Inhibition of auxin-induced ethylene production by salicylic acid in mung bean hypocotyls

Salicylic acid (SA), a common plant phenolic compound, influences diverse physiological and biochemical processes in plants. To gain insight into the mode of interaction between auxin, ethylene, and SA, the effect of SA on auxininduced ethylene production in mung bean hypocotyls was investigated. Auxin markedly induced ethylene production, while SA inhibited the auxin-induced ethylene synthesis in a dose-dependent manner. At 1 mM of SA, auxininduced ethylene production decreased more than 60% in hypocotyls. Results showed that the accumulation of ACC was not affected by SA during the entire period of auxin treatment, indicating that the inhibition of auxin-induced ethylene production by SA was not due to the decrease in ACC synthase activity, the rate-limiting step for ethylene biosynthesis. By contrast, SA effectively reduced not only the basal level of ACC oxidase activity but also the wound-and ethylene-induced ACC oxidase activity, the last step of ethylene production, in a dose-dependent manner. Northern and immuno blot analyses indicate that SA does not exert any inhibitory effect on the ACC oxidase gene expression, whereas it effectively inhibits both the in vivo and in vitro ACC oxidase enzyme activity, thereby abolishing auxin-induced ethylene production in mung bean hypocotyl tissue. It appears that SA inhibits ACC oxidase enzyme activity through the reversible interaction with Fe²⁺, an essential cofactor of this enzyme. These results are consistent with the notion that ethylene production is controlled by an intimate regulatory interaction between auxin and SA in mung bean hypocotyl tissue.     

Identification and characterization of three putative genes for 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyl segments

The polymerase chain reaction (PCR) was used to produce 3 putative clones for ACC synthase from etiolated mung bean (Vigna radiata Rwilcz cv. Berken) hypocotyls. This was accomplished by utilizing genomic DNA from mung bean and degenerate primers made from information derived from highly conserved regions of ACC synthase from different plant tissues. The total length of pMAC-1, pMAC-2 and pMAC-3 are 308, 321, and 326 bp, respectively, all of which code for 68 amino acids. The introns for pMAC-1, pMAC-2 and pMAC-3 are 92, 105, and 110 bp, respectively. The degrees of homology at the DNA level for each of these clones is ca. 80% in their coding region and ca. 50% in their respective introns. This is the first report providing evidence that there are at least 3 genes for ACC synthase in etiolated mung bean.     

Differential requirements for proliferation of CD4+ and gammadelta+ T cells to spirochetal antigens

Alphabeta+ and gammadelta+ T cells have different mechanisms of epitope recognition and are stimulated by antigens of different chemical nature. An immunization model with antigens from the spirochete Brachyspira hyodysenteriae was used to examine the requirements for proliferation of circulating porcine CD4+ and gammadelta+ T cells in mixed lymphocyte cultures. CD4+ T cells only responded to stimulation with B. hyodysenteriae antigens, whereas gammadelta+ T cells proliferated when cultures were stimulated with either spirochetal antigens or interleukin-2 (IL-2). T cells that had proliferated expressed high levels of IL-2-receptor-alpha (IL-2Ralpha). Furthermore, neutralization of IL-2 at the beginning of the culture period was more efficient in blocking gammadelta+ than CD4+ T cell proliferation. Immunization induced interferon-gamma (IFN-gamma) production by CD4+ T cells, whereas only a small fraction of the antigen-stimulated gammadelta+ T cells produced this cytokine. Our results indicate that, under the same environmental conditions, CD4+ T cell functions are more tightly regulated when compared to gammadelta+ T cells. We conclude that these differences are due, in part, to the enhanced gammadelta+ T cell responsiveness to IL-2.     

Cell-surface H+-ATP synthase as a potential molecular target for anti-obesity drugs

Here we show that the cell-surface expression of the alpha subunit of H(+)-ATP synthase is markedly increased during adipocyte differentiation. Treatment of differentiated adipocytes with small molecule inhibitors of H(+)-ATP synthase or antibodies against alpha and beta subunits of H(+)-ATP synthase leads to a decrease in cytosolic lipid droplet accumulation. Apolipoprotein A-I, which has been shown to bind to the ectopic beta-chain of H(+)-ATP synthase and inhibit the activity of cell-surface H(+)-ATP synthase, also was found to inhibit cytosolic lipid accumulation. These results suggest that the cell-surface H(+)-ATP synthase has a previously unsuspected role in lipid metabolism in adipocytes.     

