High Concentrations Of CaCl Research Articles

Optimization of the production of the lantibiotic mutacin 1140 in minimal media

Mutacin 1140 is produced by Streptococcus mutans and belongs to the type A lantibiotic family. Experiments were done to optimize production of mutacin 1140 in minimal media enabling a more cost efficient downstream purification method. The development of a small volume fermentation method enabled a rapid screen of several variables in a standard shaking incubator. This method provided a fast approach for determining components that promote mutacin 1140 production in minimal media broth. Lactose was determined to be the optimal carbon source for mutacin 1140 production. High concentrations of CaCl(2) (0.3% w/v) and MgSO(4) (0.77% w/v) promoted an increase in mutacin 1140 production, while ZnCl(2) and FeCl(3) appeared to impair production. Optimization of mutacin 1140 production in minimal media resulted in more than a 100-fold increase in production compared to the base medium used to begin our optimizations. The yield has been estimated by RP-HPLC to be ~10 mg/L.

Segregative interactions and competitive binding of Ca 2+ in gelling mixtures of whey protein isolate with Na +κ-carrageenan

Gelling mixtures of Na + κ-carrageenan with whey protein isolate (WPI) at pH 7.0 have been studied rheologically and by differential scanning calorimetry (DSC), with comparative measurements for the individual constituents of the mixtures. The concentration of WPI was held fixed at 10.0 wt% and carrageenan concentration was varied in the range 0.05–3.0 wt%. Ca 2+ cations, which have been shown previously to be particularly effective in inducing gelation of κ-carrageenan, were introduced as CaCl 2. The concentration of CaCl 2 used in most of the experiments was 8 mM, but other concentrations were also studied. Mixtures were prepared in the solution state at 45 °C, and showed no evidence of either phase separation or complex formation. Rheological changes were monitored by low-amplitude oscillatory measurements of storage modulus, G′, during (i) cooling (1 °C/min) and holding at 5 °C, to induce gelation of the carrageenan in the presence of non-gelled WPI; (ii) heating and holding at 80 °C to dissociate the carrageenan network and induce gelation of WPI; (iii) cooling and holding again at 5 °C, to give composite networks with both components gelled; and (iv) re-heating to 80 °C to dissociate the carrageenan network. Gel structure was characterised further by creep–recovery measurements at the end of each holding period, and by torsion measurements at 5 °C, before and after thermal gelation of WPI. In 8 mM CaCl 2, the presence of 10.0 wt% WPI promoted earlier gelation of κ-carrageenan during cooling, and displaced the disorder–order transition in DSC to higher temperature. These changes are attributed to segregative interaction with non-gelled WPI. The resulting networks, however, were weaker that those formed by κ-carrageenan alone, which was traced to binding of Ca 2+ cations to the WPI. At higher concentrations of CaCl 2, where availability of Ca 2+ cations was no longer a limiting factor in gelation of the κ-carrageenan, the effect of segregative interactions could be seen as an increase in gel strength. On heating to gel the WPI, the presence of κ-carrageenan led to the earlier appearance of an aggregation exotherm in DSC and to network formation at shorter times, which can similarly be explained by enhanced self-association of denatured WPI in response to segregative interactions with disordered carrageenan. An initial large (∼10 fold) increase in gel strength with increasing concentration of carrageenan to ∼0.25 wt% was followed by a reduction at higher concentrations, attributed to excessive aggregation (incipient precipitation) of the WPI. The magnitude of the changes in G′ observed on subsequent cooling suggest that gelation of κ-carrageenan within the pores of an existing network of gelled WPI gives rise to a bicontinuous co-gel structure. Comparison of moduli at 80 °C before and after cooling to 5 °C demonstrated that formation and dissociation of the carrageenan network caused no disruption of the network of gelled WPI.

