Identification of the Binding and Inhibition Sites in the Calmodulin Molecule for Ophiobolin A by Site-Directed Mutagenesis

Ophiobolin A, a fungal toxin that affects maize and rice, has previously been shown to inhibit calmodulin by reacting with the lysine (Lys) residues in the calmodulin. In the present study we mutated Lys-75, Lys-77, and Lys-148 in the calmodulin molecule by site-directed mutagenesis, either by deleting them or by changing them to glutamine or arginine. We found that each of these three Lys residues could bind one molecule of ophiobolin A. Normally, only Lys-75 and Lys-148 bind ophiobolin A. Lys-77 seemed to be blocked by the binding of ophiobolin A to Lys-75. Lys-75 is the primary binding site and is responsible for all of the inhibition of ophiobolin A. When Lys-75 was removed, Lys-77 could then react with ophiobolin A to produce inhibition. Lys-148 was shown to be a binding site but not an inhibition site. The Lys-75 mutants were partially resistant to ophiobolin A. When both Lys 75 and Lys-77 or all three Lys residues were mutated, the resulting calmodulins were very resistant to ophiobolin A. Furthermore, Lys residues added in positions 86 and/or 143 (which are highly conserved in plant calmodulins) did not react with ophiobolin A. None of the mutations seemed to affect the properties of calmodulin. These results show that ophiobolin A reacts quite specifically with calmodulin.     

Induction of Oligodendrocyte Differentiation and In Vitro Myelination by Inhibition of Rho-Associated Kinase

In inflammatory demyelinating diseases such as multiple sclerosis (MS), myelin degradation results in loss of axonal function and eventual axonal degeneration. Differentiation of resident oligodendrocyte precursor cells (OPCs) leading to remyelination of denuded axons occurs regularly in early stages of MS but halts as the pathology transitions into progressive MS. Pharmacological potentiation of endogenous OPC maturation and remyelination is now recognized as a promising therapeutic approach for MS. In this study, we analyzed the effects of modulating the Rho-A/Rho-associated kinase (ROCK) signaling pathway, by the use of selective inhibitors of ROCK, on the transformation of OPCs into mature, myelinating oligodendrocytes. Here we demonstrate, with the use of cellular cultures from rodent and human origin, that ROCK inhibition in OPCs results in a significant generation of branches and cell processes in early differentiation stages, followed by accelerated production of myelin protein as an indication of advanced maturation. Furthermore, inhibition of ROCK enhanced myelin formation in cocultures of human OPCs and neurons and remyelination in rat cerebellar tissue explants previously demyelinated with lysolecithin. Our findings indicate that by direct inhibition of this signaling molecule, the OPC differentiation program is activated resulting in morphological and functional cell maturation, myelin formation, and regeneration. Altogether, we show evidence of modulation of the Rho-A/ROCK signaling pathway as a viable target for the induction of remyelination in demyelinating pathologies.     

Two Genetically Separable Phases of Growth Inhibition Induced by Blue Light in Arabidopsis Seedlings

High fluence-rate blue light (BL) rapidly inhibits hypocotyl growth in Arabidopsis, as in other species, after a lag time of 30 s. This growth inhibition is always preceded by the activation of anion channels. The membrane depolarization that results from the activation of anion channels by BL was only 30% of the wild-type magnitude in hy4, a mutant lacking the HY4 BL receptor. High-resolution measurements of growth made with a computer-linked displacement transducer or digitized images revealed that BL caused a rapid inhibition of growth in wild-type and hy4 seedlings. This inhibition persisted in wild-type seedlings during more than 40 h of continuous BL. By contrast, hy4 escaped from the initial inhibition after approximately 1 h of BL and grew faster than wild type for approximately 30 h. Wild-type seedlings treated with 5-nitro-2-(3-phenylpropylamino)-benzoic acid, a potent blocker of the BL-activated anion channel, displayed rapid growth inhibition, but, similar to hy4, these seedlings escaped from inhibition after approximately 1 h of BL and phenocopied the mutant for at least 2.5 h. The effects of 5-nitro-2-(3-phenylpropylamino)-benzoic acid and the HY4 mutation were not additive. Taken together, the results indicate that BL acts through HY4 to activate anion channels at the plasma membrane, causing growth inhibition that begins after approximately 1 h. Neither HY4 nor anion channels appear to participate greatly in the initial phase of inhibition.     

