K+-Selective Inward-Rectifying Channels and Apoplastic pH in Barley Roots

Recent structure-function analysis of heterologously expressed K⁺-selective inward-rectifying channels (KIRCs) from plants has revealed that external protons can have opposite effects on different members of the same gene family. An important question is how the diverse response of KIRCs to apoplastic pH is reflected at the tissue level. Activation of KIRCs by acid external pH is well documented for guard cells, but no other tissue has yet been studied. In this paper we present, for the first time to our knowledge, in planta characterization of the effects of apoplastic pH on KIRCs in roots. Patch-clamp experiments on protoplasts derived from barley (Hordeum vulgare) roots showed that a decrease in external pH shifted the half-activation potential to more positive voltages and increased the limit conductance. The resulting enhancement of the KIRC current, together with the characteristic voltage dependence, strongly relates the KIRC of barley root cells to AKT1-type as opposed to AKT3-type channels. Measurements of cell wall pH in barley roots with fluorescent dye revealed a bulk apoplastic pH close to the pK values of KIRC activation and significant acidification of the apoplast after the addition of fusicoccin. These results indicate that channel-mediated K⁺ uptake may be linked to development, growth, and stress responses of root cells via the activity of H⁺-translocating systems.     

Apoplastic pH and Fe(3+) reduction in intact sunflower leaves

It has been hypothesized that under NO(3)(-) nutrition a high apoplastic pH in leaves depresses Fe(3+) reductase activity and thus the subsequent Fe(2+) transport across the plasmalemma, inducing Fe chlorosis. The apoplastic pH in young green leaves of sunflower (Helianthus annuus L.) was measured by fluorescence ratio after xylem sap infiltration. It was shown that NO(3)(-) nutrition significantly increased apoplastic pH at distinct interveinal sites (pH >/= 6.3) and was confined to about 10% of the whole interveinal leaf apoplast. These apoplastic pH increases presumably derive from NO(3)(-)/proton cotransport and are supposed to be related to growing cells of a young leaf; they were not found in the case of sole NH(4)(+) or NH(4)NO(3) nutrition. Complementary to pH measurements, the formation of Fe(2+)-ferrozine from Fe(3+)-citrate was monitored in the xylem apoplast of intact leaves in the presence of buffers at different xylem apoplastic pH by means of image analysis. This analysis revealed that Fe(3+) reduction increased with decreasing apoplastic pH, with the highest rates at around pH 5. 0. In analogy to the monitoring of Fe(3+) reduction in the leaf xylem, we suggest that under alkaline nutritional conditions at interveinal microsites of increased apoplastic pH, Fe(3+) reduction is depressed, inducing leaf chlorosis. The apoplastic pH in the xylem vessels remained low in the still-green veins of leaves with intercostal chlorosis.     

Ca2+ Homeostasis in the Endoplasmic Reticulum: Coexistence of High and Low [Ca2+] Subcompartments in Intact HeLa Cells

Two recombinant aequorin isoforms with different Ca²⁺ affinities, specifically targeted to the endoplasmic reticulum (ER), were used in parallel to investigate free Ca²⁺ homeostasis in the lumen of this organelle. Here we show that, although identically and homogeneously distributed in the ER system, as revealed by both immunocytochemical and functional evidence, the two aequorins measured apparently very different concentrations of divalent cations ([Ca²⁺]_er or [Sr²⁺]_er). Our data demonstrate that this contradiction is due to the heterogeneity of the [Ca²⁺] of the aequorin-enclosing endomembrane system. Because of the characteristics of the calibration procedure used to convert aequorin luminescence into Ca²⁺ concentration, the [Ca²⁺]_er values obtained at steady state tend, in fact, to reflect not the average ER values, but those of one or more subcompartments with lower [Ca²⁺]. These subcompartments are not generated artefactually during the experiments, as revealed by the dynamic analysis of the ER structure in living cells carried out by means of an ER-targeted green fluorescent protein. When the problem of ER heterogeneity was taken into account (and when Sr²⁺ was used as a Ca²⁺ surrogate), the bulk of the organelle was shown to accumulate free [cation²⁺]_er up to a steady state in the millimolar range. A theoretical model, based on the existence of multiple ER subcompartments of high and low [Ca²⁺], that closely mimics the experimental data obtained in HeLa cells during accumulation of either Ca²⁺ or Sr²⁺, is presented. Moreover, a few other key problems concerning the ER Ca²⁺ homeostasis have been addressed with the following conclusions: (a) the changes induced in the ER subcompartments by receptor generation of InsP₃ vary depending on their initial [Ca²⁺]. In the bulk of the system there is a rapid release whereas in the small subcompartments with low [Ca²⁺] the cation is simultaneously accumulated; (b) stimulation of Ca²⁺ release by receptor-generated InsP₃ is inhibited when the lumenal level is below a threshold, suggesting a regulation by [cation²⁺]_er of the InsP₃ receptor activity (such a phenomenon had already been reported, however, but only in subcellular fractions analyzed in vitro); and (c) the maintenance of a relatively constant level of cytosolic [Ca²⁺], observed when the cells are incubated in Ca²⁺-free medium, depends on the continuous release of the cation from the ER, with ensuing activation in the plasma membrane of the channels thereby regulated (capacitative influx).     