Cyclic AMP-Independent CGRP8–37-Sensitive Receptors Mediate Adrenomedullin-Induced Decrease of CaCl2-Contraction in Pregnant Rat Mesenteric Artery

Objectives: We tested the hypothesis that adrenomedullin reduces calcium influx independent of potassium channels in depolarized endothelium-denuded mesenteric artery from pregnant rats. Results: Adrenomedullin reduced the CaCl₂-induced contraction, while the receptor antagonist calcitonin gene-related peptide (CGRP)_8–37, but not adrenomedullin_22–52, reversed these effects. Adenylate cyclase inhibition by SQ22536 did not prevent adrenomedullin effects on CaCl₂-induced contraction. Adrenomedullin did not inhibit depolarization-induced calcium entry to isolated vascular smooth muscle. Inhibition of myosin light-chain (MLC) phosphatase by calyculin A reversed the effects of adrenomedullin on contraction caused by submillimolar concentrations of CaCl₂, while adrenomedullin still inhibited contraction caused by higher concentrations of CaCl₂. However, the ratio of phosphorylated to total myosin phosphatase target 1, the regulatory subunit of MLC phosphatase, did not change with adrenomedullin, indicating a lack of MLC phosphatase activation. Interestingly, sodium fluoride, a nonspecific protein phosphatase inhibitor, completely blocked the effect of adrenomedullin on CaCl₂-induced contraction. Adrenomedullin inhibited calcium mobilization from intracellular stores induced by thapsigargin. Conclusion: Adrenomedullin inhibits CaCl₂-induced contraction, without affecting calcium influx, through a CGRP_8–37-sensitive receptor, but not using the cyclic adenosine monophosphate pathway, probably through activation of protein phosphatases. Inhibition of intracellular calcium release is an additional role played by adrenomedullin in calcium homeostasis in vascular smooth muscle.

Genetic transformation of barley (Hordeum vulgare L.) via infection of androgenetic pollen cultures with Agrobacterium tumefaciens

A novel genetic transformation method for barley (Hordeum vulgare L.), based on infection of androgenetic pollen cultures with Agrobacterium tumefaciens, is presented. Winter-type barley cv. 'Igri' was amenable to stable integration of transgenes mediated by A. tumefaciens strain LBA4404 harbouring a vector system that confers hypervirulence, or by the non-hypervirulent strain GV3101 with a standard binary vector. The efficacy of gene transfer was substantially influenced by pollen pre-culture time, choice of Agrobacterium strain and vector system, Agrobacterium population density, medium pH and the concentrations of acetosyringone, CaCl(2) and glutamine. After co-culture, rapid removal of viable agrobacteria was crucial for subsequent development of the pollen culture. To this end, the growth of agrobacteria was suppressed by the concerted effects of appropriate antibiotics, low pH, reduced level of glutamine and high concentrations of CaCl(2) and acetosyringone. Following infection with LBA4404 and GV3101, about 31% and 69%, respectively, of the primary transgenic (T(0)) plants carried a single copy of the sequence integrated. The use of hypervirulent A. tumefaciens and hygromycin resistance as a selectable marker resulted in 3.7 T(0) plants per donor spike. About 60% of the primary transgenic plants set seed, indicating spontaneous genome doubling. An analysis of 20 T(1) populations revealed that four progenies did not segregate for reporter gene expression. This indicates that the approach pursued enables the generation of instantly homozygous primary transgenic plants. The method established will be a valuable tool in functional genomics as well as for the biotechnological improvement of barley.

The complex of xylan and iodine: the induction and detection of nanoscale order

The complex of xylan and iodine and its formation in a solution of xylan, CaCl 2, and I 2 + KI was investigated by UV/Vis, second-derivative UV/Vis, and Raman spectroscopy. The complex forms only at very high concentrations of CaCl 2, suggesting that when the water available in the solution is not sufficient to fully hydrate the calcium cation the chelation with the hydroxyl groups of the xylan can occur. The electronic spectra indicate that iodine is present in the form of three linear polyiodides I 9 3−, I 11 3−, and I 13 3− structures, which the Raman spectra show to be linear aggregates of the I 3 − and I 5 − substructures. Iodide concentration has a significant influence on the relative population of I 9 3−, I 11 3−, and I 13 3−, as well as I 3 − and I 5 −, which lead to changes in both the UV/Vis absorption maxima shifts and changes in the Raman spectra. The key difference between this system of complexes with the linear polyiodide aggregates and that of amylose is that the longest aggregate observed with the amylose system, the I 15 3− polyanion, is not observed with the xylans. This indicates that the ordered arrays in the xylan–iodine complex do not exceed 4 nm in length. It is not possible to conclude at this time whether the ordered segment of the xylan molecule is linear or helical. If it is linear the length of the longest ordered arrays would be eight xylose residues. The number would exceed eight if the xylan molecule were helically wound.