Mg2+ release coupled to Ca2+ uptake: A novel Ca2+ accumulation mechanism in rat liver

Isolated hepatocytes release 2–3 nmol Mg²⁺/mg protein or ~10% of the total cellular Mg²⁺ content within 2 minutes from the addition of agonists that increase cellular cAMP, for example, isoproterenol (ISO). During Mg²⁺ release, a quantitatively similar amount of Ca²⁺ enters the hepatocyte, thus suggesting a stoichiometric exchange ratio of 1 Mg²⁺:1Ca²⁺. Calcium induced Mg²⁺ extrusion is also observed in apical liver plasma membranes (aLPM), in which the process presents the same 1 Mg²⁺:1Ca²⁺ exchange ratio. The uptake of Ca²⁺ for the release of Mg²⁺ occurs in the absence of significant changes in Δψ as evidenced by electroneutral exchange measurements with a tetraphenylphosphonium (TPP⁺) electrode or ³H-TPP⁺. Collapsing the Δψ by high concentrations of TPP⁺ or protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) does not inhibit the Ca²⁺-induced Mg²⁺ extrusion in cells or aLPM. Further, the process is strictly unidirectional, serving only in Ca²⁺ uptake and Mg²⁺ release. These data demonstrate the operation of an electroneutral Ca²⁺/Mg²⁺ exchanger which represents a novel pathway for Ca²⁺ accumulation in liver cells following adrenergic receptor stimulation. This work was supported by National Institutes of Health Grant HL 18708.     

Differential accumulation of S-adenosylmethionine synthetase transcripts in response to salt stress

NaCl stress causes the accumulation of several mRNAs in tomato seedlings. An upregulated cDNA clone, SAM1, was found to encode a S-adenosyl-L-methionine synthetase enzyme (AdoMet synthetase). Expression of the cDNA SAM1 in a yeast mutant lacking functional SAM genes resulted in high AdoMet synthetase activity and AdoMet accumulation. We show that tomato plants contain at least four SAM isogenes. Clones corresponding to isogenes SAM2 and SAM3 have also been isolated and sequenced. they encode predicted polypeptides 95% and 92% identical, respectively, to the SAM1-encoded AdoMet Synthetase. RNA hybridization analysis showed a differential response of SAM genes to salt and other stress treatments. SAM1 and SAM3 mRNAs accumulated in the root in response to NaCl, mannitol or ABA treatments. SAM1 mRNA accumulated also in leaf tissue. These increases of mRNA level were apparent as soon as 8 h after the initiation of the salt treatment and were maintained for at least 3 days. A possible role for AdoMet synthetases in the adaptation to salt stress is discussed.     

Modulation of Mn2+ accumulation in cultured rat neuronal and astroglial cells

The effects of physiological concentrations of K⁺ on Mn²⁺ accumulation were compared in rat glial cells and neurons in culture. Increasing the K⁺ concentration in growth medium increased significantly the Mn²⁺ level of the cultivated cells, with glial cells more affected than neurons. Ethanol markedly increased the Mn²⁺ accumulation within glia but not within neurons while ouabaïn caused inhibition of Mn²⁺ uptake with neurons and glial cells. A modulation of the total protein synthesis by Mn²⁺ and ethanol level in the growth medium was observed with glial cells. These data suggest that the mechanisms involved in Mn²⁺ accumulation in glial cells are different from those present in neurons. Moreover, the results are consistent with the hypothesis that Mn²⁺ plays a regulatory role in glial cell metabolism.     

