Studies on the Germination of Pine Pollen (Pinus mugo) in vitro. II. Effects of Different Ions

Studies on the initial germination of pollen of Pinus mugo showed no significant influence of ions on O₂ uptake and uptake of ³²P-labelled phosphate. At the onset of tube growth O₂ uptake decreased in the absence of calcium. In inorganic media tube growth and ³²P uptake were reduced in the absence of calcium or boric acid. In the absence of calcium a requirement for magnesium was observed. When the medium was deprived of polyvalent ions with EDTA, growth and ³²P uptake ceased. The presence of calcium in the medium was found to be essential for the maintenance of the structural and functional integrity of the cell membranne. — The ion requirement was more pronounced when tube growth was stimulated with sucrose. Calcium, magnesium, boric acid, and nitrate (as nitrogen source) were essential constitutents of the medium. The stimulation due to calcium required either magnesium or boric acid. — A density effect was observed which can be related to diffusible substances from the pollen into the medium. This was not observed when calcium and magnesium were present in the medium. The phenomenon is explained as an enrichment of the medium with diffusible substances from non-germinated dead pollen. — Germination and the tube growth were found to be greatly dependent on a short period of equilibration of pollen at room temperature before sowing.     

Control of the Life Cycle of Methanosarcina mazei S-6 by Manipulation of Growth Conditions

The morphology of Methanosarcina mazei was controlled by magnesium, calcium, and substrate concentrations and by inoculum size; these factors allowed manipulation of the morphology and interconversions between pseudosarcinal aggregates and individual, coccoid cells. M. mazei grew as aggregates in medium with a low concentration of catabolic substrate (either 50 mM acetate, 50 mM methanol, or 10 mM trimethylamine) unless Ca²⁺ and Mg²⁺ concentrations were high. Growth in medium high in Ca²⁺, Mg²⁺, and substrate (i.e., 150 mM acetate, 150 mM methanol, or 40 mM trimethylamine) converted pseudosarcinal aggregates to individual cocci. In such media, aggregates separated into individual cells which continued to grow exclusively as single cells during subsequent transfers. Conversion of single cells back to aggregates was complicated, because conditions which supported the aggregated morphology (e.g., low calcium or magnesium concentration) caused lysis of coccoid inocula. We recovered aggregates from coccoid cells by inoculating serial dilutions into medium high in calcium and magnesium. Cells from very dilute inocula grew into aggregates which disaggregated on continued incubation. However, timely transfer of the aggregates to medium low in calcium, magnesium, and catabolic substrates allowed continued growth as aggregates. We demonstrated the activity of the enzyme (disaggregatase) which caused the dispersion of aggregates into individual cells; disaggregatase was produced not only during disaggregation but also in growing cultures of single cells. Uronic acids, the monomeric constituents of the Methanosarcina matrix, were also produced during disaggregation and during growth as coccoids.     

Determination of the concentration of free Ca2+ in the presence of magnesium (or manganese) and chelating effectors of the NAD+-linked isocitrate dehydrogenase.

An equation was formulated that enabled the calculation of the concentration of free Ca²⁺ in a reaction medium containing calcium, magnesium (or manganese), and up to five metal chelators. The concentrations of all ionic and chelating species present in the incubation mixture could also be estimated. The equation, a polynomial to the sixth power with respect to free Ca²⁺, is expressed in terms of known equilibrium (formation) constants and concentrations of reactants. The calculated values of free Ca²⁺ were compared with those determined experimentally, using a calcium ion-specific electrode, and the two were found to be in excellent agreement. To illustrate the usefulness of the technique, we examined the inhibition by calcium of NAD⁺-linked isocitrate dehydrogenase, in the presence of ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid (EGTA), ADP, ATP, isocitrate, citrate, and magnesium. The effect of manganese on the concentration of free Ca²⁺ in solutions of calcium and EGTA was also deseribed. It was found that manganese, but not magnesium, displaced calcium from the Ca-EGTA cholate resulting in a much higher than expected concentration of free Ca²⁺. This raises questions about the inhibition of the isocitrate dehydrogenase by physiological concentrations of calcium previously reported.     

Do novel cement-type biomaterials reveal ion reactivity that affects cell viability in vitro?

