Phy Signaling Research Articles

Production of tensor glueball in ultraperipheral pp, AuAu and PbPb collisions

In this work, we discuss the production of a tensor glueball in the proton–proton and nuclei–nuclei ultraperipheral collisions (UPCs). The cross section is calculated in the equivalent photon approximation (EPA). The total UPCs cross section is related to the matrix elements of gamma gamma rightarrow pi ^0 G_2 process, which can be described in terms of the neutral pion and tensor glueball G_2’s distribution amplitudes. The predictions for the rapidity distributions of UPCs cross sections and total cross sections are presented. The theoretical uncertainty of the whole calculation is considered. We consider the decay modes of the final-state tensor glueball and estimate the corresponding number of events. We estimate that there are about 10 tensor glueball G_2rightarrow phi phi signal events in PbPb collisions at the LHC. We also argue that the lead–lead UPC experiments at future HE-LHC and FCC experiments will provide larger opportunities to measure tensor glueballs.

Plant Phytochrome B Assembles an Asymmetric Dimer with Unique Signaling Potential

Phytochromes (Phy) sense red and far‐red light and reversibly interconvert between inactive Pr and active Pfr conformers. Phys typically consist of a photo sensory module (PSM) formed by sequential N‐terminal PAS, GAF, and PHY domains, followed by a C‐terminal histidine kinase‐related domain (HKRD). There are four plant isoforms Phy A through D, and they all contain two additional PAS domains (PAS1 and PAS2) inserted between PSM and HKRD. Despite extensive biochemical and structural studies, there has been a lack of a full‐length structure, hindering our understanding of plant Phy signaling. Here we report a 3.3‐Å cryo‐EM structure of Arabidopsis PhyB dimer. Instead of the widely anticipated parallel association of the two monomers resembling the prokaryotic relatives, Plant PhyB adopts a topologically complex dimeric architecture with the two PSMs in a head‐to‐tail antiparallel form while the two HKRDs in a head‐to‐head parallel form. The PAS2 mediates the antiparallel dimerization of PSM that leads to a parallelogram‐shaped platform. And a preceding ‘modulator’ loop assembles tightly with the PSM of its own protomer. We show that these inter‐ and intra‐protomer connections contribute to stabilizing of dimer and accelerate thermal reversion of Pfr back to Pr. Lean of HKRDs towards the platform results in profound asymmetry of PhyB that might enable distinct signaling potentials to the protomers. We propose that this unique structural dynamism creates an extensive photostate‐sensitive surface for interaction with their downstream signaling partners.

Crystal structure of the photosensing module from a red/far-red light-absorbing plant phytochrome.

Many aspects of plant photomorphogenesis are controlled by the phytochrome (Phy) family of bilin-containing photoreceptors that detect red and far-red light by photointerconversion between a dark-adapted Pr state and a photoactivated Pfr state. Whereas 3D models of prokaryotic Phys are available, models of their plant counterparts have remained elusive. Here, we present the crystal structure of the photosensing module (PSM) from a seed plant Phy in the Pr state using the PhyB isoform from Arabidopsis thaliana. The PhyB PSM crystallized as a head-to-head dimer with strong structural homology to its bacterial relatives, including a 5(Z)syn, 10(Z)syn, 15(Z)anti configuration of the phytochromobilin chromophore buried within the cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) domain, and a well-ordered hairpin protruding from the Phy-specific domain toward the bilin pocket. However, its Per/Arnt/Sim (PAS) domain, knot region, and helical spine show distinct structural differences potentially important to signaling. Included is an elongated helical spine, an extended β-sheet connecting the GAF domain and hairpin stem, and unique interactions between the region upstream of the PAS domain knot and the bilin A and B pyrrole rings. Comparisons of this structure with those from bacterial Phys combined with mutagenic studies support a toggle model for photoconversion that engages multiple features within the PSM to stabilize the Pr and Pfr end states after rotation of the D pyrrole ring. Taken together, this Arabidopsis PhyB structure should enable molecular insights into plant Phy signaling and provide an essential scaffold to redesign their activities for agricultural benefit and as optogenetic reagents.

