-type growth during the course of the infection.     

Cultured Alveolar Epithelial Cells From Septic Rats Mimic In Vivo Septic Lung

Sepsis results in the formation of pulmonary edema by increasing in epithelial permeability. Therefore we hypothesized that alveolar epithelial cells isolated from septic animals develop tight junctions with different protein composition and reduced barrier function relative to alveolar epithelial cells from healthy animals. Male rats (200–300g) were sacrificed 24 hours after cecal ligation and double puncture (2CLP) or sham surgery. Alveolar epithelial cells were isolated and plated on fibronectin-coated flexible membranes or permeable, non-flexible transwell substrates. After a 5 day culture period, cells were either lysed for western analysis of tight junction protein expressin (claudin 3, 4, 5, 7, 8, and 18, occludin, ZO-1, and JAM-A) and MAPk (JNK, ERK, an p38) signaling activation, or barrier function was examined by measuring transepithelial resistance (TER) or the flux of two molecular tracers (5 and 20 Å). Inhibitors of JNK (SP600125, 20 µM) and ERK (U0126, 10 µM) were used to determine the role of these pathways in sepsis induced epithelial barrier dysfunction. Expression of claudin 4, claudin 18, and occludin was significantly lower, and activation of JNK and ERK signaling pathways was significantly increased in 2CLP monolayers, relative to sham monolayers. Transepithelial resistance of the 2CLP monolayers was reduced significantly compared to sham (769 and 1234 ohm-cm², respectively), however no significant difference in the flux of either tracer was observed. Inhibition of ERK, not JNK, significantly increased TER and expression of claudin 4 in 2CLP monolayers, and prevented significant differences in claudin 18 expression between 2CLP and sham monolayers. We conclude that alveolar epithelial cells isolated from septic animals form confluent monolayers with impaired barrier function compared to healthy monolayers, and inhibition of ERK signaling partially reverses differences between these monolayers. This model provides a unique preparation for probing the mechanisms by which sepsis alters alveolar epithelium.     

Resistance and capacity in the thallus of Marchantia polymorpha

Calculations of the resistance r and capacity c of cell membrances and the resistancer ₁ of cell interiors of a community of cells in Marchantia polymorpha L. thalli are presented. These parameters of a multicellular system were determined by the adaptation of methods employed for the calculation of the resistance and capacity of single cells. The obtained results indicate that such a procedure is justified. A generally accepted resistance-capacity model of the cell was used as a basis for the determination of r, c, and r₁ (representing membrane resistance, membrane capacity, and resistance of cell interior, respectively). The calculations were based on measurements of impedance and phase shift within the frequency range of 5 Hz-1000 Hz. Stainless steel plates were employed as the measuring electrodes; polarization resistance and capacity were determined by separate measurements. The calculations confirmed the assumption that the parameters r, c, and r₁ were constant within the investigated frequency range.
The calculations of resistance and capacity for 25 plants were constant within the investigated frequency range. The calculations of resistance and capacity for 25 plants were carried out by four different methods and they yielded results of the order of : r = 0.45 kΩ± 0.15 kΩ, r₁= 1.0 kΩ± 0.45 kΩ, c = 11 μF ± 3.5 μF. Circular diagrams of impedance also confirmed the validity of the accepted model within the frequency range of 25–300 Hz.