Differentiation between Minus- and Plus-Strand Synthesis: Polymerase Activity of dsRNA Bacteriophage φ6 in an in Vitro Packaging and Replication System

Empty procapsids of the segmented dsRNA virus φ6, produced in Escherichia coli from a cloned L genome segment, package plus-strand φ6 ssRNA genomic segments, synthesize minus strands, and transcribe the newly formed dsRNA templates. Procapsids can be restricted to minus-strand synthesis by high concentrations of CaCl 2 or low concentrations of nucleotides, enabling us to separate the viral minus-strand (replication) and plus-strand (transcription) RNA-dependent RNA polymerase activities in vitro. Reaction conditions for minus-strand synthesis were optimized. Plus-strand synthesis by procapsids could be activated by binding of purine nucleoside triphosphates to a low-affinity NTP-binding site. The second 5′-terminal nucleotide of the φ6 plus-sense ssRNA L genomic segment is important for determining the level of transcription of that segment and the generation of infectious procapsids.

Ketamine relaxes rabbit femoral arteries by reducing [Ca 2+] i and phospholipase C activity

The effects of the short-acting anesthetic, ketamine, on intracellular free Ca 2+ concentrations, ([Ca 2+] i), inositol phosphate levels and force produced by contractile agonists were investigated in strips of rabbit femoral artery. In concentration-response curves, ketamine produced an insurmountable inhibition of contractions produced by KCl and the L-type Ca 2+ channel agonist, Bay k 8644. However, in K + -depolarized tissues, high concentrations of CaCl 2 could overcome the inhibition produced by ketamine, suggesting that ketamine may have competed with Ca 2+ in activated L-type Ca 2+ channels. In support of the contention that it inhibits L-type Ca 2+ channels, ketamine was found to concomitantly reduce the levels of force and [Ca 2+] i produced by 50 mM KCl. Ketamine reduced the potency, but not the maximum force, produced by phenylephrine. However, this surmountable inhibition may have been due to activation of ‘spare’ α-adrenoceptors rather than to competition of receptor binding because, after phenoxybenzamine pretreatment to reduce α-adrenoceptor numbers, phenylephrine concentration-response curves in the presence of ketamine were insurmountable. Ketamine at 0.32 mM reduced the transient contractions produced in a Ca 2+-free solution and the increase in phospholipase C activity (estimated by measuring inositol phosphate production in the presence of Li +) produced by 1 but not 10 μM phenylephrine. These data suggest that ketamine inhibited contractions produced in rabbit femoral artery by decreasing Ca 2+ channel activity and by reducing phospholipase C activation.

ω-Conotoxin binding and effects on calcium channel function in human neuroblastoma and rat pheochromocytoma cell lines

Binding of ω-conotoxin, a peptide toxin specific for some subtypes of voltage-operated calcium channels (VOCCs), was investigated in IMR32 neuroblastoma and PC12 pheochromocytoma cell lines. In both cell types, binding was specific, saturable and of high affinity. Association was rapid and dissociation almost non-existent. Dihydropyridines and verapamil failed to affect toxin binding, while high concentrations of CaCl 2 completely antagonized it. Depolarization with high K + induced a [Ca 2+] i rise (revealed by the fura-2 fluorimetric technique) that consisted of an initial (0.5–1 min) peak followed by a prolonged (several minutes) plateau phase. ω-Conotoxin blocked mainly the first phase, while the dihydropyridine Ca 2+ channel blocker, nitrendipine, primarily affected the plateau. This result suggests that in the two cell lines investigated, ω-conotoxin acts mainly on a subgroup of VOCCs that is resistant to dihydropyridines.