Seed Germination in Tomato: A Focus on Interaction between Phytochromes and Gibberellins or Abscisic Acid

Separately, it is well-documented that phytochromes (phys), gibberellin (GA) and abscisic acid (ABA) strongly control the seed germination in tomato. However, we hipothesized that phys interact with GA or ABA during this response. Thus, we make an analysis of seed germination of ABA deficient (sit), GA constitutive response (pro), phytochrome deficient (au) mutants as well as, specially, au sit and au pro double mutants of tomato incubated in the dark or light conditions during 120 h [12 h intervals (i)]. Compared to au, which severely reduced percentage germination (Gi%) and pro, which did not alter Gi%, au pro showed in the light enhanced Gi% and germination speed index (GSI) besides the reduced average germination time (AGT). Moreover, in the dark, germination of au pro was similar to pro. These results indicate that the mechanisms by which GA modulate germination in tomato are light dependent through the phy signaling, whereas intermediary values of Gi%, GSI and AGT in dark and light of au sit compared to au and sit single mutants indicate an additive effect of the au and sit mutations, suggesting that ABA and phy may act through the parallel signaling pathway.

Structure-Guided Engineering of Plant Phytochrome B with Altered Photochemistry and Light Signaling

Phytochromes (phys) encompass a diverse collection of biliproteins that enable cellular light perception by photoconverting between a red-light-absorbing ground state (Pr) and a far-red light-absorbing active state (Pfr). Based on the central role of plant phys in controlling numerous agriculturally important processes, their rational redesign offers great promise toward accelerating crop improvement. Employing as templates the available three-dimensional models of the photosensory module within bacterial phys, we report here our initial attempt to apply structure-guided mutagenesis to phy engineering using Arabidopsis (Arabidopsis thaliana) phyB, the dominant isoform in light-grown plants, as the example. A collection of phyB mutants was generated affecting the bilin-binding pocket that altered photochemistry, thermal stability, and/or nuclear localization patterns, some of which also impacted phenotypic outputs. Of particular interest are the Y361F substitution, which created Arabidopsis plants with greatly enhanced light sensitivity, mutants variably altered in Pfr-to-Pr thermal reversion and nuclear aggregation, and the D307A substitution, which failed to photoconvert from Pr to Pfr and display light-induced nuclear aggregation but retained some biological activity and accelerated turnover in red light. Taken together, this collection provides variants potentially useful to agriculture as well as new tools to better understand the molecular mechanisms underpinning phy signaling.

Phosphorylation by CK2 Enhances the Rapid Light-induced Degradation of Phytochrome Interacting Factor 1 in Arabidopsis

The phytochrome family of sensory photoreceptors interacts with phytochrome interacting factors (PIFs), repressors of photomorphogenesis, in response to environmental light signals and induces rapid phosphorylation and degradation of PIFs to promote photomorphogenesis. However, the kinase that phosphorylates PIFs is still unknown. Here we show that CK2 directly phosphorylates PIF1 at multiple sites. α1 and α2 subunits individually phosphorylated PIF1 weakly in vitro. However, each of four β subunits strongly stimulated phosphorylation of PIF1 by α1 or α2. Mapping of the phosphorylation sites identified seven Ser/Thr residues scattered throughout PIF1. Ser/Thr to Ala scanning mutations at all seven sites eliminated CK2-mediated phosphorylation of PIF1 in vitro. Moreover, the rate of degradation of the Ser/Thr to Ala mutant PIF1 was significantly reduced compared with wild-type PIF1 in transgenic plants. In addition, hypocotyl lengths of the mutant PIF1 transgenic plants were much longer than the wild-type PIF1 transgenic plants under light, suggesting that the mutant PIF1 is suppressing photomorphogenesis. Taken together, these data suggest that CK2-mediated phosphorylation enhances the light-induced degradation of PIF1 to promote photomorphogenesis.