Apoplastic pH and Ammonium Concentration in Leaves of Brassica napus L

A vacuum infiltration technique was developed that enabled the extraction of apoplastic solution with very little cytoplasmic contamination as evident from a malate dehydrogenase activity of less than 1% in the apoplastic solution relative to that in bulk leaf extracts. The volume of apoplastic water, a prerequisite for determination of the concentration of apoplastic solutes, was determined by vacuum infiltration of indigo carmine with subsequent analysis of the dilution of the dye in apoplastic extracts. Indigo carmine was neither transported across the cell membrane nor significantly adsorbed to the cell walls, ensuring reproducible (SE < 2%) and precise determination of apoplastic water. Analysis of leaves from four different positions on senescing Brassica napus plants showed a similar apoplastic pH of 5.8, while apoplastic NH4+ increased from 1.1 mM in lower leaves to 1.3 mM in upper leaves. Inhibition of glutamine synthetase in young B. napus plants resulted in increasing apoplastic pH from 6.0 to 6.8 and increasing apoplastic NH4+ concentration from 1.0 to 25.6 mM, followed by a marked increase in NH3 emission. Calculating NH3 compensation points for B. napus plants on the basis of measured apoplastic H+ and NH4+ concentrations gave values ranging from 4.3 to 5.9 nmol NH3 mol-1 air, consistent with an estimate of 5.3 [plus or minus] 3.6 nmol NH3 mol-1 air obtained by NH3 exchange experiments in growth chambers. A strong linear relationship was found between calculated NH3 compensation points and measured NH3 emission rates in glutamine synthetase-inhibited plants.     

Apoplastic Sugar Concentration and pH in Barley Leaves Infected with Brown Rust

Soluble sugars were extracted by low speed centrifugation fromthe apoplast of leaves of barley (Hordeum distichum L.) infiltratedwith water. Infection of the leaf with the brown rust fungus(Puccinia hordeii) resulted in a reduction in the concentrationof sucrose, glucose and fructose in the apoplast. Sugars werepresent in an apoplastic space occupying 12 and 17 cm³ m^–2of leaf area in healthy and infected tissue, respectively. Uptakeof hexoses by intercellular hyphae is suggested as a cause ofthis reduction. The pH of apoplastic sap extracted from rust-infectedleaves was increased to pH 7·3 from pH 6·6 incontrols. The effect of a reduced apoplastic sugar pool andincreased pH on export from infected leaves is discussed. Key words: Apoplast, barley (Hordeum distichum L.), brown rust (Puccinia hordeii Otth.), pH, sucrose, hexose     

Phosphatidylinositol 3-Kinase-mediated Endocytosis of Renal Na+,K+-ATPase α Subunit in Response to Dopamine

Dopamine (DA) inhibition of Na⁺,K⁺-ATPase in proximal tubule cells is associated with increased endocytosis of its α and β subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na⁺,K⁺-ATPase α subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na⁺,K⁺-ATPase α subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D₁ receptor subtype and the sequential activation of phospholipase A₂, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na⁺,K⁺-ATPase α subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.     

Polymorphism of Ca2+ Sparks Evoked from In-Focus Ca2+ Release Units in Cardiac Myocytes