Root Tissue Response of Two Related Soybean Cultivars to Infection by Lectin-treated Meloidogyne spp.

Treatment of second-stage juveniles (J2) of Meloidogyne incognita race 1 and M. javanica with soybean agglutinin, Concanavalin A, wheat germ agglutinin, Lotus tetragonolobus agglutinin, or Limax flavus agglutinin or the corresponding competitive sugars for each of these lectins did not alter normal root tissue response of soybean cultivars Centennial and Pickett 71 to infection by M. incognita race 1 or M. javanica. Giant cells were frequently induced in Centennial and Pickett 71 roots 5 and 20 days after inoculation of roots with untreated J2 of a population of M. incognita race 3. Treatment of J2 of M. incognita race 3 with the lectins or carbohydrates listed above caused Centennial, but not Pickett 71, root tissue to respond in a hypersensitive manner to infection by M. incognita race 3. Penetration of soybean roots by J2 of Meloidogyne spp. was strongly inhibited in the presence of 0.1 M sialic acid. Treatment of J2 with sialic acid was not lethal to nematodes, and the inhibitory activity of sialic acid was apparently not caused by low pH. These results suggest that carbohydrates may influence plant-nematode interactions.     

Control of Root-knot Nematodes on Tomato by Lectins

Significant control of tomato root knot was achieved by applications of the lectins Concanavalin A (Con A) and Limax flavus agglutinin in greenhouse, growth chamber, and microplot trials. Four consecutive weekly applications at lower concentrations of Con A yielded better control than single applications at a higher total concentration. The present state of knowledge on binding of Con A to soil nematodes and the in vitro effect of this lectin in chemotactic behavior are discussed. The mode of action of Con A on root-knot control is unknown.     

