MORPHOLOGICAL CHANGES OF CULTURED MYOCARDIAL CELLS DUE TO CHANGE IN EXTRACELLULAR CALCIUM ION CONCENTRATION

Increase in the extracellular Ca²⁺ concentration from low (≤ 10⁻⁷ M) to normal (10⁻³ M) caused morphological changes of cultured myocardial cells obtained from fetal mouse heart. The extracellular Na⁺ and K⁺ concentrations of the normal medium (10⁻³ M Ca²⁺) did not significantly affect the genesis of these morphological changes. Like Ca²⁺, Ba²⁺ and Sr²⁺, but not Mg²⁺, Co²⁺ or Ni²⁺, could induce morphological changes. Increase in the extracellular Ca²⁺ concentration from 10⁻⁸ M to 10⁻³M also caused excess uptake of ⁴⁵Ca²⁺ by cultured myocardial cells. B–16CW 1 cells, which did not show these morphological changes, did not take up excess ⁴⁵Ca²⁺ on this treatment. Treatments, such as addition of verapamil or incubation at pH 6.3, which reduced the genesis of morphological changes, reduced the rate of ⁴⁵Ca²⁺ uptake by myocardial cells. These facts show that the morphological changes of myocardial cells induced by increasing the extracellular Ca²⁺ concentration from low to normal are due to excess uptake of Ca²⁺ by the myocardial cells.
The morphological changes of cultured myocardial cells induced by increasing the extracellular Ca²⁺ concentration from low to normal were reversed on further incubation of the cells in medium with or without Ca²⁺.     

Attraction of zebrafish, Brachydanio rerio, to alanine and its suppression by copper

Preference responses of zebrafish to 10⁻³, 10⁻⁴ and 10⁻⁵M alanine (Ala) were concentration- dependent. Behavioural responses to copper (Cu) and Cu + Ala mixtures were also assessed. Zebrafish avoided 100 and 10 μg Cu l⁻¹, but not 1 μg l⁻¹. Mixtures of 10⁻³ m Ala+ 100 μg Cu l⁻¹ and 10 ⁴ M Ala + 10 μg Cu 1⁻¹ were avoided as intensely as was Cu alone. Responses to 10⁻³ M Ala + 10 or 1 μg Cu l⁻¹ and 10 ⁴ M Ala +1 μg Cu l⁻¹ did not differ statistically from controls (no detectable preference or avoidance). These results demonstrate, firstly, that a concentration of a pollutant avoided by itself (10 μg Cu l⁻¹) may not be avoided when encountered with an attractant chemical stimulus (Ala) and may suppress the preference for an attractant stimulus, and secondly, that a concentration of a pollutant not avoided by itself and not considered deleterious (1 μg Cu l⁻¹) suppresses attraction to Ala (an important constituent of prey odours for many fishes).     

Prospective isolation and characterization of mesenchymal stem cells from human placenta using a frizzled-9-specific monoclonal antibody

Abstract We have recently shown that frizzled-9 (FZD9, CD349) is expressed on the cell surface of cultured mesenchymal stromal cells (MSC) derived from the human bone marrow (BM) and chorionic placenta (PL). To study whether FZD9 is also a marker for naive mesenchymal stem cells (MSC), we analyzed the expression pattern of FZD9 on freshly isolated PL cells and determined the clonogenic potential of isolated FZD9⁺ cells using the colony-forming units-fibroblastic (CFU-F) assay. About 0.2% of isolated PL cells were positive for FZD9. Two-color analysis revealed that FZD9⁺ PL cells uniformly express CD9, CD63, and CD90, but are heterogeneous for CD10, CD13, and CD26 expression. In contrast to BM-derived MSC, PL-derived MSC expressed only low levels of CD271. Colony assays of sorted cells showed that clonogenic CFU-F reside exclusively in the FZD9⁺ but not in the FZD9⁻ fraction. Further analysis revealed that CFU-F were enriched by 60-fold in the FZD9⁺CD10⁺CD26⁺ fraction but were absent in the FZD9⁺CD10⁻CD26⁻ population. Cultured FZD9⁺ cells expressed the embryonic stem cell makers Oct-4 and nanog as well as SSEA-4 and TRA1-2-49/6E. In addition, they could be differentiated into functional adipocytes and osteoblasts. This report describes for the first time that FZD9 is a novel and specific marker for the prospective isolation of MSC from human term PL.     