Unique phytochrome responses of the holoparasitic plant Orobanche minor

Holoparasitic plants such as Orobanche spp. have lost their photosynthetic ability, so photoresponses to optimize photosynthesis are not necessary in these plants. Photoresponses are also involved in the regulation of plant development but the photoresponses of holoparasites have not been characterized in detail. In this study, the phytochrome (phy)-related photoresponse of Orobanche minor was investigated. Its photoreceptor, phytochrome A (OmphyA), was also characterized. Light effects on germination, shoot elongation, anthocyanin biosynthesis, and OmphyA expression and subcellular localization were analyzed. Red light (R):far-red light (FR) reversible inhibition of O. minor seed germination demonstrated that phy-mediated responses are retained in this holoparasite. Shoot elongation was inhibited by FR but not by R. This pattern is unique among known patterns of plant photoresponses. Additionally, molecular analysis showed that OmphyA is able to respond to the light signals. Interestingly, the unique pattern of photoresponses in O. minor seems to have been modified for adaptation to its parasitic life cycle. We hypothesize that this alteration has resulted from the loss or alteration of some phy-signaling components. Elucidation of altered components in phy signaling in this parasite will provide useful information not only about its physiological characteristics but also about general plant photoreception systems.

Phytochrome‐regulated Gene Expression

AbstractIdentification of all genes involved in the phytochrome (phy)‐mediated responses of plants to their light environment is an important goal in providing an overall understanding of light‐regulated growth and development. This article highlights and integrates the central findings of two recent comprehensive studies in Arabidopsis that have identified the genome‐wide set of phy‐regulated genes that respond rapidly to red‐light signals upon first exposure of dark‐grown seedlings, and have tested the functional relevance to normal seedling photomorphogenesis of an initial subset of these genes. The data: (a) reveal considerable complexity in the channeling of the light signals through the different phy‐family members (phyA to phyE) to responsive genes; (b) identify a diversity of transcription‐factor‐encoding genes as major early, if not primary, targets of phy signaling, and, therefore, as potentially important regulators in the transcriptional‐network hierarchy; and (c) identify auxin‐related genes as the dominant class among rapidly‐regulated, hormone‐related genes. However, reverse‐genetic functional profiling of a selected subset of these genes reveals that only a limited fraction are necessary for optimal phy‐induced seedling deetiolation.

PhyA dominates in transduction of red‐light signals to rapidly responding genes at the initiation of Arabidopsis seedling de‐etiolation

Contrary to expectations based on the visible phenotypic behavior of seedlings undergoing de-etiolation in response to continuous red light (Rc), previous gene expression profiling showed that one or more of the five-membered phytochrome (phy) family of Arabidopsis, other than phyB, is predominantly responsible for transducing the Rc signals to light-responsive genes. To begin to identify which phys are involved, and to define potential primary targets of phy signaling, we have examined the genome-wide expression profiles of genes responding to Rc within 1 h (early response genes) of initial exposure of dark-grown wild-type, phyA, phyB and phyAphyB double mutant seedlings to the light signal. The data show that phyA has a quantitatively dominant role in Rc-induced expression of these early response genes, that phyB has minimal detectable regulatory activity in the presence of phyA, but assumes a quantitatively larger role in its absence, and that phyA and phyB combined are responsible for the full extent of Rc responsiveness of 96% of these genes. No evidence was obtained of a significant role for the remaining family members, phyC, phyD or phyE, in this process. In striking contrast, Rc-imposed repression of early response gene expression remains quantitatively strong in the phyAphyB double mutant, as well as the monogenic mutants, suggesting a significant role for one or more of the other three phys in this response. Examination of the established or predicted functional roles of the early response genes indicates that genes encoding transcription factors represent the largest single category, at a frequency three times their prevalence genome-wide. This dominance is particularly striking among those genes responding most robustly to the Rc signal, where >50% are classified as involved in transcriptional regulation, suggesting that these may have potentially primary regulatory roles at the interface between phy signaling and the light-responsive transcriptional network. Integration of the present data with those of a previous genome-scale transcriptional analysis of a pif3 mutant, suggests a complex network involving perception and transduction of inductive Rc signals by both phyA and phyB through both PIF3 and other undefined signaling partners to early response genes.

