A hot pepper cDNA encoding a pathogenesis-related protein 4 is induced during the resistance response to tobacco mosaic virus

Hot pepper (Capsicum annuum) plants exhibit a hypersensitive response (HR) against infection by many tobamoviruses. A clone (CaPR-4) encoding a putative pathogenesis-related protein 4 was isolated by differential screening of a cDNA library prepared from resistant pepper plant leaves inoculated with tobacco mosaic virus (TMV) pathotype P0. The predicted amino acid sequence of CaPR-4 is very similar to those of other plant PR-4s. Southern blot analysis showed that small gene families of PR-4-related sequences were present in the pepper genome. Hot pepper cultivar Bugang, resistant to TMV-P0 and susceptible to TMV-P1.2, induced CaPR-4 expression by pathotype P0 inoculation in inoculated and systemic leaves, but not by pathotype P1.2. Effects of exogenously applied abiotic elicitors upon the CaPR-4 expression were also examined. The expression of the CaPR-4 gene was stimulated by methyl jasmonate (MeJA), ethephon and wounding treatment. However, application of salicylic acid (SA) did not trigger the expression. Evidence is emerging that jasmonic acid and ethylene play key roles in the SA-independent pathways of plant-pathogen interaction. Taken together, these results suggest that the CaPR-4 gene is one of the defense-related genes conferring resistance on pepper plants by the SA-independent pathway and the cross-talk between signaling compounds, jasmonic acid and ethylene could have a great regulatory potential in a plant's defense against TMV.     

Studies on the mechanisms of ozone tolerance: Cytokinin-like activity of N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N'-phenylurea, a compound protecting against ozone injury

The cytokinin-like activity of the growth regulating chemical EDU, N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N'-phenylurea, was determined and compared with the actitivity of kinetin using the tobacco callus bioassay. EDU has a pronounced stimulatory effect on callus growth at concentrations of 5 × 10⁻⁴ and 1 × 10⁻³ M but was 5 000 times less potent than the synthetic cytokinin, kinetin. Senescence regulation and oxidant resistance induced by EDU and kinetin were also studied. EDU retarded the breakdown of chlorophyll, protein and RNA in 0₃-sensitive tobacco leaf discs during senescence. EDU was much more effective in arresting senescence and in protecting against 0₃ injury than kinetin. Results indicate the EDU-induced plant tolerance to 0₃ phytotoxicity may be indirect through enzyme induction regulation.     

Effects of Increased γ-Aminobutyric Acid Levels on GAD67 Protein and mRNA Levels in Rat Cerebral Cortex

Abstract: Rats were injected with saline or the γ-aminobutyric acid (GABA) transaminase inhibitor γ-vinyl-GABA for 7 days and the effects on GABA content and glutamic acid decarboxylase (GAD) activity, and the protein and mRNA levels of the two forms of GAD (GAD₆₇ and GAD₆₅) in the cerebral cortex were studied. γ-Vinyl-GABA induced a 2.3-fold increase in GABA content, whereas total GAD activity decreased by 30%. Quantitative immunoblotting showed that the decline in GAD activity was attributable to a 75–80% decrease in GAD₆₇ levels, whereas the levels of GAD₆₅ remained unchanged. RNA slot-blotting with a ³²P-labeled GAD₆₇ cDNA probe demonstrated that the change in GAD₆₇ protein content was not associated with a change in GAD₆₇ mRNA levels. Our results suggest that GABA specifically controls the level of GAD₆₇ protein. This effect may be mediated by a decreased translation of the GAD₆₇ mRNA and/or a change in the stability of the GAD₆₇ protein.     

