Rhythmic Fluctuations in the Concentration of Intracellular Mg2+ in Association with Spontaneous Rhythmic Contraction in Cultured Cardiac Myocytes

Magnesium ions (Mg²⁺) play a fundamental role in cellular function, but the cellular dynamic changes of intracellular Mg²⁺ remain poorly delineated. The present study aims to clarify whether the concentration of intracellular Mg²⁺ possibly changes cyclically in association with rhythmic contraction and intracellular Ca²⁺ oscillation in cultured cardiac myocytes from neonatal rats. To do this, we performed a noise analysis of fluctuations in the concentration of intracellular Mg²⁺ in cardiac myocytes. The concentration was estimated by loading cells with either Mg‐fluo4/AM or KMG‐20/AM. Results revealed that the intensity of Mg‐fluo‐4 or KMG‐20 fluorescence fluctuated cyclically in association with the rhythmic contraction of cardiac myocytes. In addition, the simultaneous measurement of Fura2 and Mg‐fluo‐4 fluorescence revealed phase differences between the dynamics of the two signals, suggesting that the cyclic changes in the Mg‐fluo‐4 or KMG‐20 fluorescent intensity actually reflected the changes in intracellular Mg²⁺. The complete termination of spontaneous rhythmic contractions did not abolish Mg²⁺ oscillations, suggesting that the rhythmic fluctuations in intracellular Mg²⁺ did not result from mechanical movements. We suggest that the concentration of intracellular Mg²⁺ changes cyclically in association with spontaneous, cyclic changes in the concentration of intracellular Ca²⁺ of cardiac myocytes. A noise analysis of the fluctuation of subtle changes in fluorescence intensity could contribute to the elucidation of novel functional roles of Mg²⁺ in cells.     

Correlations between changes in membrane capacitance induced by changes in ionic environment and the conductance of channels incorporated into bilayer lipid membranes

The action of metal polycations and pH on ionic channels produced in bilayer lipid membranes (BLM) by three different toxins was studied by measuring membrane capacitance and channel conductance. Here, we show that critical concentrations of Cd²⁺, La³⁺ or Tb³⁺ induce complex changes in membrane capacitance. The time course of capacitance changes is similar to the time course of channel blocking by these ions at low concentration. No changes in BLM capacitance or conductance were observed in the range of pH 5.8–9.0. A pH shift from 7.4 to 3–4 or 11–12 induced large changes in BLM capacitance and channel conductance. For all studied channel-forming proteins, the initial capacitance increase preceded the conductance decrease caused by addition of polycations or by a change in pH. A close relationship between membrane lipid packing and ion channel protein is suggested.     

Genetic instability of anthocyanin production in Impatiens balsamina

Summary It is established that in a naturally occurring variegated Impatiens balsamina the phenotype is determined by a mutable allele p ^m, of an anthocyanin-governing gene P ^r. The special allele produces an acyanic phenotype like the stable recessive p but undergoes frequent changes to P ^r in somatic and germinal cells (causing a variegated phenotype in the former) when a controlling element M is also present in the genome. It is suggested that p ^m is a repressed p ^r and M acts either by removing or inactivating whatever causes that repression. Such changes proceed in a unique fashion: either p ^m changes to p ^r or to an intermediate labile condition P which then changes to p ^r, resulting either in dark or pale, or dark super-imposed on pale, sectors; a reverse situation was not observed. Colourless plants which occasionally appear in unstable lines seem to be due to loss of M although changes ofp ^m itself cannot be ruled out at present.     

Limited breakdown of cytoskeletal proteins by an endogenous protease controls Ca-induced membrane fusion events in chicken erythrocytes

The profound morphological changes which follow the treatment of chicken erythrocytes with the ionophore A23187 and Ca²⁺ are associated with a concomitant breakdown of certain membrane-associated proteins including α-spectrin, goblin and microtubule-associated proteins (MAPS) which undergo a limited proteolysis to give large, well-defined fragments. The Ca²⁺-sensitive protease responsible for these changes appears to be present in the soluble fraction of the cells. Treatment with TLCK or iodoacetamide inhibits both the major morphological changes and the proteolytic events but these agents do not prevent the dissociation of microtubules or the activation of endogenous sphingomyelinase which occur in cells with raised levels of intracellular Ca²⁺. It is suggested that the sphingomyelinase is activated as a consequence of a Ca²⁺-induced loss of phospholipid asymmetry in the plasma membrane.     