Functional Profiling Reveals That Only a Small Number of Phytochrome-Regulated Early-Response Genes inArabidopsisAre Necessary for Optimal Deetiolation

In previous time-resolved microarray-based expression profiling, we identified 32 genes encoding putative transcription factors, signaling components, and unknown proteins that are rapidly and robustly induced by phytochrome (phy)-mediated light signals. Postulating that they are the most likely to be direct targets of phy signaling and to function in the primary phy regulatory circuitry, we examined the impact of targeted mutations in these genes on the phy-induced seedling deetiolation process in Arabidopsis thaliana. Using light-imposed concomitant inhibition of hypocotyl and stimulation of cotyledon growth as diagnostic criteria for normal deetiolation, we identified three major mutant response categories. Seven (22%) lines displayed statistically significant, reciprocal, aberrant photoresponsiveness in the two organs, suggesting disruption of normal deetiolation; 13 (41%) lines displayed significant defects either unidirectionally in both organs or in hypocotyls only, suggesting global effects not directly related to photomorphogenic signaling; and 12 (37%) lines displayed no significant difference in photoresponsiveness from the wild type. Potential reasons for the high proportion of rapidly light-responsive genes apparently unnecessary for the deetiolation phenotype are discussed. One of the seven disrupted genes displaying a significant mutant phenotype, the basic helix-loop-helix factor-encoding PHYTOCHROME-INTERACTING FACTOR3-LIKE1 gene, was found to be necessary for rapid light-induced expression of the photomorphogenesis- and circadian-related PSEUDO-RESPONSE REGULATOR9 gene, indicating a regulatory function in the early phy-induced transcriptional network.

Identification of Primary Target Genes of Phytochrome Signaling. Early Transcriptional Control during Shade Avoidance Responses in Arabidopsis

The phytochrome (phy) photoreceptors modulate plant development after perception of light. Upon illumination of etiolated seedlings, phys initiate a transcriptional cascade by directly transducing light signals to the promoters of genes encoding regulators of morphogenesis. In light-grown plants, however, little is known about the transcriptional cascade modulated by phys in response to changes in light. The phy entry points in this cascade are completely unknown. We are particularly interested in the shade avoidance syndrome (SAS). Here we describe a subset of six genes whose expression is rapidly modulated by phys during both deetiolation and SAS in Arabidopsis (Arabidopsis thaliana). Using cycloheximide, we provide evidence that four of these phy rapidly regulated (PAR) genes are direct targets of phy signaling during SAS, revealing these genes as upstream components of the transcriptional cascade. Promoter-beta-glucuronidase fusions confirmed that PAR genes are photoregulated at the transcriptional level. Analysis of gene expression in light signal transduction mutants showed that COP1 and DET1 (but not DET2 or HY5) play a role in modulating PAR expression in response to shade in light-grown seedlings. Moreover, genetic analyses showed that one of the genes identified as a direct target of phy signaling was phy-interacting factor 3-like-1 (PIL1). PIL1 has previously been implicated in SAS in response to transient shade, but we show here that it also plays a key role in response to long-term shade. The action of PIL1 was particularly apparent in a phyB background, suggesting an important negative role for PIL1 under dense vegetation canopies.

The phytochrome-interacting transcription factor, PIF3, acts early, selectively, and positively in light-induced chloroplast development

The phytochrome (phy) family of sensory photoreceptors transduce informational light signals to selected nuclear genes, inducing plant growth and developmental responses appropriate to the environment. Existing data suggest that one signaling pathway by which this occurs involves direct, intranuclear interaction of the photoactivated phy molecule with PIF3, a basic helix-loop-helix transcription factor. Here, we provide evidence from recently identified pif3 mutant alleles that PIF3 is necessary for early chloroplast greening and rapid phy-induced expression of nuclear genes encoding chloroplast components upon first exposure of seedlings to light. Therefore, these data indicate that PIF3 functions to transduce phy signals to genes involved in a critical facet of the early seedling deetiolation process, the generation of a functional photosynthetic apparatus. When transgenically expressed GUS:PIF3 fusion protein constructs were used, we found that PIF3 protein levels are rapidly and reversibly modulated by the photoreceptor over diurnal cycles in Arabidopsis seedlings. The PIF3 protein declines rapidly to a basal steady-state level upon initial light exposure, but reaccumulates to preirradiation levels in darkness during the subsequent night period. These data suggest that PIF3 may function in early phy signaling at the dark-to-light transition, not only during initial seedling deetiolation, but daily at dawn under diurnal light-dark cycles.