Sodium Transport from Blood to Brain: Inhibition by Furosemide and Amiloride

Abstract: Brain sodium uptake in vivo was studied using a modified intracarotid bolus injection technique in which the uptake of ²²Na ⁺ was compared with that of the relatively impermeable molecule, [³H]l-glucose. At a Na ⁺ concentration of 1.4 m M , Na ⁺ uptake was 1.74 ± 0.07 times greater than l -glucose uptake. This decreased to 1.34 ± 0.04 at 140 m M Na ⁺, indicating saturable Na ⁺ uptake. Relative Na ⁺ extraction was not affected by pH but was inhibited by amiloride ( K _i= 3 ± 10⁻⁷ M ) and by 1 m M furosemide. The effects of these two inhibitors were additive. Brain uptake of ⁸⁶Rb ⁺, a K ⁺ analogue, was measured to study interaction of K ⁺ with Na ⁺ transport systems. Relative ⁸⁶Rb ⁺ extraction was also inhibited by amiloride; however, it was not inhibited by furosemide. The results suggest the presence of two distinct transport systems that allow Na ⁺ to cross the luminal membrane of the brain capillary endothelial cell. These transport systems could play an important role in the movement of Na ⁺ from blood to brain.     

F-plasmid gene srnB+ complements the function of the S gene of phage lambda

Abstract The srnB ⁺ gene located on the F plasmid was assayed for its capacity to facilitate the release from infected cells of phage λ lacking the usual lytic activity. The srnB ⁺ plasmid pOY54, carrying the 1.4–2.5F fragment in the Eco RI- Bam HI fragment of pBR322, induced bacteriolysis and the release of progeny phage of the λcI 857 susS 7 lysogen in the presence of rifampin at 42°C. An srnB 1 mutant plasmid, pOY541, did not promote bacteriolysis. These results suggest that the srnB ⁺ gene of the F plasmid complements the function of the λ S gene in the nonpermissive host strain.     

Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA

Abstract Bacillus anthracis can be identified on the basis of the detection of virulence factor genes located on two plasmids, pXO1 and pXO2. Thus isolates lacking both pXO1 and pXO2 are indistinguishable from closely related B. cereus group bacteria. We developed a multiplex PCR assay for characterization of B. anthracis isolates, and simultaneous confirmation of the species identity independent of plasmid content. The assay amplifies lef, cya, pag (pXO1) and cap (pXO2) genes, and a B. anthracis specific chromosomal marker, giving an easy-to-read profile. This system unambiguously identified virulent (pXO1⁺/2⁺) and avirulent (pXO1⁺/2⁻, pXO1⁻/2⁺ and pXO1⁻/2⁻) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria.     

PHOTOSYNTHETIC BICARBONATE UTILIZATION BY A TERRESTRIAL CYANOBACTERIUM, NOSTOC FLAGELLIFORME (CYANOPHYCEAE)

The photosynthetic characteristics of the terrestrial cyanobacterium, Nostoc flagelliforme , after complete recovery by rewetting, was investigated to see whether it could use bicarbonate as the external inorganic carbon source when submerged. The photosynthesis–pH relationship and high pH compensation point suggested that the terrestrial alga could use bicarbonate to photosynthesize when submerged. The photosynthetic oxygen evolution rates were significantly inhibited in Na ⁺ -free and Na ⁺ + Li ⁺ media but were not affected by the absence of Cl ⁻ , implying that the bicarbonate uptake was associated with Na ⁺ / HCO₃ ⁻ symport rather than Cl ⁻ /HCO₃ ⁻ exchange system.     

Calcium ion transport across discs of the cortical flesh of apple fruit in relation to fruit development

Transport of Ca²⁺ through discs of apple fruit tissue was examined in tissue taken at different stages of fruit development. Transport rates decreased with fruit development when cation exchange was the predominant influence on transport (with 10⁻⁶ M ⁴⁵CaCl₂ as the source solution). This decrease was associated with a reduction in relative cell wall surface area, cation exchange capacity and cell wall yield that occurred during fruit growth. When diffusion was the major transport force, and when transport was influenced by solution infiltration of the tissue disc (10⁻² M ⁴⁵CaCl₂ in the source solution), transport rates increased during fruit growth. This increment was related to increases in air space of the tissue. Ca²⁺ transport through apple fruit tissue is influenced by the extent and nature of the cell wall, changing proportions of air space and Ca²⁺ concentration in the extracellular solution.     