Calcium phosphate bioceramics have been studied as bone filler materials for years and have become a component of many commercial products. It is widely known that surface-reactive biomaterials may cause changes in the concentration of crucial ions in the surrounding environment, thereby affecting cell metabolism and viability. The aim of this study was to produce five cement-type biomaterials and characterize their phase composition using X-ray diffraction method, and porosity and pore size distribution using mercury intrusion porosimeter. We then evaluated ion interactions of the novel biomaterials with the surrounding environment (culture medium). A commercially available bone substitute, HydroSet? (Stryker®), was used as a reference. MTT and NRU cytotoxicity tests were performed to assess the effect of changes in the concentration of crucial ions (calcium, magnesium, phosphate) on osteoblast metabolism and viability in vitro. Our study clearly indicated that various biomaterials demonstrated different ion reactivity and consequently may cause changes in ion concentration in the local environment. Critically low or high values of calcium, magnesium, and phosphate concentrations in the medium exerted cytotoxic effects on the cultured cells. Moreover, we discovered that the chemical composition of the culture medium had a substantial influence on ion interactions with biomaterials.     

Inhibition of active strontium transport from erythrocyte ghosts by internal calcium: Evidence for a specificity controlling site

Summary The inhibition of strontium transport from erythrocyte ghosts by internal calcium was investigated. When active strontium transport was measured in the presence of increasing levels of internal calcium it was found that the inhibition of strontium transport started at an internal calcium level of 0.3mm and was virtually complete when this concentration reached 1.0mm. It was also noted that calcium transport was virtually constant between concentrations of 0.3 and 1.0mm. This experiment indicated that calcium did not inhibit strontium transport by competing for the active site of the transport system. This inhibition was partially reversed by increasing the internal magnesium concentration from 1 to 4mm. A higher level of magnesium at the time of lysis and during incubation enhanced strontium transport. However, the inhibition remained noncompetitive with respect to calcium. Manganese was also found to support calcium and strontium transport. However, it could not reverse the inhibition of strontium transport by internal calcium at any concentration tested. In fact, manganese restored the inhibition of strontium transport by calcium in ghosts that were prepared and incubated in solutions that had high magnesium levels.     

Seasonal ionic fluctuations and annual growth rates in stands of Pinus silvestris L. and Picea abies Karst. (L.)

Seasonal variations in potassium, calcium, nitrogen, phosphorus, and magnesium contents of needles from two different stands of Pinus silvestris L. and Picea abies (L.) Karst. were determined monthly during a period of two years, and the growth rates once a year during five years. The stands were growing on sand or on clay. The supposed transport of potassium to the shoot during autumn could not be demonstrated. The nutrient status of pine and spruce growing on sand and clay was very similar, except for calcium and magnesium. There were only small differences in ion contents between pine and spruce, except for calcium. On soils with low water-holding capacity, the available water in dry years is the limiting factor affecting the growth rate of spruce but not of pine. It is concluded that the nutrient status of the leaves, the growth rates and the available water in the soil are factors that should have an important part in any discussion of fertilization of coniferous forests.     

Requirement of a Relatively High Threshold Level of Mg2+ for Cell Growth of a Rhizoplane Bacterium, Sphingomonas yanoikuyae EC-S001

Mg²⁺ is one of the essential elements for bacterial cell growth. The presence of the magnesium cation (Mg²⁺) in various concentrations often affects cell growth restoration in plant-associating bacteria. This study attempted to determine whether Mg²⁺ levels in Sphingomonas yanoikuyae EC-S001 affected cell growth restoration in the host plant and what the threshold level is. S. yanoikuyae EC-S001, isolated from the rhizoplane of spinach seedlings grown from surface-sterilized seeds under aseptic conditions, displayed uniform dispersion and attachment throughout the rhizoplane and phylloplane of the host seedlings. S. yanoikuyae EC-S001 did not grow in potato-dextrose broth medium but grew well in an aqueous extract of spinach leaves. Chemical investigation of the growth factor in the spinach leaf extract led to identification of the active principle as the magnesium cation. A concentration of ca. 0.10 mM Mg²⁺ or more allowed S. yanoikuyae EC-S001 to grow in potato-dextrose broth medium. Some saprophytic and/or diazotrophic bacteria used in our experiment were found to have diverse threshold levels for their Mg²⁺ requirements. For example, Burkholderia cepacia EC-K014, originally isolated from the rhizoplane of a Melastoma sp., could grow even in Mg²⁺-free Hoagland's no. 2 medium with saccharose and glutamine (HSG medium) and requires a trace level of Mg²⁺ for its growth. In contrast, S. yanoikuyae EC-S001, together with Bacillus subtilis IFO12113, showed the most drastic restoring responses to subsequent addition of 0.98 mM Mg²⁺ to Mg²⁺-free HSG medium. Our studies concluded that Mg²⁺ is more than just the essential trace element needed for cell growth restoration in S. yanoikuyae EC-S001 and that certain nonculturable bacteria may require a higher concentration of Mg²⁺ or another specific essential element for their growth.     