Limited breakdown of cytoskeletal proteins by an endogenous protease controls Ca2+-induced membrane fusion events in chicken erythrocytes

The profound morphological changes which follow the treatment of chicken erythrocytes with the ionophore A23187 and Ca²⁺ are associated with a concomitant breakdown of certain membrane-associated proteins including α-spectrin, goblin and microtubule-associated proteins (MAPS) which undergo a limited proteolysis to give large, well-defined fragments. The Ca²⁺-sensitive protease responsible for these changes appears to be present in the soluble fraction of the cells. Treatment with TLCK or iodoacetamide inhibits both the major morphological changes and the proteolytic events but these agents do not prevent the dissociation of microtubules or the activation of endogenous sphingomyelinase which occur in cells with raised levels of intracellular Ca²⁺. It is suggested that the sphingomyelinase is activated as a consequence of a Ca²⁺-induced loss of phospholipid asymmetry in the plasma membrane.     

Renal adenosine triphosphatase in hypothermically stored canine kidneys.

After 24-hr storage of canine kidneys with extracellular (Ringer's lactate) or intracellular (Ursol) solutions, the cortical and medullary renal ATPase enzymes (total (Na⁺ + K⁺)- and Mg-ATPase, (Na⁺ + K⁺)-ATPase, and Mg-ATPase) were examined. It was found that storage with extracellular solution decreased all cortical enzymes. This was not the case with intracellular solution or in kidneys cooled and stored without any solution. A decrease in the potassium concentration of the Ursol solution decreased also the cortical (Na⁺ + K⁺)-ATPase enzymatic activity. The medullary enzymatic changes were similar in the different groups and lower than in unstored controls.It appears that the changes on the level of the ATPase enzyme system, which is related to the cation transport system, might play a significant role in explaining the different results seen in clinical or experimental renal preservation systems. These changes can be related to the injury of preservation due to environmental effects of the cation concentrations and to a lesser degree to damage to the enzymatic system which provides the energy for the cation transport system.     

PROTONATION AND CHLOROPLAST MEMBRANE STRUCTURE

Light changes the structure of chloroplasts. This effect was investigated by high resolution electron microscopy, photometric methods, and chemical modification. (a) A reversible contraction of chloroplast membrane occurs upon illumination, dark titration with H⁺, or increasing osmolarity. These gross structural changes arise from a flattening of the thylakoids, with a corresponding decrease in the spacing between membranes. Microdensitometry showed that illumination or dark addition of H⁺ resulted in a 13–23% decrease in membrane thickness. Osmotically contracted chloroplasts do not show this effect. (b) Rapid glutaraldehyde fixation during actual experiments revealed that transmission changes are closely correlated with the spacing changes and therefore reflect an osmotic mechanism, whereas the light scattering changes have kinetics most similar to changes in membrane thickness or conformation. (c) Kinetic analysis of light scattering and transmission changes with the changes in fluorescence of anilinonaphthalene sulfonic acid bound to membranes revealed that fluorescence preceded light scattering or transmission changes. (d) It is concluded that the temporal sequence of events following illumination probably are protonation, changes in the environment within the membrane, change in membrane thickness, change in internal osmolarity accompanying ion movements with consequent collapse and flattening of thylakoid, change in the gross morphology of the inner chloroplast membrane system, and change in the gross morphology of whole chloroplasts.     