Vitamin D3 affects growth and Ca2+ uptake by Phaseolus vulgaris roots cultured in vitro

Vitamin D₃ at low concentration (10⁻⁹ M) inhibited the growth of Phaseolus vulgaris L. (cv. Contrancha) roots in vitro as measured by elongation (14 h) and [³H]-leucine incorporation into protein (2 h), and increased their labelling with ⁴⁵Ca²⁺ (2 h). Cycloheximide and puromycin (50 u.M) blocked vitamin D₃ stimulation of root ⁴⁵Ca²⁺ labelling, indicating that it is mediated by de novo protein synthesis. The calcium ionophore X-537A (10⁻⁵JW) induced similar changes both in root elongation and ⁴⁵Ca²⁺ uptake (14 h). This may indicate that the inhibitory effects of the sterol on root growth are mediated by changes in Ca²⁺ fluxes. However, this interpretation should be further strengthened by additional studies as the ionophore may have acted on root growth, affecting physiological processes other than Ca²⁺ transport.     

KINETICS OF [3H]ACETYLCHOLINE SYNTHESIS AND RELEASE IN PRIMARY CELL CULTURES FROM MAMMALIAN CNS

Abstract— The present study was undertaken to characterize the cholinergic system of primary cell cultures of mouse and rat CNS.
In confirmation of previous reports, primary cultures were found to contain choline acetyltransferase (ChAc). Furthermore they contain acetylcholine (ACh) as measured by two different bioassays. They also synthesize [³H]ACh from [³H]Choline offered to the cultures.
The formation of [³H]ACh is inhibited in the presence of hemicholinium-3 (10⁻⁶ m ) to 50% or ouabain (10⁻³ m ) to 20% of the values found in untreated cultures. Omission of Na ⁺ from the incubation solution also diminishes the [³H]ACh formation of the cells.
[³H]ACh is released upon depolarisation by K⁺ ions in a concentration dependent manner. The release can be prevented by lack of Ca²⁺ ions in the incubation solution.     

Cyclic AMP-Elevating Agents Inhibit Proliferation of Keratinizing Guinea Pig Epidermal Cells

Cultured guinea pig epidermal cells and dermal fibroblasts were chosen as model systems to study possible growth inhibition by cyclic AMP (cAMP)-elevating drugs. The rate of DNA synthesis was used to assay growth rate in control cultures and those treated with agents which increase intracellular cAMP, including dibutyryl cAMP, the phosphodiesterase inhibitors papaverine and theophylline and agents which stimulate adenylate cyclase, iso-proterenol and prostaglandin E₂ methyl ester. Treatment for 24 h with dibutyryl cAMP (10⁻⁴ to 10⁻² M) inhibited cell growth by 50 to 95%, whereas butyrate(10⁻⁴M) showed essentially no effect. This inhibition could not be attributed to decreased precursor transport or to drug toxicity. Papaverine (10⁻⁶ to 10⁻⁴ M) and theophylline (10⁻⁴ to 10⁻³ M) also gave dose-dependent growth inhibition as did isoproterenol and prostaglandinE₂methyl ester. Radioautographic analysis of grain density after dibutyryl cAMP treatment and ³H-thymidine incorporation indicated no S-phase inhibition. Cyclic AMP-elevating drugs appear to inhibit growth of guinea-pig epidermal cells and dermal flbroblasts by blocking the cell cycle in G₋₂, M₁, or G. ₋₁     

Conjugal transfer at natural population densities in a microcosm simulating an estuarine environment

Abstract Estuarine microcosms were used to follow conjugal transfer of a broad host range IncP1 plasmid from a Pseudomonas putida donor to indigenous bacteria. Donor cells were added at a concentration similar to the natural abundance of bacteria in the water column (10⁶ cells ml⁻¹). Transfer was not detected in any of the test microcosms (calculated limit of detection of 10⁻⁷ and 10⁻⁴ transconjugants donor⁻¹ in water column and sediment, respectively), with the exception of transfer to an isogenic recipient (added at 10⁵ cells ml⁻¹) in sediments of controls that had been inoculated with both donors and recipients. The same plasmid was transferred with high efficiencies (10⁻¹ to 10⁻³) to a variety of recipients in filter and broth matings. These results suggest that if conjugal gene transfer occurred, it was at efficiencies that were not detectable in estuarine microcosms simulating natural population densities.     