Mechanistic Insight into Control of CFTR by AMPK

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP and protein kinase A (PKA)-regulated Cl^– channel in the apical membrane of epithelial cells. The metabolically regulated and adenosine monophosphate-stimulated kinase (AMPK) is colocalized with CFTR and attenuates its function. However, the sites for CFTR phosphorylation and the precise mechanism of inhibition of CFTR by AMPK remain obscure. We demonstrate that CFTR normally remains closed at baseline, but nevertheless, opens after inhibition of AMPK. AMPK phosphorylates CFTR in vitro at two essential serines (Ser⁷³⁷ and Ser⁷⁶⁸) in the R domain, formerly identified as “inhibitory” PKA sites. Replacement of both serines by alanines (i) reduced phosphorylation of the R domain, with Ser⁷⁶⁸ having dramatically greater impact, (ii) produced CFTR channels that were partially open in the absence of any stimulation, (iii) significantly augmented their activation by IBMX/forskolin, and (iv) eliminated CFTR inhibition post AMPK activation. Attenuation of CFTR by AMPK activation was detectable in the absence of cAMP-dependent stimulation but disappeared in maximally stimulated oocytes. Our data also suggest that AMP is produced by local phosphodiesterases in close proximity to CFTR. Thus we propose that CFTR channels are kept closed in nonstimulated epithelia with high baseline AMPK activity but CFTR may be basally active in tissues with lowered endogenous AMPK activity.The cystic fibrosis transmembrane regulator (CFTR)² gene is mutated in patients with cystic fibrosis. CFTR has an adapted ABC transporter structural motif thereby creating an anion channel at the apical surface of secretory epithelia (1). The consequent CFTR-mediated ion transport is tightly controlled by ATP binding and phosphorylation by protein kinase A (PKA). However, a number of other protein kinases including PKC, Ca²⁺/calmodulin-dependent kinase, and cGMP-dependent kinase also control the activity of CFTR (2–4). These kinases converge on the regulatory domain of CFTR that is unique not only within the large ABC transporter family but among all known sequences, and may be considered as a “phosphorylation control module” (3). Regulation of CFTR by an inhibitory kinase, the adenosine monophosphate-dependent kinase (AMPK), has been described recently but the regulatory sites within CFTR, the mechanism of regulation, and the physiological relevance have all remained obscure (5–8). Additionally, CFTR mutation is linked to inflammation and a lack of functional CFTR expression has itself been suggested to up-regulate AMPK activity in epithelial cells carrying the cystic fibrosis (CF) defect. Pharmacologic AMPK activation was shown to inhibit secretion of inflammatory mediators (9). Thus AMPK may play multiple roles in CF pathophysiology making the mechanism of interaction an important problem in biology.AMPK is a ubiquitous serine/threonine kinase that exists as a heterotrimer with a catalytic α subunit and regulatory β and γ subunits, each with multiple isoforms. In response to metabolic depletion and a consequent increase in the cellular AMP to ATP ratio, AMPK phosphorylates numerous proteins and activates catabolic pathways that generate ATP, whereas inhibiting cell growth, protein biosynthesis, and a number of other ATP-consuming processes, thereby operating as a cellular “low-fuel” sensor (10, 11). AMPK also controls signaling pathways involved in apoptosis, cell cycle, and tissue inflammation (12). Because AMPK is a cellular metabolic sensor that inhibits CFTR and limits cAMP activated Cl^– secretion, a coupling of membrane transport by CFTR to the cellular metabolism has been proposed (13). However, AMPK activity can also increase without detectable changes in the cytosolic AMP to ATP ratio, suggesting a contribution of additional AMP-independent signals for regulation of CFTR by AMPK (14). Drugs used to combat type 2 diabetes, such as phenformin and metformin, act in this manner to activate AMPK, AMP-independently. It is also likely that cytosolic AMP is compartmentalized depending on the distribution of AMP generating enzymes such as phosphodiesterases that convert cAMP to AMP. The concept of spatiotemporal control of cAMP signaling by anchored protein complexes is well established (15). CFTR is known to form such macromolecular complexes with a number of interacting partners (16–18). For example, competitive interaction of EBP50-PKA and Shank2-PDE4D with CFTR has been demonstrated recently (19). In addition, Barnes and co-workers (20) demonstrated that phosphodiesterase 4D generates a cAMP diffusion barrier local to the apical membrane of the airway epithelium. It is therefore likely that activator pathways through cAMP and inhibitory AMP/AMPK signaling occur in a local CFTR-organized compartment. Here we explore the functional links between CFTR, inhibition of phosphodiesterases, and AMPK focusing on the effects of mutating putative AMPK targets within the R domain on CFTR function.     

Nodulation of Soybeans as Affected by Half-root Infection with Heterodera glycines

A split-root technique was applied to soybean, Glycine max (L.) Merr. cv. Lee 68, to characterize the nature of the nodulation suppression by race 1 of the soybean cyst nematode (SCN), Heterodera glycines. Root-halves of each split-root plant were inoculated with Rhizobium japonicum, and one root-half only was inoculated with various numbers of SCN eggs. Nodulation (indicated by nodule number, nodule weights, and ratio of nodule weight to root weight) and nitrogen-fixing capacity (indicated by rate of acetylene reduction) were systemically and variously suppressed on both root-halves of the split-root plant 5 weeks after half-root inoculation with 12,500 SCN eggs. Inoculation with 500 eggs caused this suppression only on the SCN-infected (+NE) root-half; nodulation on the companion uninfected (-NE) root-half was stimulated slightly. The +NE root-halves inoculated with 5,000 eggs were excised at 2-week intervals; nodulation on the remaining -NE root-halves was not different from that of the noninoculated control when measured 6 weeks after the SCN inoculation. Thus, the systemic suppression of nodulation was reversible upon the removal of the SCN. Similarly, application of various levels of KNO₃ to the -NE root-halves of the split-root plant did not alleviate the suppressed nodulation on the companion +NE root-halves, even though plant growth was much improved at certain levels of nitrogen (125 μg N/g soil). This indicated that the localized suppression of nodulation by SCN was caused by factors in addition to poor plant growth.     