In vitro antiviral activity of Melaleuca alternifolia essential oil

Aims:  To investigate the in vitro antiviral activity of Melaleuca alternifolia essential oil (TTO) and its main components, terpinen-4-ol, α-terpinene, γ-terpinene, p -cymene, terpinolene and α-terpineol.
Methods and Results:  The antiviral activity of tested compounds was evaluated against polio type 1, ECHO 9, Coxsackie B1, adeno type 2, herpes simplex (HSV) type 1 and 2 viruses by 50% plaque reduction assay. The anti-influenza virus assay was based on the inhibition of the virus-induced cytopathogenicity. Results obtained from our screening demonstrated that the TTO and some of its components (the terpinen-4-ol, the terpinolene, the α-terpineol) have an inhibitory effect on influenza A/PR/8 virus subtype H1N1 replication at doses below the cytotoxic dose. The ID₅₀ value of the TTO was found to be 0·0006% (v/v) and was much lower than its CD₅₀ (0·025% v/v). All the compounds were ineffective against polio 1, adeno 2, ECHO 9, Coxsackie B1, HSV-1 and HSV-2. None of the tested compounds showed virucidal activity. Only a slight virucidal effect was observed for TTO (0·125% v/v) against HSV-1 and HSV-2.
Conclusions:  These data show that TTO has an antiviral activity against influenza A/PR/8 virus subtype H1N1 and that antiviral activity has been principally attributed to terpinen-4-ol, the main active component.
Significance and Impact of the Study:  TTO should be a promising drug in the treatment of influenza virus infection.     

Differences in the Cyclic AMP-Dependent Phosphorylation of Plasma Membrane Proteins of Differentiated and Undifferentiated L6 Myogenic Cells

Differences in the cyclic AMP-dependent plasma membrane phosphorylation system of undifferentiated and differentiated L₆ myogenic cells have been detected. Endogenous plasma membrane protein phosphorylation in undifferentiated L₆ myoblasts was stimulated more than three fold by 5 × 10⁻⁵ M cyclic AMP, whereas no statistically significant cyclic AMP-dependent phosphorylation of endogenous plasma membrane proteins was observed in differentiated L₆ cells. In undifferentiated cells cyclic AMP promoted the phosphorylation of several proteins, the most prominent of which had a molecular weight of 110,000. In differentiated cells cyclic AMP did not selectively promote the phosphorylation of specific plasma membrane proteins. Both differentiated and undifferentiated L₆ cells, however, contain a cyclic AMP-dependent protein kinase capable of catalyzing the phosphorylation of exogenous substrates, such as histone f₂b. Therefore, the data show that differentiation in L₆ cells is associated with a selective change in the activity of a plasma membrane cyclic AMP-dependent protein kinase which employs endogenous membrane proteins as substrate.     

Inhibition of viral protein translation by indomethacin in vesicular stomatitis virus infection: role of eIF2α kinase PKR

Indomethacin, a cyclooxygenase‐1 and ‐2 inhibitor widely used in the clinic for its potent anti‐inflammatory/analgesic properties, possesses antiviral activity against several viral pathogens; however, the mechanism of antiviral action remains elusive. We have recently shown that indomethacin activates the double‐stranded RNA (dsRNA)‐dependent protein kinase R (PKR) in human colon cancer cells. Because of the important role of PKR in the cellular defence response against viral infection, herein we investigated the effect of indomethacin on PKR activity during infection with the prototype rhabdovirus vesicular stomatitis virus. Indomethacin was found to activate PKR in an interferon‐ and dsRNA‐independent manner, causing rapid (< 5 min) phosphorylation of eukaryotic initiation factor‐2 α‐subunit (eIF2α). These events resulted in shutting off viral protein translation and blocking viral replication (IC₅₀ = 2 μM) while protecting host cells from virus‐induced damage. Indomethacin did not affect eIF2α kinases PKR‐like endoplasmic reticulum‐resident protein kinase (PERK) and general control non‐derepressible‐2 (GCN2) kinase, and was unable to trigger eIF2α phosphorylation in the presence of PKR inhibitor 2‐aminopurine. In addition, small‐interfering RNA‐mediated PKR gene silencing dampened the antiviral effect in indomethacin‐treated cells. The results identify PKR as a critical target for the antiviral activity of indomethacin and indicate that eIF2α phosphorylation could be a key element in the broad spectrum antiviral activity of the drug.     