First Evidence for Polyamine Conjugation Mediated by an Enzymic Activity in Plants

An enzyme activity, found for the first time in plants, mainly located in the 22,000g supernatant of the crude extract of sprout apices of Helianthus tuberosus L. cv OB1 tubers, is able in vitro to covalently bind polyamines to endogenous substrates of different molecular weights. The major assay parameters, such as pH, dithiothreitol, and extract concentrations were optimized. The time course of the reaction, the dependence on putrescine concentration, its competition with histamine, the capacity to bind spermidine and spermine better than putrescine, the stability of the reaction product and analysis of the latter by sodium dodecyl sulfate polyacrylamide gel electrophoresis and thin-layer chromatography suggest that putrescine is linked to endogenous substrates by means of an enzyme reaction that shows some similarities with transglutaminase activities detected in animals. However, the activities of the crude extract and of a fraction eluted from a Sephadex G25 column were not affected by CaCl(2) concentrations lower or equal to 5 millimolar; 4 or 10 millimolar EGTA caused a very small reduction; higher concentrations of CaCl(2) and 15 millimolar or more of EDTA were inhibitory. N,N'-dimethylcasein was not recognized as a substrate. These data indicate that this activity also displays some characteristics which are different from those of animal transglutaminases.

Studies on preparation and viability of Phytophthora parasitica spheroplasts

Spheroplasts were obtained from Phytophthora parasitica mycelium by digestion with Novozym 234 using 035 m CaCl 2 (pH 5.7) as initial osmotic stabilizer. Spheroplast yield decreased rapidly with increasing age of the mycelium. Young mycelium gave highest yields of 9.3±4.5 × 10 6 cells per Petri dish. Regeneration was investigated on synthetic and complex media in the presence of different osmotic stabilizers. Survival on solid and in liquid media was enhanced in the presence of CaCl 2 in addition to mannitol as osmotic stabilizer. When spheroplasts were spread on agar containing 0.1 M CaCl 2 plus 0.4 M mannitol as osmotica the regeneration rate was 12±4 %. After embedding in soft agarose stabilized with 0.8 M mannitol spheroplast regeneration was 19±6%. Incubation in osmotically stabilized liquid medium resulted in a rapid loss of viability. High concentrations of CaCl 2 as osmotic stabilizer caused abnormal growth of spheroplasts which eventually gave rise to normal mycelium.

Ionic strength effects on the electrophoretic mobility of casein-coated polystyrene latex particles

Electrophoretic mobilities of polystyrene particles coated with three casein proteins (α sl, β, and κ) have been measured at 25°C in 20 m M imidazole buffer (pH 7.5) as a function of ionic strength of added sodium chloride (0.01–0.15 mole dm −3) or calcium chloride (0.00003–0.09 mole dm −3), with protein concentrations sufficient to ensure saturation coverage as shown by measured adsorption isotherms. Particle mobilities were found to lie in the order α sl > κ ≳ β in NaCl and at low concentrations of CaCl 2, but in the order κ > α sl ≳ β at higher concentrations of CaCl 2, reflecting the different calcium-binding properties of the three proteins. Qualitatively similar results for bare and coated particles show that the large difference between sodium and calcium counterions is not due to specific calcium ion-binding on the adsorbed protein. For these biocolloidal particles, the two techniques of laser Doppler electrophoresis and particle microelectrophoresis give essentially identical results.