Ca²⁺ sparks are the elementary release events in many types of cells. Here we present a morphometric analysis of Ca²⁺ sparks (i.e., amplitude and kinetic parameters) using an approach that minimizes the confounding factor of the detection of out-of-focus events. By activation and visualization of Ca²⁺ sparks from Ca²⁺ release units under loose-seal patch-clamp conditions, we found that the amplitude and rising rate of in-focus sparks exhibited a broad modal distribution, whereas spark rise time and spatial width appeared to be stereotyped. Spark morphometrics were constant irrespective of the latency of spark production and the time-dependent L-type Ca²⁺ channel activation. Polymorphism of Ca²⁺ sparks in terms of variable amplitude and rising rate was evident for events from the same release units, and intra- and interrelease unit variability contributed equally to the overall variability. The rising rate, a reporter of the underlying Ca²⁺ release flux, displayed a strong positive correlation with spark amplitude, but a negative correlation with spark rise time, an index of Ca²⁺ release duration. On the basis of Ca²⁺ spark morphometrics measured here, we suggested a model in which cohorts of variable number of ryanodine receptors are activated in the genesis of Ca²⁺ sparks, and the ensuing negative feedback overrides the regenerative Ca²⁺-induced Ca²⁺ release to extinguish the ongoing Ca²⁺ spark.     

Modulation of the Local SR Ca2+ Release by Intracellular Mg2+ in Cardiac Myocytes

In cardiac muscle, Ca²⁺-induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum (SR) defines the amplitude and time course of the Ca²⁺ transient. The global elevation of the intracellular Ca²⁺ concentration arises from the spatial and temporal summation of elementary Ca²⁺ release events, Ca²⁺ sparks. Ca²⁺ sparks represent the concerted opening of a group of ryanodine receptors (RYRs), which are under the control of several modulatory proteins and diffusible cytoplasmic factors (e.g., Ca²⁺, Mg²⁺, and ATP). Here, we examined by which mechanism the free intracellular Mg²⁺ ([Mg²⁺]_free) affects various Ca²⁺ spark parameters in permeabilized mouse ventricular myocytes, such as spark frequency, duration, rise time, and full width, at half magnitude and half maximal duration. Varying the levels of free ATP and Mg²⁺ in specifically designed solutions allowed us to separate the inhibition of RYRs by Mg²⁺ from the possible activation by ATP and Mg²⁺-ATP via the adenine binding site of the channel. Changes in [Mg²⁺]_free generally led to biphasic alterations of the Ca²⁺ spark frequency. For example, lowering [Mg²⁺]_free resulted in an abrupt increase of spark frequency, which slowly recovered toward the initial level, presumably as a result of SR Ca²⁺ depletion. Fitting the Ca²⁺ spark inhibition by [Mg²⁺]_free with a Hill equation revealed a K_i of 0.1 mM. In conclusion, our results support the notion that local Ca²⁺ release and Ca²⁺ sparks are modulated by Mg²⁺ in the intracellular environment. This seems to occur predominantly by hindering Ca²⁺-dependent activation of the RYRs through competitive Mg²⁺ occupancy of the high-affinity activation site of the channels. These findings help to characterize CICR in cardiac muscle under normal and pathological conditions, where the levels of Mg²⁺ and ATP can change.     

pkl1+and klp2+: Two Kinesins of the Kar3 Subfamily in Fission Yeast Perform Different Functions in Both Mitosis and Meiosis

We have identified Klp2p, a new kinesin-like protein (KLP) of the KAR3 subfamily in fission yeast. The motor domain of this protein is 61% identical and 71% similar to Pkl1p, another fission yeast KAR3 protein, yet the two enzymes are different in behavior and function. Pkl1p is nuclear throughout the cell cycle, whereas Klp2p is cytoplasmic during interphase. During mitosis Klp2p enters the nucleus where it forms about six chromatin-associated dots. In metaphase-arrested cells these migrate back and forth across the nucleus. During early anaphase they segregate with the chromosomes into two sets of about three, fade, and are replaced by other dots that form on the spindle interzone. Neither klp2(+) nor pkl1(+) is essential, and the double deletion is also wild type for both vegetative and sexual reproduction. Each deletion rescues different alleles of cut7(ts), a KLP that contributes to spindle formation and elongation. When either or both deletions are combined with a dynein deletion, vegetative growth is normal, but sexual reproduction fails: klp2 Delta,dhc1-d1 in karyogamy, pkl1 Delta,dhc1-d1 in multiple phases of meiosis, and the triple deletion in both. Deletion of Klp2p elongates a metaphase-arrested spindle, but pkl1 Delta shortens it. The anaphase spindle of klp2 Delta becomes longer than the cell, leading it to curl around the cell's ends. Apparently, Klp2p promotes spindle disassembly and contributes to the behavior of mitotic chromosomes.     

Structural classification of αββ and ββα supersecondary structure units in proteins

We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α–β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α–β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193–212, 1998. © 1998 Wiley-Liss, Inc.     