A novel pathogenesis-related protein (SsPR10) from Solanum surattense with ribonucleolytic and antimicrobial activity is stress- and pathogen-inducible

A cDNA clone (designated as SsPR10, GenBank Accession Number AY660753 ) encoding a PR10 protein from yellow-fruit nightshade (Solanum surattense) was isolated and characterized. SsPR10 encoded a 160-amino-acid polypeptide with a predicted molecular mass of 17.58 kDa and pI of 5.29. Sequence alignments showed that SsPR10 had high identity (68.1%) with CaPR10, but had only about 31.7% identity with JIOsPR10 at the amino acid level. Genomic DNA gel blot analysis indicated that SsPR10 belonged to a multigene family. The constitutively expressed SsPR10 was detected to be the highest in roots of the sterile seedlings cultured in jars, while SsPR10 expression was the highest in old yellow leaves from the seedlings incubated with sap containing TMV. SsPR10 always expressed at slightly higher level in senescent leaves than in tender ones under both conditions. Further expression analysis revealed that the signaling components of defense/stress pathways (MeJA, SA, ABA, GA3, H2O2 and Cu2+) up-regulated significantly the SsPR10 mRNA levels over the control. However, darkness failed to induce SsPR10 expression and its expression was also inhibited by cold treatment. The SsPR10 was successfully expressed in Eschericha coli and the expressed protein was purified to near homogeneity. The dialytically renatured SsPR10 protein without phosphorylation exhibited ribonucleolytic activity against S. surattense leaf total RNA preparations and could inhibit hyphal growth of Pyricularia oryzae. Our findings suggest that the novel stress- and pathogen-inducible SsPR10 with ribonucleolytic and antimicrobial activity participates not only in the defense/stress response pathways but also in plants' growth, development and senescence.     

Functional Coupling of the δ-, μ-, and κ-Opioid Receptors to Mitogen-Activated Protein Kinase and Arachidonate Release in Chinese Hamster Ovary Cells

Abstract: To examine whether the mitogen-activated protein kinase (MAPK) cascade and phospholipase A₂ (PLA₂) are involved in the signal transduction mechanism of the opioid receptor, the δ-, μ-, and κ-opioid receptors were stably expressed from cDNA in Chinese hamster ovary cells. Activation of the δ-, μ-, and κ-receptors by agonists induced a rapid and transient increase in MAPK activity accompanied by reduced electrophoretic mobility of the 42-kDa isoform of MAPK (p42), probably owing to phosphorylation. The opioid receptor-mediated increase in MAPK activity was suppressed not only by pretreatment with genistein, a tyrosine protein kinase inhibitor, but also by prolonged exposure to phorbol 12-myristate 13-acetate and pretreatment with GF 109203X, a selective protein kinase C (PKC) inhibitor, suggesting the involvement of PKC as well as tyrosine protein kinase. Furthermore, stimulation of the δ-, μ-, and κ-receptors with opioid agonists in the presence of A23187, a calcium ionophore, resulted in an increase in arachidonate release, suggesting that PLA₂ is activated by the opioid receptors when the intracellular Ca²⁺ concentration is elevated. Both MAPK activation and increase in arachidonate release mediated by the opioid receptors were abolished by pretreatment with pertussis toxin, suggesting that these responses are mediated by G_i or G_o types of GTP-binding regulatory proteins.     

Therapeutic effects of coenzyme Q10 (CoQ10) and reduced CoQ10 in the MPTP model of Parkinsonism