The effects of CaCl 2 on limestone sulfation in fluidized-bed coal combustion

In an investigation into the effects of CaCl 2 on the sulfation of limestones in a laboratory furnace simulating fluidized-bed coal combustion conditions, small additions of CaCl 2 (<1 mole%) to the limestone prior to calcination were found to increase the extent of sulfation by changing the limestone pore structure. These changes were effected by trace amounts of liquid in the system in a synthetic SO 2/O 2 flue gas at 850°C. At much higher concentrations of CaCl 2, large amounts of a liquid phase are produced, containing a substantial quantity of dissolved CaO and leading to greatly enhanced sulfation when exposed to SO 2/O 2. The use of CaCl 2 additive in fluidized-bed combustion would reduce the quantity of limestones required to meet air pollution standards for SO 2 and also reduce the quantity of solid waste generated.

Reevaluation of the Effect of Calcium Ions on Auxin-induced Elongation

The mechanism by which calcium ions inhibit cell elongation has been reinvestigated. Growth-inhibiting levels of calcium, when applied to isolated walls (in vitro treatment) do not decrease cell wall extensibility as measured by the Instron technique. Thus, the hypothesis that calcium inhibits growth by forming wall-stiffening calcium bridges must be abandoned. Treatment of living auxin-treated sections with calcium (in vivo treatment) does cause a decrease in the subsequently measured wall extensibility, but this decline appears to be simply a consequence of the growth inhibition rather than its cause. Growth-inhibiting levels of calcium do not appreciably reduce the rate of auxin-enhanced H(+) excretion. Pretreatment with calcium does not reduce the capacity of walls to undergo acid-activated wall loosening in the absence of calcium. High concentrations of CaCl(2) (0.02 m) cause an initial elastic shrinkage of Avena sections comparable to that caused by the same osmolarity of mannitol, but the subsequent growth inhibition is too great to be explained by an osmotic inhibition. Calcium ions do inhibit H(+)-induced extension of frozen-thawed sections under tension. The growth-inhibitory effects of calcium, then, may be ascribed to a direct inhibition exerted by calcium ions on the H(+)-induced wall-loosening process.

Über die Salzsekretion hei Halimione portulacoides (L.) Aellen

Salt secretion was studied in Halimione portulacoiaes cultured in nutrient solutions. The organ of secretion are bladders, found on both sides of the epidermis of the leaf. They are built similar to the bladders of the genus Atriplex , consisting of a small stalk cell, filled with protoplasm and a larger vacuolated final cell. At high salinity of the nutrient solution the secreted sap of the bladders always contains a higher concentration of ions than the sap (pressed sap) of the mesophyll cells. The content of ions both of the pressed sap of the leaf tissue and the secreted sap of the bladder cells is influenced by the concentration of salt of the nutrient solution. High concentrations of NaCl promote the accumulation and secretion of Na + - and Cl — -ions and high concentrations of KCl those of K + - and Cl — -ions. Between the Na + - and K + -ions antagonistic relations are to be found. The greatest differences in the concentration of the pressed sap and that of the secreted sap are to be observed at the Na + - and HCO 3 — -ions. Analysis of the procentual composition of the secreted sap showed, that among the cations Na + - and K + -ions and among the anions HCO 3 — - and Cl — -ions are dominant. Apart from these ions only Mg ++ -ions seem to be secreted against a concentration gradient. Ca ++ -, NO 3 — -, SO 4 —— - and HPO 4 —— -ions in the secreted sap are always to be found with smaller procentual shares than in the pressed sap of the leaf tissue. Anantomic analysis of the leaves showed numerous cristals of oxalate. Furthermore the chemical analysis also showed a high oxalat content of the leaf tissue, which is increased by a high concentration of NaCl of the nutrient solution, enhancing, however, the watersoluble oxalate only. High concentrations of CaCl 2 in the nutrient solution effects the total oxalate to be waterinsoluble.