Improvement in cell yield of Methylobacterium sp. by reducing the inhibition of medium components for poly-β-hydroxybutyrate production

The inhibitory effect of the concentrations of medium components on the growth of Methylobacterium sp. for poly--hydroxybutyrate production was investigated by measuring the specific growth rates for various concentrations of each medium component. When the methanol concentration was increased, the cell growth decreased and was strongly inhibited above 6% (v/v) methanol. Ammonia, calcium and iron ion did not significantly inhibit the cell growth while there were some inhibitory effects at high concentrations of sodium, potassium, and magnesium. In particular, phosphate gave most significant inhibition at concentrations higher than 75 mM. By using an automatic feeding control system of methanol, ammonia, phosphate, and minerals, their concentrations were maintained within the level necessary to reduce the inhibition of medium components. The finial dry cell weight of Methylobacterium sp. in such a system was 172 g/l at 84 h.     

Effect of enhanced CaCl2, MgSO4, and KH2PO4 on improved in vitro growth of potato

Potato (Solanum tuberosum L.) is a major global food crop. Contemporary potato production largely utilizes micropropagation to produce healthy seed potatoes. The micropropagation of potatoes is widely achieved through nodal explants using the conventional Murashige and Skoog (MS) medium. Currently, effective culture media that can facilitate rapid propagation are increasingly required for new cultivars that have been developed to possess improved traits. In this study, we evaluated the effect of enhanced meso nutrients (CaCl₂.2H₂O, MgSO₄, and KH₂PO₄) in MS medium on the growth of S. tuberosum. The cultivars used in this study were representative of Japanese, European, and Peruvian lines. Enhanced meso nutrients improved the overall quality of all cultivars, as indicated by longer shoots and larger leaves with dark color, compared with MS medium only. Shoots grown on enhanced mesos were approximately 1.5 times longer than on MS medium. Quantitative ion analysis revealed that plantlets with improved shoot length and leaf quality in most cultivars had increased calcium, magnesium, potassium, and phosphorus uptake than plantlets on MS medium. The results suggest that the reduced iron uptake on 3.0×MS, compared with 2.0× or 2.5×MS mesos, reduced plant growth. This study revealed for the first time that mesos concentrations higher than MS medium concentrations, complemented by enhanced calcium, magnesium, potassium, phosphorus, and iron uptake, play a significant role in improving the in vitro growth of potato.     

Factors in the inactivation of postjunctional membrane receptors of frog skeletal muscle

Several factors which influence the rate of inactivation of muscle postjunctional membrane (PJM) receptors during the sustained application of carbamylcholine (CARB) have been studied by two methods. The rate of inactivation was increased by elevating the tonicity of the bathing medium, by increasing the CARB concentration, by raising the calcium ion concentration, and by substituting SO₄ ⁼ for Cl^- ions in the extracellular fluid. The relative effectiveness of calcium and other divalent cations in receptor inactivation was compared. In the absence of calcium, other divalent cations such as magnesium, strontium, or manganese were not efficient substitutes for calcium. In the presence of calcium, the addition of strontium or manganese ions accelerated the rate of receptor inactivation, but the addition of magnesium (up to 12 mM) inhibited this process. The inactivation of the membrane receptors in denervated muscle fibers was found to be similar to that in innervated muscle fibers. Various factors in PJM receptor inactivation are discussed. It is suggested that PJM receptor inactivation is influenced by the binding of calcium ions to sites on the internal surface of the PJM.     

The beneficial effect EDTA on development of mouse one-cell embryos in chemically defined medium.

An improved methology for culturing noninbred (ICR) mouse one-cell embryos is described. The successful development of one-cell embryos into blastocysts in chemically defined (Whitten's) medium was significantly enhanced by the presence of EDTA. More than 70% of ICR one-cell embryos developed into blastocysts in Whitten's medium in the presence of 10.8 μM EDTA, while, without EDTA, only 15–30% of embryos reached blastocyst stage. A concentration of 10.8 μM EDTA also promoted the development of 65–90% of inbred $\text{C57BL}\text{6}$ one-cell embryos in Whitten's medium. This beneficial role of EDTA is probably related to the chelation of some metal ion(s) other than Ca²⁺ or Mg²⁺.     