Coenzyme Q₁₀ (CoQ₁₀) is a promising agent for neuroprotection in neurodegenerative diseases. We tested the effects of various doses of two formulations of CoQ₁₀ in food and found that administration in the diet resulted in significant protection against loss of dopamine (DA), which was accompanied by a marked increase in plasma concentrations of CoQ₁₀. We further investigated the neuroprotective effects of CoQ₁₀, reduced CoQ₁₀ (ubiquinol), and CoQ₁₀ emulsions in the (MPTP) model of Parkinson's disease (PD). We found neuroprotection against MPTP induced loss of DA using both CoQ₁₀, and reduced CoQ₁₀, which produced the largest increases in plasma concentrations. Lastly, we administered CoQ₁₀ in the diet to test its effects in a chronic MPTP model induced by administration of MPTP by Alzet pump for 1 month. We found neuroprotective effects against DA depletion, loss of tyrosine hydroxylase neurons and induction of alpha-synuclein inclusions in the substantia nigra pars compacta. The finding that CoQ₁₀ is effective in a chronic dosing model of MPTP toxicity, is of particular interest, as this may be more relevant to PD. These results provide further evidence that administration of CoQ₁₀ is a promising therapeutic strategy for the treatment of PD.     

Activation of Phospholipase A2 by the Nociceptin Receptor Expressed in Chinese Hamster Ovary Cells

Abstract: To gain insight into the molecular mechanism for nociceptin function, functional coupling of the nociceptin receptor expressed in Chinese hamster ovary (CHO) cells with phospholipase A₂ (PLA₂) was examined. In the presence of A23187, a calcium ionophore, activation of the nociceptin receptor induced time- and dose-dependent release of arachidonate, which was abolished by pretreatment of the cells with pertussis toxin (PTX). Immunoblot analysis using anti-Ca²⁺-dependent cytosolic PLA₂ (cPLA₂) monoclonal antibody demonstrates that activation of the nociceptin receptor induces a time- and dose-dependent electrophoretic mobility shift of cPLA₂, suggesting that phosphorylation of cPLA₂ is induced by the nociceptin receptor. Pretreatment of the cells with PD98059, a specific mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 inhibitor, or staurosporine, a potent inhibitor of serine/threonine protein kinases and tyrosine protein kinases, partially inhibited the nociceptin-induced cPLA₂ phosphorylation and arachidonate release. These results indicate that the nociceptin receptor expressed in CHO cells couples with cPLA₂ through the action of PTX-sensitive G proteins and suggest that cPLA₂ is activated by phosphorylation induced by the nociceptin receptor via mechanisms partially dependent on p44 and p42 mitogen-activated protein kinases.     

Formation of a Disulfide Bond in the Immunoglobulin Domain of the Myelin P0 Protein Is Essential for Its Adhesion

Abstract: It is widely accepted, although never demonstrated, that the formation of a disulfide bond in the majority of immunoglobulin (Ig)-like domains stabilizes their final conformation and thus is essential to their functioning as adhesion/recognition molecules. The myelin P₀ protein, which has been shown directly to behave as a homophilic adhesion molecule, contains a single Ig-like domain, stabilized by a putative Cys²¹-Cys⁹⁸ disulfide bond. To test if this bond is indeed necessary to the adhesive function of P₀, the nucleotides in the P₀ cDNA coding for Cys²¹ were altered to code for an alanine. The mutated P₀ cDNA was transfected into Chinese hamster ovary cells, expression of the mutated P₀ protein was characterized, and the adhesiveness of Cys²¹-mutated P₀-expressing cells and that of cells expressing equivalent surface amounts of the unmutated protein were compared. It was found, as we previously reported, that incubation of a single cell suspension of the unmutated P₀-expressing cells resulted in the rapid formation of large aggregates. In contrast, after a similar incubation the cells expressing the Cys²¹-mutated P₀ were still mostly single cells, a result indistinguishable from that observed with the control transfected cells. This suggests that the P₀ protein, when mutated at Cys²¹, does not behave as a homophilic adhesion molecule, which in turn implies that the formation of an Ig domain disulfide bond is essential to the functioning of this molecule.     