CQESTR Simulated Changes in Soil Organic Carbon under Residue Management Practices in Continuous Corn Systems

Soil organic carbon (SOC) is an important soil property and is strongly influenced by management. Changes in SOC stocks are difficult to measure through direct sampling, requiring both long time periods and intensive sampling to detect small changes in the large, highly variable pool. Models have the potential to predict management-induced changes in SOC stocks, but require long-term data sets for validation. CQESTR is a processed-based C model that uses site weather, management, and crop data to estimate changes in SOC stocks. Crop residue removal for livestock feed or future biofuel feedstock use is a management practice that potentially affects SOC stocks. Simulated changes in SOC using CQESTR were compared to measured SOC changes over 10 years for two contrasting residue removal studies in eastern Nebraska. The rainfed study compared SOC changes in no-tillage continuous corn grown under two N fertilizer rates (120 or 180 kg N ha^?1) and two residue removal rates (0 or 50 %). The irrigated study compared SOC changes in continuous corn grown under no-tillage or disk tillage and three residue removal rates (0, 35, or 70 %). After 10 years under these management scenarios, CQESTR-estimated SOC stocks agreed well with the measured SOC stocks at both sites (r ²?=?0.93 at the rainfed site and r ²?=?0.82 at the irrigated site). These results are consistent with other CQESTR validation studies and demonstrate that this process-based model can be a suitable tool for supporting current management and long-term planning decisions.     

Direct effects of angiotensin II on glomerular ultrastructure in the rainbow trout,Salmo gairdneri

Summary Slices from the kidneys of the rainbow trout which were exposed to 10^-6 or 10^-5 M angiotensin II (AII) and isolated glomeruli exposed to 10^-7 or 10^-5 M AII showed ultrastructural changes compared to control tissues incubated without AII. The studies indicate that angiotensin II has a direct action on glomerular ultrastructure, flattening the epithelial podocytes and broadening the primary processes with fusion of pedicels in extreme cases. These changes suggest a probable effect of AII on water permeability of the trout glomerulus, an intrarenal action which is believed to form an essential part of the antidiuretic adaptation to increased environmental salinities.     

Adaptational changes in the lipids and fatty acid profile of the cell and thylakoid membrane of rice plants exposed to sunlight

Adaptational changes occurring in the lipids and fatty acids of the cell and the thylakoid membrane in response to high light treatment, was studied in 30 days old rice (Oryza sativa L. cv. Jyothi) plants grown under low (150–200 μmol m⁻² s⁻¹) or moderate (600–800 μmol m⁻² s⁻¹) light conditions. Results were compared with rice plants grown in high (1200–2200 μmol m⁻² s⁻¹) light conditions. Exposure of rice plants and isolated chloroplast to high light, resulted in an increase in the amount of malonaldehyde, indicating oxidation of membrane lipids. Qualitative and quantitative changes in the phosphoglycolipids and quantitative changes in neutral lipids were observed in rice plants grown under the different growth conditions. A few of the phosphoglycolipids and neutral lipids were present exclusively in plants grown at low or moderate or high light, indicating requirement of different type of lipid composition of rice plants in response to their different growth irradiances. However, no significant quantitative changes were observed in the different saturated and unsaturated fatty acid groups of total lipids in low, moderate and high light grown rice plants, as a result of exposure to high light. No qualitative changes in the fatty acid composition due to difference in growth irradiance or high light treatment were seen. The changes observed in the phosphoglycolipids and neutral lipid composition of cell and thylakoid membrane of low, moderate and high light grown rice plants in response to high light, are probably the result of physiological changes in the rice plants, to sustain optimum structure and function of the cell and thylakoid membrane to maintain active physiological functions to endure high light conditions.     

Cation dependent conformations of brain CA2+-dependent regulator protein detected by nuclear magnetic resonance.

The 250MHz NMR spectrum of the brain Ca²⁺-dependent regulator protein was examined in the absence of cations and in the presence of Ca²⁺ or Mg²⁺. The Ca²⁺-saturated regulator protein and Mg²⁺-saturated regulator protein exhibited several spectral differences in the aromatic and aliphatic regions of their spectra. Certain spectral changes observed to occur upon addition of metal ions are qualitatively similar to those which have been observed in the spectrum of skeletal troponin-C. These results suggest that the large sequence homology between skeletal troponin-C and the regulator protein results in similar conformational changes due to the binding of Ca²⁺ or Mg²⁺.     