Revisiting the role of H+ in chemotactic signaling of sperm.

Chemotaxis of sperm is an important step toward fertilization. During chemotaxis, sperm change their swimming behavior in a gradient of the chemoattractant that is released by the eggs, and finally sperm accumulate near the eggs. A well established model to study chemotaxis is the sea urchin Arbacia punctulata. Resact, the chemoattractant of Arbacia, is a peptide that binds to a receptor guanylyl cyclase. The signaling pathway underlying chemotaxis is still poorly understood. Stimulation of sperm with resact induces a variety of cellular events, including a rise in intracellular pH (pHi) and an influx of Ca2+; the Ca2+ entry is essential for the chemotactic behavior. Previous studies proposed that the influx of Ca2+ is initiated by the rise in pHi. According to this proposal, a cGMP-induced hyperpolarization activates a voltage-dependent Na+/H+ exchanger that expels H+ from the cell. Because some aspects of the proposed signaling pathway are inconsistent with recent results (Kaupp, U.B., J. Solzin, J.E. Brown, A. Helbig, V. Hagen, M. Beyermann, E. Hildebrand, and I. Weyand. 2003. Nat. Cell Biol. 5:109–117), we reexamined the role of protons in chemotaxis of sperm using kinetic measurements of the changes in pHi and intracellular Ca2+ concentration. We show that for physiological concentrations of resact (<25 pM), the influx of Ca2+ precedes the rise in pHi. Moreover, buffering of pHi completely abolishes the resact-induced pHi signal, but leaves the Ca2+ signal and the chemotactic motor response unaffected. We conclude that an elevation of pHi is required neither to open Ca2+-permeable channels nor to control the chemotactic behavior. Intracellular release of cGMP from a caged compound does not cause an increase in pHi, indicating that the rise in pHi is induced by cellular events unrelated to cGMP itself, but probably triggered by the consumption and subsequent replenishment of GTP. These results show that the resact-induced rise in pHi is not an obligatory step in sperm chemotactic signaling. A rise in pHi is also not required for peptide-induced Ca2+ entry into sperm of the sea urchin Strongylocentrotus purpuratus. Speract, a peptide of S. purpuratus may act as a chemoattractant as well or may serve functions other than chemotaxis.

Arabidopsis PSEUDO-RESPONSE REGULATOR7 is a signaling intermediate in phytochrome-regulated seedling deetiolation and phasing of the circadian clock.

To identify new components in the phytochrome (phy) signaling network in Arabidopsis, we used a sensitized genetic screen for deetiolation-defective seedlings. Two allelic mutants were isolated that exhibited reduced sensitivity to both continuous red and far-red light, suggesting involvement in both phyA and phyB signaling. The molecular lesions responsible for the phenotype were shown to be mutations in the Arabidopsis PSEUDO-RESPONSE REGULATOR7 (PRR7) gene. PRR7 is a member of a small gene family in Arabidopsis previously suggested to be involved in circadian rhythms. A PRR7-beta-glucuronidase fusion protein localized to the nucleus, implying a possible function in the regulation of photoresponsive gene expression. Consistent with this suggestion, prr7 seedlings were partially defective in the regulation of the rapidly light-induced genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), observable as a premature increase in expression level during the second peak of the biphasic induction profile that is elicited upon initial exposure of dark-grown seedlings to light. A similar 3- to 6-h coordinated advance in peak free-running expression of CCA1, LHY, and TIMING-OF-CAB1, which are considered to encode the molecular components of the circadian oscillator in Arabidopsis, was observed in entrained fully green prr7 seedlings compared with wild-type seedlings. Collectively, these data suggest that PRR7 functions as a signaling intermediate in the phytochrome-regulated gene expression responsible for both seedling deetiolation and phasing of the circadian clock in response to light.

GIGANTEA is a nuclear protein involved in phytochrome signaling in Arabidopsis.