Effect of rigidity of gel medium on benzyladenine-induced adventitious bud formation and vitrification in vitro in Picea abies

Observations on the effects of different degrees of rigidity of both an agar (Tayio) and a non-agar (Gelrite) gel on the uptake of radiolabelled N⁶-benzyladenine (¹⁴C-BA) were also extended to mode of application and positioning of the explant. Regression analysis showed a highly significant inverse correlation between ¹⁴C-BA accumulation and degree of gel stiffness. Significantly greater numbers of adventitious buds per explant were induced at low to medium levels of rigidity (2.5–10 g Tayio 1⁻¹, 1–5 g Gelrite 1⁻¹); this advantage was almost completely nullified at the lower levels (2.5 and 5.0 g Tayio 1⁻¹, 1 and 1.5 g Gelrite 1⁻¹) as a result of the high incidence of vitrification. In addition to turgor distension, vitrified buds displayed cellular damage. Explants with their cotyledons flattened onto the agar surface accumulated less ¹⁴C-BA after 96 h than upright explants, but produced greater numbers of adventitious buds, pseudobuds and phylloids. It was suggested that BA was taken up only by "target" cells, presumably the differentiating subsidiary cells of those stomatal complexes in surface contact with the medium. Pulse treatments of relatively short durations (2 h) with optimal concentrations of BA (ca 125 μ M ), followed by subculturing on hormone-free media gelled with 10 g agar 1⁻¹, produced a satisfactory balance between yield and competence of adventitiously-induced buds.     

Dipeptidyl aminopeptidase yspI mutants of Schizosaccharomyces pombe: Genetic mapping of dpa1+ on chromosome III

Abstract A mutant strain of Schizosaccharomyces pombe lacking dipeptidyl aminopeptidase yspI was isolated from a strain already defective in aminopeptidase activity by means of a staining technique with the chromogenic substrate ala-pro-4-methoxy-β-naphthylamide to screen colonies for the absence of the enzyme. The defect segregated 2⁺ :2⁻ in meiotic tetrads, indicating a single chromosomal gene mutation, which was shown to be recessive. Gene dosage experiments indicated that the mutation resides in the structural gene of dipeptidyl aminopeptidase yspI, dpa 1⁺. The dpa 1⁺ gene was located on chromosome III by using l m- fluorophen-ylalanine-induced haploidization and mitotic analysis. dpa1 mutants did not show any obvious phenotype under a variety of conditions tested.     

In Human Fetal Astrocytes Exposure to Interleukin-1β Stimulates Acquisition of the GD3+ Phenotype and Inhibits Cell Division

Abstract: In human astrocyte cultures established from second-trimester fetal brain tissue, ∼5–10% of total astrocyte population in unstimulated cultures were GD3⁺/glial fibrillary acidic protein (GFAP)⁺. The GD3⁺ cells were always GFAP⁺ and grew as flat, highly spread cells but changed to process-bearing cells after interleukin-1β (IL-1β) stimulation. It is interesting that IL-1β, a known mitogen for rat astrocytes, suppressed human fetal astrocyte proliferation as determined by [³H]thymidine incorporation, bromodeoxyuridine (BrdU) labeling, and cell counting. The GD3⁺ population, however, consistently increased in absolute number after IL-1β stimulation, in a dose- and time-dependent manner. The IL-1β-mediated increase in number of GD3⁺ astrocytes was independent of initial cell density or serum concentration. By flow cytometry, IL-1β enhanced both the mean fluorescence intensity and the percentage of GD3⁺ cells. To investigate whether the increase in GD3⁺ astrocyte cell number was due to proliferation of preexisting GD3⁺ astrocytes or due to conversion of GD3⁻ to GD3⁺ cells, we performed BrdU/GD3 double immunocytochemistry. BrdU/GD3 double-positive cells were extremely rare in both control and IL-1β-stimulated cultures. Moreover, an increase in number of GD3⁺ astrocytes was still observed in control and IL-1β-stimulated cultures where GD3⁺ cells had been initially eliminated by cell sorting. These results indicate that GD3⁺ astrocytes in human fetal culture may represent a postmitotic, differentiated, distinct phenotype.     