Longitudinal changes in δ13C of riffle macroinvertebrates from mountain to lowland sections of a gravel‐bed river

Summary 1. To understand longitudinal changes in the trophic base of benthic macroinvertebrates from mountain to lowland river sections, we investigated carbon stable isotopic compositions (δ¹³C) of macroinvertebrates and their food resources in riffles for four seasons at 14 sites along the main stem of the Toyo River, Japan. 2. At each site, δ¹³C was usually highest or nearly highest for periphyton (epilithic biofilm) and was lowest for transported leaf materials. Among macroinvertebrate groups, grazers usually had higher δ¹³C values than filterers or predators. 3. During all seasons, δ¹³C of periphyton and all macroinvertebrate groups increased downstream from mountain to upland sections, but decreased downstream from upland to lowland sections. In addition, the difference between grazer δ¹³C and filterer δ¹³C decreased from mountain to upland sections, but increased from upland to lowland sections. 4. The observed changes in δ¹³C of periphyton and macroinvertebrates from mountain to upland sections agree with previous reports: the δ¹³C of periphyton and consumers increased with stream size and productivity. The decrease in δ¹³C of periphyton and macroinvertebrates from upland to lowland sections has not been reported previously, and this may have resulted from an increased importance of terrestrial detritus relative to periphyton production in the lowland section, where riffles were infrequent and pools dominated the reach. 5. A simple mixing model of δ¹³C showed that grazers rely mostly on periphyton at all sites, whereas the importance of periphyton for filterers changed longitudinally increasing from mountain to upland sections and decreasing from upland to lowland sections. This longitudinal trend for filterers is possibly associated with the changes in the availability or quality of terrestrial detritus in transported particulate organic matter. 6. Longitudinal changes in the relative importance of autochthonous production and allochthonous detritus appear to be reflected in δ¹³C of riffle benthic communities. The longitudinal changes were not monotonic, and specific reach characteristics may be responsible for the greater importance of allochthonous detritus in mountain and lowland sections.     

Stress-Induced Grey Matter Loss Determined by MRI Is Primarily Due to Loss of Dendrites and Their Synapses

Stress, unaccompanied by signs of post-traumatic stress disorder, is known to decrease grey matter volume (GMV) in the anterior cingulate cortex (ACC) and hippocampus but not the amygdala in humans. We sought to determine if this was the case in stressed mice using high-resolution magnetic resonance imaging (MRI) and to identify the cellular constituents of the grey matter that quantitatively give rise to such changes. Stressed mice showed grey matter losses of 10 and 15 % in the ACC and hippocampus, respectively but not in the amygdala or the retrosplenial granular area (RSG). Concurrently, no changes in the number or volumes of the somas of neurons, astrocytes or oligodendrocytes were detected. A loss of synaptic spine density of up to 60 % occurred on different-order dendrites in the ACC and hippocampus (CA1) but not in the amygdala or RSG. The loss of spines was accompanied by decreases in cumulative dendritic length of neurons of over 40 % in the ACC and hippocampus (CA1) giving rise to decreases in volume of dendrites of 2.6 mm³ for the former and 0.6 mm³ for the latter, with no change in the amygdala or RSG. These values are similar to the MRI-determined loss of GMV following stress of 3.0 and 0.8 mm³ in ACC and hippocampus, respectively, with no changes in the amygdala or RSG. This quantitative study is the first to relate GMV changes in the cortex measured with MRI to volume changes in cellular constituents of the grey matter.     