The Kinetics of Zeaxanthin Formation Is Retarded by Dicyclohexylcarbodiimide

The de-epoxidation of violaxanthin to antheraxanthin (Anth) and zeaxanthin (Zeax) in the xanthophyll cycle of higher plants and the generation of nonphotochemical fluorescence quenching in the antenna of photosystem II (PSII) are induced by acidification of the thylakoid lumen. Dicyclohexylcarbodiimide (DCCD) has been shown (a) to bind to lumen-exposed carboxy groups of antenna proteins and (b) to inhibit the pH-dependent fluorescence quenching. The possible influence of DCCD on the de-epoxidation reactions has been investigated in isolated pea (Pisum sativum L.) thylakoids. The Zeax formation was found to be slowed down in the presence of DCCD. The second step (Anth → Zeax) of the reaction sequence seemed to be more affected than the violaxanthin → Anth conversion. Comparative studies with antenna-depleted thylakoids from plants grown under intermittent light and with unstacked thylakoids were in agreement with the assumption that binding of DCCD to antenna proteins is probably responsible for the retarded kinetics. Analyses of the DCCD-induced alterations in different antenna subcomplexes showed that Zeax formation in the PSII antenna proteins was predominantly influenced by DCCD, whereas Zeax formation in photosystem I was nearly unaffected. Our data support the suggestion that DCCD binding to PSII antenna proteins is responsible for the observed alterations in xanthophyll conversion.     

Quantification of Intermediates Formed during the Reduction of Nitrite by Deoxyhemoglobin

Nitric oxide (NO) plays a crucial role in human physiology by regulating vascular tone and blood flow. The short life-span of NO in blood requires a mechanism to retain NO bioactivity in the circulation. Recent studies have suggested a mechanism involving the reduction of nitrite back to NO by deoxyhemoglobin in RBCs. A role for RBCs in transporting NO must, however, bypass the scavenging of NO in RBCs by hemoglobin. To understand how the nitrite reaction can deliver bioactive NO to the vasculature, we have studied the intermediates formed during the reaction. A reliable measure of the total concentration of heme-associated nitrite/NO intermediates formed was provided by combining filtration to measure free nitrite by chemiluminescence and electron paramagnetic resonance to measure the final product Hb(II)NO. By modifying the chemiluminescence method used to detect NO, we have been able to identify two intermediates: 1) a heme-associated nitrite complex that is released as NO in acid solution in the presence of ascorbate and 2) an intermediate that releases NO at neutral pH in the presence of ferricyanide when reacted with an Fe(III) ligand like azide. This species designated as “Hb(II)NO⁺ ⇆ Hb(III)NO” has properties of both isomeric forms resulting in a slower NO dissociation rate and much higher stability than Hb(III)NO, but provides a potential source for bioactive NO, which can be released from the RBC. This detailed analysis of the nitrite reaction with deoxyHb provides important insights into the mechanism for nitrite induced vasodilation by RBCs.Nitric oxide (NO), also known as the endothelium-derived relaxing factor, is an important messenger molecule involved in the regulation of vascular tone and blood flow (1). The primary source for the synthesis of NO in the circulatory system involves endothelial nitric-oxide synthase (2). This enzyme requires oxygen for the synthesis of NO and is, therefore, less effective in the microcirculation where hypoxic vasodilation regulates the delivery of oxygen. Because nitric oxide has a life-time in blood of <2 ms (3), a mechanism is required to allow for more distal and sustained effects of NO at the reduced oxygen pressures found in the microcirculation. Recent studies have suggested that the bioactivity of NO can be conserved in the blood by the uptake of NO and/or nitrite by red blood cells (RBCs)² and its interaction with hemoglobin (4–7). However, any role for the red cell in transporting nitric oxide must be able to avoid the very efficient scavenging of nitric oxide by both oxyhemoglobin (oxyHb) and deoxyhemoglobin (deoxyHb) that destroy and trap NO, respectively, preventing a physiological role for RBC NO.In a series of studies, Stamler and co-workers (7–10) have hypothesized that NO can bypass this difficulty by being transferred to the β-93 thiol group of hemoglobin (Hb) forming S-nitrosylated hemoglobin (SNO-Hb) when partially heme nitrosylated hemoglobin (Hb(II)NO) is oxygenated. The allosteric quaternary conformational change of hemoglobin at low oxygen pressure destabilizes the β-93 nitrosylated thiol and results in the transfer of NO to membrane thiol groups facilitating the release of the NO to the plasma and the vasculature. However, the extremely low levels of SNO-Hb (11) found in human blood and its instability (12) as a result of intracellular reducing conditions within the RBCs do not support the SNO-Hb hypothesis as the major mechanism for NO transport (11–13).The 2003 studies by Rifkind and Gladwin and their collaborators (4, 5, 14, 15) proposed an alternative mechanism that involved the reduction of nitrite, formed by the oxidation of NO, back to NO by a reaction with deoxyHb. Nitrite is present in the blood at fairly high levels (0.1–0.5 μmol/liter) (4, 16–18), and it is much more stable than NO or S-nitrosothiols (6), making nitrite an ideal storage pool that can be converted to NO. However, the mechanism by which the NO produced in the red cell by nitrite reduction is exported without being trapped or destroyed is still unclear. Recent studies by Rifkind and co-workers (5, 13, 19) have suggested that the trapping of NO by deoxyHb and/or oxyHb can be bypassed by the formation of a metastable intermediate(s) that retains the NO in a state that is not quenched by reacting with oxyHb or deoxyHb.In this report, we quantitate the two intermediate species that are formed during the reduction of nitrite by deoxyHb when an excess of hemoglobin is present. We also demonstrate that one of the intermediate species designated as “Hb(II)NO⁺ ⇆ Hb(III)NO” has properties of Hb(II)NO⁺ and Hb(III)NO, respectively. This species has a slower NO dissociation rate and a much higher stability than Hb(III)NO. This intermediate is a potential source for bioactive NO that can be released from RBCs.     