Ni2+ Transport and Accumulation in Rhodospirillum rubrum

The cooCTJ gene products are coexpressed with CO-dehydrogenase (CODH) and facilitate in vivo nickel insertion into CODH. A Ni(2+) transport assay was used to monitor uptake and accumulation of (63)Ni(2+) into R. rubrum and to observe the effect of mutations in the cooC, cooT, and cooJ genes on (63)Ni(2+) transport and accumulation. Cells grown either in the presence or absence of CO transported Ni(2+) with a K(m) of 19 +/- 4 microM and a V(max) of 310 +/- 22 pmol of Ni/min/mg of total protein. Insertional mutations disrupting the reading frame of the cooCTJ genes, either individually or all three genes simultaneously, transported Ni(2+) the same as wild-type cells. The nickel specificity for transport was tested by conducting the transport assay in the presence of other divalent metal ions. At a 17-fold excess Mn(2+), Mg(2+), Ca(2+), and Zn(2+) showed no inhibition of (63)Ni(2+) transport but Co(2+), Cd(2+), and Cu(2+) inhibited transport 35, 58, and 66%, respectively. Nickel transport was inhibited by cold (50% at 4 degrees C), by protonophores (carbonyl cyanide m-chlorophenylhydrazone, 44%, and 2,4-dinitrophenol, 26%), by sodium azide (25%), and hydroxyl amine (33%). Inhibitors of ATP synthase (N, N'-dicyclohexylcarbodiimide and oligomycin) and incubation of cells in the dark stimulated Ni(2+) transport. (63)Ni accumulation after 2 h was four times greater in CO-induced cells than in cells not exposed to CO. The CO-stimulated (63)Ni(2+) accumulation coincided with the appearance of CODH activity in the culture, suggesting that the (63)Ni(2+) was accumulating in CODH. The cooC, cooT, and cooJ genes are required for the increased (63)Ni(2+) accumulation observed upon CO exposure because cells containing mutations disrupting any or all of these genes accumulated (63)Ni(2+) like cells unexposed to CO.     

Regulation of Apoplastic pH in Source Leaves of Vicia faba by Gibberellic Acid

Leaf discs of broad bean (Vicia faba L.), peeled on the spongy mesophyll side, rapidly altered the pH of the surrounding medium (apoplast). Using pH indicator paper appressed against the leaf, immediately after peeling, initial apoplastic pH was estimated to be 4.5. Changes in the apoplastic pH were measured with a microelectrode placed into a 100-microliter drop of an unbuffered solution (2 millimolar KCl, 0.5 millimolar CaCl₂, and 200 millimolar mannitol) on the peeled surface. Discs acidified the medium until the pH stabilized at about 5.0 (about 10 minutes). Acidification was inhibited by 50 micromolar sodium vanadate, an inhibitor of the plasmalemma H⁺-ATPase and attenuated by omitting the osmoticum or potassium ions from the medium. Fusicoccin (10 micromolar) greatly enhanced the rate of acidification. The presence of 0.1 to 1 micromolar gibberellic acid resulted in a slower rate of medium acidification. Gibberellic acid appeared to modulate the activity of the H⁺-translocating ATPase located at the plasma membrane of the mesophyll cells.     

CD4+-T-Cell and CD20+-B-Cell Changes Predict Rapid Disease Progression after Simian-Human Immunodeficiency Virus Infection in Macaques

Simian-human immunodeficiency virus 89.6PD (SHIV_89.6PD) was pathogenic after intrarectal inoculation of rhesus macaques. Infection was achieved with a minimum of 2,500 tissue culture infectious doses of cell-free virus stock, and there was no evidence for transient viremia in animals receiving subinfectious doses by the intrarectal route. Some animals experienced rapid progression of disease characterized by loss of greater than 90% of circulating CD4⁺ T cells, sustained decreases in CD20⁺ B cells, failure to elicit virus-binding antibodies in plasma, and high levels of antigenemia. Slower-progressing animals had moderate but varying losses of CD4⁺ T cells; showed increases in circulating CD20⁺ B cells; mounted vigorous responses to antibodies in plasma, including neutralizing antibodies; and had low or undetectable levels of antigenemia. Rapid progression led to death within 30 weeks after intrarectal inoculation. Plasma antigenemia at 2 weeks after inoculation (P ≤ 0.002), B- and T-cell losses (P ≤ 0.013), and failure to seroconvert (P ≤ 0.005) were correlated statistically with rapid progression. Correlations were evident by 2 to 4 weeks after intrarectal SHIV inoculation, indicating that early events in the host-pathogen interaction determined the clinical outcome.