Primary photochemical processes in isolated reaction centers of Rhodopseudomonas viridis

Picosecond and nanosecond spectroscopic techniques have been used to study the primary electron transfer processes in reaction centers isolated from the photosynthetic bacterium Rhodopseudomonas viridis. Following flash excitation, the first excited singlet state (P1) of the bacteriochlorophyll complex (P) transfers an electron to an intermediate acceptor (I) in less than 20 ps. The radical pair state (P⁺I^?) subsequently transfers an electron to another acceptor (X) in about 230 ps. There is an additional step of unknown significance exhibiting 35 ps kinetics. P⁺ subsequently extracts an electron from a cytochrome, with a time constant of about 270 ns. At low redox potential (X reduced before the flash), the state P⁺I^? (or P^F) lives approx. 15 ns. It decays, in part, into a longer lived state (P^R), which appears to be a triplet state. State P^R decays with an exponential time of approx. 55 μs. After continuous illumination at low redox potential (I and X both reduced), excitation with an 8-ps flash produces absorption changes reflecting the formation of the first excited singlet state, P1. Most of P1 then decays with a time constant of 20 ps. The spectra of the absorbance changes associated with the conversion of P to P1 or P⁺ support the view that P involves two or more interacting bacteriochlorophylls. The absorbance changes associated with the reduction of I to I^? suggest that I is a bacteriopheophytin interacting strongly with one or more bacteriochlorophylls in the reaction center.     

RAPID CHANGES IN NUCLEOSIDE TRANSPORT INDUCED BY GROWTH INHIBITORS : Studies with Neoplastic Mast Cells

Aqueous extracts of murine embryonic or uterine tissue, or [⁶N]O²'-dibutyryl 3',5'-adenosine monophosphate (dbc-AMP) which were cytostatic for the murine mastocytoma P815Y in vitro also induced rapid changes in the incorporation of exogenous nucleosides into acid-insoluble material. However, these alterations were not a consequence of growth arrest. Different dose-response curves were obtained for cytostasis and inhibition of [³H]-nucleoside incorporation, and changes in [³H]thymidine uptake were detected within 15 min of treatment with the inhibitors. Also, there were differential effects of each inhibitor on the incorporation of ³H-labeled thymidine, uridine, adenosine, or choline into acid-insoluble material.     

Development of (Na+-K+)-ATPase in rat cerebrum: correlation with Na+-dependent phosphorylation and K+-paranitrophenylphosphatase.

Abstract— The activities of (Na⁺ K⁺)-ATPase and its proposed partial reactions, K ⁺-pNPPase and Na ⁺-dependent phosphorylation, all increase tenfold relative to microsomal protein between 5 days prior to birth and 60 days postnatally in NaI-treated rat cerebral microsomes, and all reach half of their adult values between the fifth and tenth postnatal day. These increases are concurrent with the most rapid changes in cerebral wet weight. Increases in the amount of the related phosphorylatable polypeptide during development. as estimated by densitometry of Coomassie-stained polyacrylamide gels after electrophoresis of constant amounts of microsomal protein dissolved in sodium dodecylsulfate, parallel the increments in levels of Na ⁺-dependent phosphorylation. The fraction of total phosphorylation that is Na ⁺-dependent increases steadily during development. suggesting a precursor role for some of the Na ⁺-independent fraction. The results are consistent with a single biosynthetic control for the enzymatic sites critical to the partial reactions of (Na ⁺-K ⁺)-ATPase. No changes in turnover number or affinity for substrate or ligands were found during development. Little similarity was noted among the age-related changes of Mg ²⁺ -ATPase activity. Mg ²⁺ -paranitrophenylphosphatase activity, and Na⁺-independent phosphorylation levels. The most rapid changes in (Na⁺-K⁺)-ATPase take place during the period corresponding to glial proliferation and neuronal arborization.     

Effects of divalent cations and glucose on mitotic-like events in fused interphase-metaphase cells