Effects of Arachidonic Acid on Dopamine Synthesis, Spontaneous Release, and Uptake in Striatal Synaptosomes from the Rat

Abstract: Arachidonic acid (AA) markedly stimulated, in a dose-dependent manner, the spontaneous release of [³H]dopamine ([³H]DA) continuously synthesized from [³H]tyrosine in purified synaptosomes from the rat striatum. As estimated by simultaneous measurement of the rate of [³H]H₂O formation (an index of [³H]tyrosine conversion into [³H]DOPA), the AA response was associated with a progressive and dose-dependent reduction of [³H]DA synthesis. In contrast to AA, arachidic acid, oleic acid, and the methyl ester of AA (all at 10⁻⁴ M ) did not modify [³H]DA release. The AA (3 × 10⁻⁵ M )-evoked release of [³H]DA was not affected by inhibiting AA metabolism, with either 5,8,11,14-eicosatetraynoic acid or metyrapone, suggesting that AA acts directly and not through one of its metabolites. AA also inhibited in a dose-dependent manner [³H]DA uptake into synaptosomes, with a complete blockade observed at 10⁻⁴ M . However, AA (10⁻⁴ M ) still stimulated [³H]DA spontaneous release in the presence of either nomifensine or other DA uptake inhibitors, indicating that AA both inhibits DA reuptake and facilitates its release process. Finally, the AA (10⁻⁴ M )-evoked release of [³H]DA was not affected by protein kinase A inhibitors (H-89 or Rp -8-Br-cAMPS) but was markedly reduced in the presence of protein kinase C inhibitors (Ro 31-7549 or chelerythrine).     

Photosynthesis and photoassimilation of glucose during photomixotrophic growth of Marchantia polymorpha cells in suspension culture

Even in the presence of glucose the growth of Marchantia polymorpha L. (cell line HYH-2F) requires light, and growth is more sensitive to 10⁻⁶ M 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea than to 10⁻⁴ Antimycin A. The inability of the cells to grow in the dark is due to the low level of respiration. The respiration rate under light increased to four times the dark value. The values of the compensation ratio (the photosyntehtic rate/the respiration rate) for the oxygen exchange were below 1.0 daring the growth period, although oxygen evolution was found. At the early exponential phase, oxygen evolution was 0.373 μmol (mg cell dry weight)⁻¹ h⁻¹ [61.7 μmol (mg chlorophyll)⁻¹ h⁻¹]. M. polymorpha cells are unable to grow anaerobically in the light without a supply of carbon dioxide. When 1% carbon dioxide in nitrogen is supplied, photochemically produced oxygen and energy are sufficient for sustained growth although at significantly reduced yields in both cell dry weight and chlorophyll. Photosyntehtic CO₂ assimilation rate was 0.13 μmol (mg cell dry weight)⁻¹ h⁻¹[11.3 μmol (mg chlorophyll)⁻¹ h⁻¹]. At least one-third of the carbon atoms in cellular constituents seem to be derived from atmospheric carbon dioxide, which indicates that M. polymorpha cells grow photomixotrophicaily.     

Soluble fractions of Solanum tuberosum enchnce call and pigment production of semi-continous cultures of the microlga Phaeodactylum tricornutum

J. FÁBREGAS, E. MORALES, N. POLANCO, M. PATINO AND A. OTERO. 1996. Soluble fractions of Solanum tuberosum extracted with different methods were tested on semicontinuous mixotrophic cultures of the marine microalga Phaeodactylum tricornutum maintained with renewal rate at 15%. The highest stabilization cell density, 78.2 x 10⁶ cells ml⁻¹, was obtained with an autoclaved non-fermented soluble fraction obtained with distilled water. Highest productivities and carotenoids, 0.9 mg 1⁻¹ d⁻¹, and chlorophyll, 2.9 mg 1⁻¹, d⁻¹, were obtained with a non-autoclaved nonfermented fraction extracted in sea water. The bacterial population associated to the microalgal cultures changed depending on the nutrient availability of each of the potato-soluble fractions.     

Detection of the response regulator AgrA in the cytosolic fraction of Staphylococcus aureus by monoclonal antibodies

Abstract The interaction of salivary lysozyme with the surface protein antigen (PAc) of Streptococcus mutans and the interaction of lysozyme with the pathogen were examined by ELISA using S. mutans MT8148 (PAc⁺) and the PAc-defective mutant EM-2 (PAc⁻). The lysozyme clearly bound to the S. mutans wild type but not to the S. mutans mutant. Furthermore, lysozyme bound directly in the fluid phase to the rPAc, of which the binding kinetics were determined ( K _on= 3.63 ± 0.04 × 10³M⁻¹ s ⁻¹, K _off= 1.72 ± 0.04 × 10⁻⁵s⁻¹ and K_on / K_off= 2.11 × 10⁸M⁻¹) using surface plasmon resonance. The kinetics of both association and dissociation were relatively slow. In addition, anti-lysozyme antibody significantly inhibited the binding of salivary components to the rPAc. The present findings indicate that lysozyme is one of the major salivary components interacting with S. mutans PAc.