Enhancement of adventitious root formation in mung bean cuttings by 3,5-dihalo-4-hydroxybenzoic acids and 2,4-dinitrophenol

3,5-Dihalo-4-hydroxybenzoic acids enhanced adventitious root formation in mung bean (Vigna radiata L.) cuttings. 3,5-Diiodo-4-hydroxybenzoic acid was more active than 3,5-dichloro-4-hydroxybenzoic acid, increasing the number of roots formed by about 4-fold. 2,4-Dinitrophenol also enhanced significantly adventitious root formation in mung bean cuttings. The phenolic compounds were active with or without indole-3-acetic acid. The possible mechanism by which these phenolic compounds enhance rooting is discussed.Abbreviations CCCP carbonyl cyanide 3-chlorophenylhydrazone - DIHB 3,5-diiodo-4-hydroxybenzoic acid - DNP 2,4-dinitrophenol     

Isolation and characterization of cDNAs encoding vacuolar H+-pyrophosphatase isoforms from rice (Oryza sativa L.)

The vacuolar H⁺-pyrophosphatase (V-PPase) is an electrogenic H⁺ pump, which was found in the plant vacuolar membrane. Two cDNA clones (OVP1 and OVP2) encoding the V-PPase were isolated from cultured rice (Oryza sativa L.) cells and subsequently sequenced. The sequence analysis has revealed thatOVP1 contains 2316 nucleotides of open reading frame (ORF) and 362 nucleotides of the 3-untranslated region, whereasOVP2 comprises 2304 nucleotides of ORF and 312 nucleotides of the 3-untranslated region. The nucleotide sequences of ORF ofOVP1 andOVP2 are 80.7% identical, and their 5- and 3-untranslated regions have 39.4% and 48.4% identity, respectively. The polypeptides encoded by the ORF ofOVP1 andOVP2 contain 771 and 767 amino acids, respectively, and the sequences of the OVP proteins are very similar to those of other V-PPases, which are shown to have 85–91% homology. Chromosomal mapping by RFLP techniques demonstrates that OVP1 and OVP2 are isoforms encoded by different genes. BothOVP1 andOVP2 are mapped on the same chromosome (chromosome 6) to a distance of ca. 90 cM. Northern analysis indicates that theOVP1 andOVP2 are also expressed in intact rice plants andOVP2 shows higher expression in the calli than the roots and shoots, compared toOVP1. These results show that at least two genes encoding the V-PPases are present in rice genome and their expressions are probably regulated in a different manner.     

Differential response of BDCA-1+ and BDCA-3+ myeloid dendritic cells to respiratory syncytial virus infection

Background

Respiratory syncytial virus (RSV) is the leading cause of respiratory infections in children, elderly, and immunocompromised individuals. Severe infection is associated with short- and long-term morbidity including pneumonia, recurrent wheezing, and abnormal pulmonary function, and several lines of evidence indicate that impaired adaptive immune responses during infection are critical in the pathophysiology of RSV-mediated disease. Myeloid Dendritic cells (mDCs) play a pivotal role in shaping antiviral immune responses in the respiratory tract; however, few studies have examined the interactions between RSV and individual mDC subsets. In this study, we examined the effect of RSV on the functional response of primary mDC subsets (BDCA-1⁺ and BDCA-3⁺) isolated from peripheral blood.

Methods

BDCA-1⁺ and BDCA-3⁺ mDCs were isolated from the peripheral blood of healthy adults using FACS sorting. Donor-matched BDCA-1⁺ and BDCA-3⁺ mDCs were infected with RSV at a multiplicity of infection (MOI) of 5 for 40 hours. After infection, cells were analyzed for the expression of costimulatory molecules (CD86, CD80, and PD-L1), cytokine production, and the ability to stimulate allogenic CD4⁺ T cell proliferation.

Results

Both BDCA-1⁺ and BDCA-3⁺ mDCs were susceptible to infection with RSV and demonstrated enhanced expression of CD86, and the inhibitory costimulatory molecules CD80 and PD-L1. Compared to BDCA-3⁺ mDCs, RSV-infected BDCA-1⁺ mDC produced a profile of cytokines and chemokines predominantly associated with pro-inflammatory responses (IL-1β, IL-6, IL-12, MIP-1α, and TNF-α), and both BDCA-1⁺ and BDCA-3⁺ mDCs were found to produce IL-10. Compared to uninfected mDCs, RSV-infected BDCA-1⁺ and BDCA-3⁺ mDCs demonstrated a reduced capacity to stimulate T cell proliferation.