The trypsin-catalyzed hydrolysis of monomolecular films of lysylphosphatidylglycerol

The hydrolysis by trypsin of the bacterial phospholipid, lysylphosphatidyl-glycerol has been studied at the air-water interface. High specific activity [ 14C]-lysylphosphatidylglycerol was prepared biosynthetically and the trypsin action followed by measuring the loss of surface radioactivity from a monolayer of the phospholipid spread on a water surface. After a lag phase, hydrolysis proceeded at a constant rate proportional to the enzyme concentration until 50–85% of the substrate had been hydrolysed. Maximum hydrolysis occurred between 10–35 dynes/cm surface pressure and at pH 7.5–8.5. A slight fall in the surface pressure occurred but this was less than predicted from the force-area curves of lysylphosphatidylglycerol and phosphatidylglycerol. Ca 2+ (0.5 mM) and to a lesser extent Mg 2+ and Sr 2+ stimulated the reaction. During the reaction 45Ca 2+ became bound on the monolayer in proportion to the extent of the enzymic hydrolysis. The stimulation by calcium required the synergic presence of low concentrations of Tris buffer. High concentrations of CaCl 2 (2 mM), NaCl (25 mM), Tris buffer (47 mM) almost completely inhibited the reaction. Chlorpromazine, which removes Ca 2+ from phospholipid membranes, markedly stimulated hydrolysis at 2.5–6.0 μM, but only in the presence of added calcium: higher concentrations of chlorpromazine (0.125 mM) completely inhibited the reaction.

Effect of divalent cations and pH on intrinsic factor-mediated attachment of vitamin B12 to intestinal microvillous membranes

Calcium, but not other divalent cations, is required for optimal uptake of intrinsic factor-bound (57)Co-labeled cyanocobalamin (IFB(12)) by microvillous membranes isolated from hamster ileal-absorptive cells. Chelation of divalent cations by disodium ethylenediaminetetraacetate (EDTA) promptly removes IFB(12) previously attached to microvillous membranes. High concentrations of CaCl(2) or MgCl(2) also markedly inhibit membrane uptake of IFB(12) and rapidly remove previously attached IFB(12). Similarly, reduction of pH to below 5.4 prevents membrane attachment of IFB(12) and removes virtually all IFB(12) already bound to microvillous membranes. The effects of calcium depletion, increased salt concentrations, and acidification on membrane uptake of IFB(12) were completely reversible. These findings are consistent with the concept that the formation of calcium salt bridges is essential for attachment of IFB(12) to the ileal-absorptive surface.

The effect of electrolytes of the general formula XCl 2 on the response of the type 1 labellar chemoreceptor of the blowfly, Phormia

An analysis has been made of the electrical responses of the type 1 labellar (‘salt’) chemoreceptor cell of Phormia regina when stimulated with solutions of divalent metal salts. Electrophysiological measurements were made by side-wall penetration of the receptor process with glass microcapillary electrodes. A mean, unstimulated response frequency of 3·3 impulses/sec is totally suppressed by all concentrations of CaCl 2 between 0·05 mM and 100 mM. The characteristic off-discharges following CaCl 2 stimuli (concentrations 0·8 mM to 100 mM) have similar time-courses of spike frequency, but at 0·6 mM, and below, little or no off-discharge can be detected. Suppression of response, with no off-discharge, is attributed to high electrical resistance of the receptor tipchamber rather than to any specific effect of the solute. The time course of off-discharge remains unaffected by the duration of CaCl 2 application between 70 and 500 msec, but is scarcely elicited, or absent, after stimuli of 50 msec or less. Concentrations of CaCl 2≥1 mM are needed to cause hyperpolarization of the receptor membrane. Above 1 mM, the hyperpolarization potential is proportional to the logarithm of the excess concentration of CaCl 2 and may be due to excess Cl − ion on the outside of the receptor membrane. Much higher concentrations of CaCl 2 are needed in mixtures with KCl to suppress the response completely than will arrest unstimulated discharge, e.g. 0·9 M CaCl 2 with 0·8 M KCl. Equal activities of Ba 2+, Sr 2+, Ca 2+, Mg 2+ and Be 2+ as chlorides mixed with KCl in the stimulus solution are able to depress discharge frequency to differing extents. Calcium is most effective, the other cations exerting a diminishing effect as their atomic numbers diverge from that of calcium. Other calcium salts (nitrate, iodide, and acetate) fully suppress the unstimulated response of the type 1 cell at 2, 20, and 100 mM concentrations, further emphasizing the importance of the cation in this effect.