The effects of Ca²⁺, Mg²⁺ and glucose on the mitotic-like events of prophasing and telophasing were studied in Sendai virus-fused interphase-metaphase (I-M) Chinese hamster binucleate cells. At normal extracellular ion concentrations and neutral pH, about 80–90% of I-M binucleates show prophasing (nuclear envelope dissolution and chromatin condensation) of the I nucleus and 10–15% show telophasing (nuclear envelope reformation and chromatin decondensation) of the M nucleus. To study the effects of cellular divalent cations, cells, depleted of about 77 % of exchangeable cell Ca²⁺ as determined by ⁴⁵Ca²⁺ studies, were incubated in different concentrations of Ca²⁺ or Mg²⁺ for 30 min prior to cell fusion. We found that relatively high concentrations of Ca²⁺ or Mg²⁺ (0.84 mM) were essential for prophasing and that in the presence of 10-fold less Ca²⁺ or Mg²⁺ (0.084 mM) the majority of binucleates showed telophasing. In contrast to a differential effect of divalent cations on the nuclear changes, we found that glucose metabolism was required for both prophasing and telophasing. Additionally, interruption of glucose metabolism in the M cell, but not in the I cell, prior to cell fusion depressed the prophasing frequency about 70%. Although we do not know how divalent cations and glucose function in prophasing and telophasing, we will discuss evidence which suggests that the effects are not mediated through secondary effects on membrane potential, by changes in intracellular concentrations of Na⁺ or K⁺, by simple osmotic changes, or through inhibition of protein synthesis.     

The effect of chloroplast coupling factor removal on thylakoid membrane ion permeability

Removal of coupling factor protein (CF₁) from spinach thylakoid membranes results in an enhancement of proton permeability but has no effect on chloride or potassium permeability. Anion permeability was measured by the rate of thylakoid packed volume changes. Potassium permeability was monitored by turbidity changes, packed thylakoid volume changes and ion flux studies using ⁸⁶Rb⁺ as a tracer. ⁴⁵Ca²⁺ was used to measure divalent cation fluxes. CF₁-depleted chloroplasts had an unaltered rate of Ca²⁺ uptake, but the rate of Ca²⁺ efflux appeared to be increased. Calcium efflux rates could also be increased by the addition of a proton specific uncoupler, FCCP.     

The membrane potential modulates thrombin-stimulated Ca mobilization and platelet aggregation

G protein-coupled receptors can be directly modulated by changes in transmembrane voltage in a variety of cell types. Here we show that, while changes in the membrane voltage itself do not induce detectable modifications in the cytosolic Ca²⁺ concentration, platelet stimulation with thrombin or the PAR-1 and PAR-4 agonist peptides SFLLRN and AYPGKF, respectively, results in Ca²⁺ release from intracellular stores that is sensitive to the membrane depolarisation. Direct activation of G proteins or phospholipase C by AlF₄⁻ and m-3M3FBS, respectively, leads to Ca²⁺ release that is insensitive to changes in the membrane potential. Thapsigargin-, as well as OAG-induced Ca²⁺ entry are affected by the membrane voltage, probably as a result of the modification in the driving force for Ca²⁺ influx; however, hyperpolarisation does not enhance thrombin- or OAG-evoked Ca²⁺ entry probably revealing the presence of a voltage-sensitive regulatory mechanism. Transmembrane voltage also modulates the activity of the plasma membrane Ca²⁺-ATPase (PMCA) most likely due to a decrease in the phosphotyrosine content of the pump. Thrombin-stimulated platelet aggregation is modulated by membrane depolarisation by a mechanism that is, at least partially, independent of Ca²⁺. These observations indicate that PAR-1 and PAR-4 receptors are modulated by the membrane voltage in human platelets.     

The influence of pH on the absorption spectrum of arsenazo III

The absorption spectrum of arsenazo III in media containing K⁺, Mg²⁺ and Ca²⁺ is sharply influenced by pH in the range of 7.5–5.0. The effect of pH is particularly pronounced in the wavelength range 532–602 nm due to the large pH dependence of the dissociation constant of Mg-arsenazo III complex. Therefore absorption changes at these wavelengths during muscle contraction cannot be used as reliable indicators of free ionized Ca²⁺ concentration in the cell. The effect of pH is less pronounced, but still noticeable at the wavelength pairs 575–650 or 660–685 nm.Multiple layers of muscle cells grown on polystyrene coils permit measurement of absorption changes of arsenazo III, introduced into the cells, by equilibration with 0.5 nM arsenazo III under routine culture conditions. The absorbance changes recorded at 660–685 nm are probably related to changes in intracellular free Ca²⁺ concentration.