Regulation of Intracellular Cholesterol Distribution by Na/K-ATPase

Recent studies have ascribed many non-pumping functions to the Na/K-ATPase. We show here that graded knockdown of cellular Na/K-ATPase α1 subunit produces a parallel decrease in both caveolin-1 and cholesterol in light fractions of LLC-PK1 cell lysates. This observation is further substantiated by imaging analyses, showing redistribution of cholesterol from the plasma membrane to intracellular compartments in the knockdown cells. Moreover, this regulation is confirmed in α1^+/– mouse liver. Functionally, the knockdown-induced redistribution appears to affect the cholesterol sensing in the endoplasmic reticulum, because it activates the sterol regulatory element-binding protein pathway and increases expression of hydroxymethylglutaryl-CoA reductase and low density lipoprotein receptor in the liver. Consistently, we detect a modest increase in hepatic cholesterol as well as a reduction in the plasma cholesterol. Mechanistically, α1^+/– livers show increases in cellular Src and ERK activity and redistribution of caveolin-1. Although activation of Src is not required in Na/K-ATPase-mediated regulation of cholesterol distribution, the interaction between the Na/K-ATPase and caveolin-1 is important for this regulation. Taken together, our new findings demonstrate a novel function of the Na/K-ATPase in control of the plasma membrane cholesterol distribution. Moreover, the data also suggest that the plasma membrane Na/K-ATPase-caveolin-1 interaction may represent an important sensing mechanism by which the cells regulate the sterol regulatory element-binding protein pathway.The Na/K-ATPase, also called the sodium pump, is an ion transporter that mediates active transport of Na⁺ and K⁺ across the plasma membrane by hydrolyzing ATP (1, 2). The functional sodium pump is mainly composed of α and β subunits. The α subunit is the catalytic component of the holoenzyme; it contains both the nucleotide and the cation binding sites (3). So far, four isoforms of α subunit have been discovered, and each one shows a distinct tissue distribution pattern (4, 5). Interestingly, studies during the past few years have uncovered many non-pumping functions of Na/K-ATPase (6–10). Recently, we have demonstrated that more than half of the Na/K-ATPase may actually perform cellular functions other than ion pumping at least in LLC-PK1 cells (11). Moreover, the non-pumping pool of Na/K-ATPase mainly resides in caveolae and interacts with a variety of proteins such as Src, inositol 1,4,5-trisphosphate receptor, and caveolin-1 (12–14). While the interaction between Na/K-ATPase and inositol 1,4,5-trisphosphate receptor facilitates Ca²⁺ signaling (13) the dynamic association between Na/K-ATPase and Src appears to be an essential step for ouabain to stimulate cellular kinases (15). More recently, we report that the interaction between the Na/K-ATPase and caveolin-1 plays an important role for the membrane trafficking of caveolin-1. Knockdown of the Na/K-ATPase leads to altered subcellular distribution of caveolin-1 and increases the mobility of caveolin-1-containing vesicles (16).Caveolin is a protein marker for caveolae (17). Caveolae are flask-shaped vesicular invaginations of plasma membrane and are enriched in cholesterol, glycosphingolipids, and sphingomyelin (18). There are three genes and six isoforms of caveolin. Caveolin-1 is a 22-kDa protein and is expressed in many types of cells, including epithelial and endothelial cells. In addition to their role in biogenesis of caveolae (19), accumulating evidence has implicated caveolin proteins in cellular cholesterol homeostasis (20). For instance, caveolin-1 directly binds to cholesterol in a 1:1 ratio (21). It was also found to be an integral member of the intracellular cholesterol trafficking machinery between internal membranes and plasma membrane (22, 23). The expression of caveolin-1 appears to be under control of SREBPs,² the master regulators of intracellular cholesterol level (24). Furthermore, knockout of caveolin-1 significantly affected cholesterol metabolism in mouse embryonic fibroblasts and mouse peritoneal macrophages (25). Because we found that the Na/K-ATPase regulates cellular distribution of caveolin-1, we propose that it may also affect intracellular cholesterol distribution and metabolism. To test our hypothesis, we have investigated whether sodium pump α1 knockdown affects cholesterol distribution and metabolism both in vitro and in vivo. Our results indicate that sodium pump α1 expression level plays a role in the proper distribution of intracellular cholesterol. Down-regulation of sodium pump α1 not only redistributes cholesterol between the plasma membrane and cytosolic compartments, but also alters cholesterol metabolism in mice.     