In a genetic screen of available T-DNA-mutagenized Arabidopsis populations for loci potentially involved in phytochrome (phy) signaling, we identified a mutant that displayed reduced seedling deetiolation under continuous red light, but little if any change in responsiveness to continuous far-red light. This behavior suggests disruption of phyB, but not phyA signaling. We have cloned the mutant locus by using the T-DNA insertion and found that the disrupted gene is identical to the recently described GIGANTEA (GI) gene identified as being involved in control of flowering time. The encoded GI polypeptide has no sequence similarity to any known proteins in the database. However, by using beta-glucuronidase-GI and green fluorescent protein-GI fusion constructs, we have shown that GI is constitutively targeted to the nucleus in transient transfection assays. Optical sectioning by using the green fluorescent protein-GI fusion protein showed green fluorescence throughout the nucleoplasm. Thus, contrary to previous computer-based predictions that GI would be an integral plasma membrane-localized polypeptide, the data here indicate that it is a nucleoplasmically localized protein. This result is consistent with the proposed role in phyB signaling, given recent evidence that early phy signaling events are nuclear localized.

Sensitivity to abscisic acid of guard-cell K+ channels is suppressed by abi1-1, a mutant Arabidopsis gene encoding a putative protein phosphatase.

Abscisic acid (ABA) modulates the activities of three major classes of ion channels--inward- and outward-rectifying K+ channels (IK,in and IK,out, respectively) and anion channels--at the guard-cell plasma membrane to achieve a net efflux of osmotica and stomatal closure. Disruption of ABA sensitivity in wilty abi1-1 mutants of Arabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein (de)-phosphorylation contributes to guard-cell transport control by ABA. To pinpoint the role of ABI1, the abi1-1 dominant mutant allele was stably transformed into Nicotiana benthamiana and its influence on IK,in, IK,out, and the anion channels was monitored in guard cells under voltage clamp. Compared with guard cells from wild-type and vector-transformed control plants, expression of the abi1-1 gene was associated with 2- to 6-fold reductions in IK,out and an insensitivity of both IK,in and IK,out to 20 microM ABA. In contrast, no differences between control and abi1-1 transgenic plants were observed in the anion current or its response to ABA. Parallel measurements of intracellular pH (pHi) using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) in every case showed a 0.15- to 0.2-pH-unit alkalinization in ABA, demonstrating that the transgene was without effect on the pHi signal that mediates in ABA-evoked K+ channel control. In guard cells from the abi1-1 transformants, normal sensitivity of both K+ channels to and stomatal closure in ABA was recovered in the presence of 100 microM H7 and 0.5 microM staurosporine, both broad-range protein kinase antagonists. These results demonstrate an aberrant K+ channel behavior--including channel insensitivity to ABA-dependent alkalinization of pHi--as a major consequence of abi1-1 action and implicate AB11 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell K+ channels to ABA-evoked signal cascades.

OXIDANTS AUGMENT ENDOTOXIN INDUCED ACTIVATION OF ALVEOLAR MACROPHAGES

Endotoxin (lipopolysaccharide) stimulation of macrophages (M phi) induces the generation of toxic reactive oxygen intermediates (ROI); however, recent studies implicate intracellular redox changes in signal transduction pathways for cytokines. To test whether oxidant stress modulates M phi activation, rabbit alveolar M phi were exposed to the following: diamide (oxidizes intracellular glutathione); glucose oxidase (generates hydrogen peroxide); or xanthine oxidase (generates superoxide), before lipopolysaccharide. Supernatants were assayed for tumor necrosis factor (TNF) and cell lysates were assayed for procoagulant activity (PCA). TNF mRNA was analyzed by Northern blot. M phi exposure to diamide and glucose oxidase augmented TNF production, PCA expression, and TNF mRNA accumulation; however, xanthine oxidase exposure inhibited TNF production while augmenting PCA expression. M phi signal transduction can be enhanced by increasing cellular oxidant stress. The differential response of TNF versus PCA suggests the existence of distinct redox-sensitive signal transduction pathways. These data define a mechanism by which oxidants generated during inflammation may modulate M phi function.

Effect of changes in extracellular potassium on intracellular pH in principal cells of frog skin.