Conclusions

RSV infection induces a distinct pattern of costimulatory molecule expression and cytokine production by BDCA-1⁺ and BDCA-3⁺ mDCs, and impairs their ability to stimulate T cell proliferation.The differential expression of CD86 and pro-inflammatory cytokines by highly purified mDC subsets in response to RSV provides further evidence that BDCA-1⁺ and BDCA-3⁺ mDCs have distinct roles in coordinating the host immune response during RSV infection. Findings of differential expression of PD-L1 and IL-10 by infected mDCs, suggests possible mechanisms by which RSV is able to impair adaptive immune responses.     

Nitric Oxide Links the Apical Na+ Transport to the Basolateral K+ Conductance in the Rat Cortical Collecting Duct

We have used the patch clamp technique to study the effects of inhibiting the apical Na⁺ transport on the basolateral small-conductance K⁺ channel (SK) in cell-attached patches in cortical collecting duct (CCD) of the rat kidney. Application of 50 μM amiloride decreased the activity of SK, defined as nP_o (a product of channel open probability and channel number), to 61% of the control value. Application of 1 μM benzamil, a specific Na⁺ channel blocker, mimicked the effects of amiloride and decreased the activity of the SK to 62% of the control value. In addition, benzamil reduced intracellular Na⁺ concentration from 15 to 11 mM. The effect of amiloride was not the result of a decrease in intracellular pH, since addition 50 μM 5-(n-ethyl-n-isopropyl) amiloride (EIPA), an agent that specifically blocks the Na/H exchanger, did not alter the channel activity. The inhibitory effect of amiloride depends on extracellular Ca²⁺ because removal of Ca²⁺ from the bath abolished the effect. Using Fura-2 AM to measure the intracellular Ca²⁺, we observed that amiloride and benzamil significantly decreased intracellular Ca²⁺ in the Ca²⁺-containing solution but had no effect in a Ca²⁺-free bath. Furthermore, raising intracellular Ca²⁺ from 10 to 50 and 100 nM with ionomycin increased the activity of the SK in cell-attached patches but not in excised patches, suggesting that changes in intracellular Ca²⁺ are responsible for the effects on SK activity of inhibition of the Na⁺ transport. Since the neuronal form of nitric oxide synthase (nNOS) is expressed in the CCD and the function of the nNOS is Ca²⁺ dependent, we examined whether the effects of amiloride or benzamil were mediated by the NO-cGMP–dependent pathways. Addition of 10 μM S-nitroso-n-acetyl-penicillamine (SNAP) or 100 μM 8-bromoguanosine 3′:5′-cyclic monophosphate (8Br-cGMP) completely restored channel activity when it had been decreased by either amiloride or benzamil. Finally, addition of SNAP caused a significant increase in channel activity in the Ca²⁺-free bath solution. We conclude that Ca²⁺-dependent NO generation mediates the effect of inhibiting the apical Na⁺ transport on the basolateral SK in the rat CCD.     

Nitric oxide synthase inhibition by L-NAME prevents the decrease of Na+,K+-ATPase activity in midbrain of rats subjected to arginine administration

In the present study we investigated the effect of acute administration of L-arginine on Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities and on some parameters of oxidative stress (chemiluminescence and total radical-trapping antioxidant parameter-TRAP) in midbrain of adult rats. We also tested the effect of L-NAME on the effects produced by arginine. Sixty-day-old rats were treated with an acute intraperitoneal injection of saline (group I, control), arginine (0.8 g/kg) (group II), L-NAME (2 mg/kg) (group III) or arginine (0.8 g/kg) plus L-NAME (2 mg/kg) (group IV). Na⁺,K⁺-ATPase activity was significantly reduced in the arginine-treated rats, but was not affected by other treatments. In contrast, Mg²⁺-ATPase activity was not altered by any treatment. Furthermore, chemiluminescence was significantly increased and TRAP was significantly decreased in arginine-treated rats, whereas the simultaneous injection of L-NAME prevented these effects. These results demonstrate that in vivo arginine administration reduces Na⁺,K⁺-ATPase activity possibly through free radical generation induced by NO formation.