Tests for Transmission of Prunus Necrotic Ringspot and Two Nepoviruses by Criconemella xenoplax

In two of three trials, detectable color reactions in ELISA for Prunus necrotic ringspot virus (PNRSV) were observed for Criconemella xenoplax handpicked from the root zone of infected peach trees. Criconemella xenoplax (500/pot) handpicked from root zones of peach trees infected with PNRSV failed to transmit the virus to cucumber or peach seedlings. The nematode also failed to transmit tomato ringspot (TomRSV) or tobacco ringspot viruses between cucumbers, although Xiphinema americanum transmitted TomRSV under the same conditions. Plants of peach, cucumber, Chenopodium quinoa, and Catharanthus roseus were not infected by PNRSV when grown in soil containing C. xenoplax collected from root zones of PNRSV-infected trees. Shirofugen cherry scions budded on Mazzard cherry seedling rootstocks remained symptomless when transplanted into root zones of PNRSV-infected trees. Virus transmission was not detected by ELISA when C. xenoplax individuals were observed to feed on cucumber root explants that were infected with PNRSV and subsequently fed on roots of Prunus besseyi in agar cultures. Even if virus transmission by C. xenoplax occurs via contamination rather than by a specific mechanism, it must be rare.     

Decline and Death of Pinus spp. in Delaware Caused by Bursaphelenchus xylophilus

Etiological studies to determine the cause of decline and death of Pinus spp. in Delaware were initiated in 1980. The pinewood nematode, Bursaphelenchus xylophilus, was found to be the major canse of mortality in Japanese black pine (Pinus thunbergii). When inoculated into healthy 5-yr-old Japanese black pines, B. xylophilus produced typical decline symptoms observed in the field. The xylophilous fungi most often associated with declining trees, Rhizosphaera pini, Fusarium spp., and Pestalotia funerea, were not pathogenic to Japanese black pine in greenhouse tests. Mineral analyses of soil and foliage showed no significant differences between healthy and infested trees. B. xylolyhilus was also found on loblolly pine (P. taeda), scrub pine (P. virginiana), Scots pine (P. sylvestris), red pine (P. resinosa), Eastern white pine (P. strobus), and pitch pine (P. rigida).