Intracellular pH (pHi), apical membrane potential (Va), and fractional apical membrane resistance (FRa) were measured in principal cells of isolated frog skin (Rana pipiens) with double-barreled microelectrodes under short-circuit and open-circuit conditions. Basolateral exposure to high K+ concentration or Ba2+ depolarized V(a), decreased short-circuit current, and increased FRa and pHi. However, an increase in K+ subsequent to Ba2+ application did not induce additional changes in these parameters. High basolateral K+, previously shown to increase apical K+ secretion (N. S. Bricker, T. Biber, and H. H. Ussing. J. Clin. Invest. 41: 88-99, 1963), also increased apical Na+ conductance. The depolarization and intracellular alkalinization induced by high K+ were also observed in absence of Na+, Cl-, and HCO3- and in presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Under all these conditions pHi moved toward electrochemical equilibrium. Reduced basolateral K+ hyperpolarized V(a) and decreased pHi. The data suggest that depolarization and hyperpolarization of the apical and/or basolateral membrane are associated with an increase and decrease, respectively, in pHi without involvement of Na(+)-H+, Cl(-)-HCO3-, or K(+)-H+ exchange and are apparently also independent of an active H+ secretion pathway. This indicates the presence of a potential-dependent H+ and/or OH- conductance in the apical and/or basolateral cell membrane that may play an important role in pHi regulation and signal transduction.

A cyclin-abundance cycle-independent p34cdc2 tyrosine phosphorylation cycle in early sea urchin embryos.

The activity of the cell cycle control protein p34cdc2 is post-translationally regulated in a variety of cell types. Using anti-phosphotyrosine antibodies, we find that p34cdc2-directed tyrosine kinase activity increases at fertilization in sea urchin eggs, leading to a gradual accumulation of phosphotyrosine on p34 during the early part of the cell cycle. Loss of phosphotyrosine from p34 accompanies entry into mitosis and phosphotyrosine reaccumulates as the embryo enters the next cell cycle. A similar pattern is seen when eggs are parthenogenetically activated with ammonium chloride. Tyrosine phosphorylation and phosphorylation/dephosphorylation cycles are suppressed when embryos are treated with the tyrosine kinase inhibitor genistein. On the other hand, a cycle persists when protein synthesis is inhibited with emetine, indicating that it is independent of the synthesis of another class of cell cycle control proteins, the cyclins. Additional experiments with the phorbol ester, phorbol myristate acetate, demonstrate that activating protein synthesis alone in unfertilized eggs does not result in stimulation of p34cdc2 tyrosine kinase activity. Our results indicate that p34 tyrosine phosphorylation cycles are triggered by the fertilization Cai transient. The first cycle is independent of the fertilization pHi signal, confirming that, in sea urchin embryos, the cycle is not tightly coupled to the cycle of cyclin abundance that is a prominent feature of the eukaryotic cell division cycle.

Relationship between intracellular pH and ammonia metabolism in LLC-PK1 cells

Previous studies from our laboratory have confirmed that cultures of LLC-PK1 cells exhibit pH-responsive alterations in ammonia metabolism produced by changes in media bicarbonate concentration. To further elucidate the mechanism of ammonia regulation, studies were carried out using parallel cultures of still and rocked LLC-PK1 cells subjected to acute alterations in media pH by either metabolic or respiratory acid-base manipulations. When media pH was altered by modifying PCO2 levels, the response of ammonia and alanine production by rocked culture was identical to the changes observed with metabolic acid-base maneuvers. Furthermore, both metabolic and respiratory acute acidosis resulted in a fall of intracellular alpha-ketoglutarate concentrations in these cells. In contrast, standard still cultures subjected to acute acidosis/alkalosis by metabolic and respiratory manipulations did not exert any significant change in ammonia and alanine production or in intracellular alpha-ketoglutarate concentration. Measurements of intracellular pH (pHi) by the 5,5-[2-14C]dimethyloxazolidine-2,4-dione method in rocked cells demonstrated changes in pHi parallel to media pH changes induced by both metabolic and respiratory acid-base maneuvers. Despite the absence of pH-responsive ammonia-genesis in still cultured cells the pHi values were altered in a fashion similar to their rocked counterparts, indicating the lack of an effect of the pHi signal on ammonia metabolism.