Nutrition Requirements of Pleurotus flabellatus

The mycelium of Pleurotus flabellatus was grown in a synthetic medium to obtain accurate information on its nutritional requirements. Among various carbon sources tried, the organism was found to utilize hexose sugars more readily than other sugars. Ammonium citrate was found to be the best source of nitrogen. The yield of dry matter increased as the concentration of nitrogen was increased up to a certain stage beyond which there was no increase in the yield, but the crude protein content of the mycelium increased. Detailed studies on the effect of varying the concentrations of other major nutrients, i.e., potassium, phosphorus, calcium, and magnesium, on the growth and crude protein content of the mycelium were also carried out. Optimal pH range was fairly broad, lying between 4.5 to 7.5.     

The in vitro release of prothoracicotropic hormone (PTTH) from the brain—corpus cardiacum—corpus allatum complex of silkworm,Bombyx mori

An in vitro system for evaluation of the exact PTTH release from the brain-CC-CA complex of Bombyx mori was developed. The PTTH release induced with a high potassium ion concentration reached the maximal level in 30 min and was dependent on the concentration of the potassium ion. The PTTH release was also dependent on the calcium ion and was induced with a calcium ionophore, indicating that increasing intracellular calcium ion might be essential for its release. Carbachol (acetylcholine agonist) induced the PTTH release fastest, followed by dopamine and serotonin in that order, while norepinephrine exerted no effect. Carbachol might act in the brain of brain-CC-CA complex.     

Effect of some mineral ions on pollen tube growth and release of proteins in culture

In the absence of cations, the release of proteins from pollen tubes ofNicotiana tabacum in culture is greatly dependent on boron concentration and inversely related to growth stimulation. The minimum of proteins in the medium occurs at 100 mg 1^? of boric acid, which is the optimum concentration for growth. The shift of boron level to this optimum further increases the proportion of proteins bound to the insoluble pollen tube fraction; on the other hand the amount of soluble proteins is not affected inside pollen tubes, but greatly decreased in the medium. The loss of proteins into the medium is considerably reduced by oalcium and also at the optimal boron concentration and in the presence of K⁺ and Mg²⁺ ions. The rate of tube elongation, however, is slightly decreased and the duration of pollen growth activity is not extended. The release of proteins is not affected by potassium and is slightly reduced by magnesium. The possible mechanism of calcium and boron action on protein release is discussed in relation to the exocytotic function of secretory vesicles and it is suggested that boron supports the incorporation of proteins transported to the growing tip into the pollen tube wall structures.     

CO(2) Fixation Rate and RuBisCO Content Increase in the Halotolerant Cyanobacterium, Aphanothece halophytica, Grown in High Salinities

The growth of the halotolerant cyanobacterium Aphanothece halophytica, previously adapted to 0.5 molar NaCl, was optimal when NaCl concentration in culture medium was in the range 0.5 to 1.0 molar. The growth was delayed at either too low or too high salinities with lag time of ca. 0.5 day in 0.25 molar NaCl and ca. 2 days in 2 molar NaCl under the experimental conditions. However, the growth rates at the logarithmic phase were similar in the culture media containing NaCl in the range 0.25 to 2.0 molar. The capacity of photosynthetic CO₂ fixation increased 3.7-fold in the cells at the logarithmic phase as NaCl concentration in the culture medium increased from 0.25 to 2.0 molar. The protein level of ribulose 1,5-bisphosphate carboxylase/oxygenase was also found to increase with increasing salinity using both an immunoblotting method and protein A-gold immunoelectron microscopy. These results indicate that high photosynthetic capacity and high ribulose 1,5-bisphosphate carboxylase/oxygenase content may entail an important role in betaine synthesis and adaptation of the A. halophytica cells to high NaCl level.     

Implications of calcium nutrition on the response of Phaseolus vulgaris L. to salinity

The effect of Ca²⁺ level in the growth medium on the response of germination and early seedling growth of Phaseolus vulgaris to NaCl salinity was investigated. When NaCl concentration was increased germination and early seedling growth was decreased. The addition of Ca²⁺ to the media increased both germination percentage and seedling growth. Chloride concentrations were not affected by the level of Ca²⁺. Potassium and Ca²⁺ concentrations and transport from roots to shoots were decreased by NaCl, but were restored by increasing Ca²⁺ in the medium. The opposite was true for Na⁺. Leakage of NO₃ ^- and H₂PO₄ ^- was increased by salinity and reduced by high Ca²⁺ in the medium. The results are discussed in terms of the beneficial effects of calcium for plant growth under saline conditions.     

Boron deficiency in cultured pine cells : quantitative studies of the interaction with ca and mg

A pronounced interaction between calcium, magnesium, and boron was found in growth studies with Pinus radiata cell cultures. Quantitative isoactivity data for the interaction was analyzed in terms of selected simple and plausible theoretical models. The data was found to be consistent with a model in which a critical acceptor molecule is activated only by binding both Ca and B at separate sites; Mg competitively displaces Ca to inactivate the acceptor. It was found that B is, surprisingly, not bound strongly (K_diss = 450 ± 80 micromolar) and that the affinity for Ca is two orders of magnitude stronger than for Mg. Therefore only a small proportion of the acceptor will be boronated under natural conditions. Moderate levels of mannitol were found to aggravate B deficiency due to its effective removal by direct chemical complexation. At higher concentrations of mannitol (or other sugars), where osmotic contribution is significant, little B was needed to overcome growth inhibition—a result consistent with B having a primary role in cell wall biosynthesis.     

Simultaneous modeling of enzyme production and biomass growth in recombinant Escherichia coli using artificial neural networks

In this work, the biomass growth and the TaqI endonuclease production by recombinant Esherichia coli were studied using artificial neural networks. The effects of the medium components on biomass growth and enzyme yield were modeled by various networks. After the most successful networks were statistically determined, they were used to extract additional knowledge such as the possible correlations between the biomass growth and the enzyme yield, and the relative significance of the medium components. It was found that the change of the biomass growth and the enzyme yield with the change of KH₂PO₄ concentration was strongly correlated with an R-value of −0.954. Some mild correlations were also observed for the other components. It was also found that the relative significances of the medium components were in the same order for both outputs; (NH₄)₂HPO₄ concentration was determined as the most important parameter followed by the glucose, KH₂PO₄ and MgSO₄ concentrations.     

A Novel Acidic Matrix Protein,PfN44, Stabilizes Magnesium Calcite to Inhibit the Crystallization of Aragonite

Magnesium is widely used to control calcium carbonate deposition in the shell of pearl oysters. Matrix proteins in the shell are responsible for nucleation and growth of calcium carbonate crystals. However, there is no direct evidence supporting a connection between matrix proteins and magnesium. Here, we identified a novel acidic matrix protein named PfN44 that affected aragonite formation in the shell of the pearl oyster Pinctada fucata. Using immunogold labeling assays, we found PfN44 in both the nacreous and prismatic layers. In shell repair, PfN44 was repressed, whereas other matrix proteins were up-regulated. Disturbing the function of PfN44 by RNAi led to the deposition of porous nacreous tablets with overgrowth of crystals in the nacreous layer. By in vitro circular dichroism spectra and fluorescence quenching, we found that PfN44 bound to both calcium and magnesium with a stronger affinity for magnesium. During in vitro calcium carbonate crystallization and calcification of amorphous calcium carbonate, PfN44 regulated the magnesium content of crystalline carbonate polymorphs and stabilized magnesium calcite to inhibit aragonite deposition. Taken together, our results suggested that by stabilizing magnesium calcite to inhibit aragonite deposition, PfN44 participated in P. fucata shell formation. These observations extend our understanding of the connections between matrix proteins and magnesium.     

Intracellular divalent cation homeostasis and developmental competence in the hamster preimplantation embryo

The intracellular magnesium and calcium ion concentrations of in vivo-developed 2-cell hamster embryos were measured using ratiometric fluorometry. Intracellular magnesium and calcium ion concentrations were found to be 0.369 ± 0.011 mM and 129.3 ± 7.5 nM respectively. Culture of 1-cell hamster embryos for 24 hr to the 2-cell stage in control medium containing 0.5 mM magnesium and 2.0 mM calcium resulted in approximately a threefold increase to 343.5 ± 8.0 nM in intracellular calcium ion concentration, while magnesium ion levels were not altered (0.355 ± 0.007 mM). Increasing medium magnesium concentrations to 2.0 mM significantly increased intracellular magnesium ion concentrations of cultured 2-cell embryos with a concomitant reduction in intracellular calcium ion concentrations. Furthermore, increasing the medium magnesium concentration to 2.0 mM significantly increased development of 1-cell embryos collected at either 3 or 9 hr post-egg activation to the morula/blastocyst and blastocyst stages. Resultant blastocysts had an increased total cell number and increased development of the inner cell mass. Most important, however, culture with 2.0 mM magnesium increased the fetal potential of cultured 1-cells twofold. Therefore, because highest rates of development were observed in a medium that resulted in reduced intracellular calcium ion concentrations, it appears that altered calcium homeostasis is associated with impaired developmental competence of 1-cell embryos in culture. Mol. Reprod. Dev. 50:443–450, 1998. © 1998 Wiley-Liss, Inc.