Functional Modulation of P2×2 Receptors by Cyclic AMP-Dependent Protein Kinase

Abstract: It is generally believed that protein phosphorylation is an important mechanism through which the functions of voltage- and ligand-gated channels are modulated. The intracellular carboxyl terminus of P_2×2 receptor contains several consensus phosphorylation sites for cyclic AMP (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC), suggesting that the function of the P_2×2 purinoceptor could be regulated by the protein phosphorylation. Whole-cell voltage-clamp recording was used to record ATP-evoked cationic currents from human embryonic kidney (HEK) 293 cells stably transfected with the cDNA encoding the rat P_2×2 receptor. Dialyzing HEK 293 cells with phorbol 12-myristate 13-acetate, a PKC activator, failed to affect the amplitude and kinetics of the ATP-induced cationic current. The role of PKA phosphorylation in modulating the function of the P_2×2 receptor was investigated by internally perfusing HEK 293 cells with 8-bromo-cAMP or the purified catalytic subunit of PKA. Both 8-bromo-cAMP and PKA catalytic subunit caused a reduction in the magnitude of the ATP-activated current without affecting the inactivation kinetics and the value of reversal potential. Site-directed mutagenesis was also performed to replace the intracellular PKA consensus phosphorylation site (Ser⁴³¹) with a cysteine residue. In HEK 293 cells expressing (S431C) mutant P_2×2 receptors, intracellular perfusion of 8-bromo-cAMP or purified PKA catalytic subunit did not affect the amplitude of the ATP-evoked current. These results suggest that as with other ligand-gated ion channels, protein phosphorylation by PKA could play an important role in regulating the function of the P_2×2 receptor and ATP-mediated physiological effects in the nervous system.     

Anti-Candida activity of four antifungal benzothiazoles

Abstract Anti- Candida activity of 6-amino-2- n -pentylthiobenzothiazole (I), benzylester of (6-amino-2-benzothiazolylthio)acetic acid (II) and of 3-butylthio-(1,2,4-triazolo)-2,3-benzothiazole (III) was followed and compared to that of 2-mercaptobenzothiazole (IV). I and II exhibited good activity against the C. albicans yeast form, similar to IV. They were inhibitorily active against other Candida strains, IC₅₀ values being of the order of 10⁻⁵ M, which means better activity than IV. Compound I also exhibited inhibitory activity on germ-tube formation and mycelial growth in the C. albicans strains, while II, III and IV were not active in these tests. III was the least active form of the compounds tested, IC₅₀ values being of the order of 10⁻⁴ M. All the compounds tested were highly active on a nystatin-resistant C. albicans mutant, with IC₅₀s of the order of 10⁻⁶ M−10⁻⁵ M.     

Broad-spectrum antiviral effect of Agrimonia pilosa extract on influenza viruses

Influenza virus continues to emerge and re-emerge, posing new threats for humans. Here we tested various Korean medicinal plant extracts for potential antiviral activity against influenza viruses. Among them, an extract of Agrimonia pilosa was shown to be highly effective against all three subtypes of human influenza viruses including H1N1 and H3N2 influenza A subtypes and influenza B virus. The EC₅₀ value against influenza A virus, as tested by the plaque reduction assay on MDCK cells, was 14–23 μg/ml. The extract also exhibited a virucidal effect at a concentration of 160–570 ng/ml against influenza A and B viruses when the viruses were treated with the extract prior to plaque assay. In addition, when tested in embryonated chicken eggs the extract exhibited a strong inhibitory effect in ovo on the H9N2 avian influenza virus at a concentration of 280 ng/ml. Quantitative RT-PCR analysis data showed that the extract, to some degree, suppressed viral RNA synthesis in MDCK cells. HI and inhibition of neuraminidase were observed only at high concentrations of the extract. And yet, the extract's antiviral activity required direct contact between it and the virus, suggesting that its antiviral action is mediated by the viral membrane, but does not involve the two major surface antigens, HA and NA, of the virus. The broad-spectrum antiviral activity of Agrimonia pilosa extract on various subtypes of influenza viruses merits further investigation as it may provide a means of managing avian influenza infections in poultry farms and potential avian-human transmission.     

Antiviral Activity of the Glucan Lichenan (Poly-β(→ 3,1 → 4)D-anhydroglucose)

Lichenan a β(1→ 3,1→ 4)β-glucan exhibited a broad antiviral activity against mechanically transmitted viruses of different taxonomic groups in different Nicoliana spp. As shown for tobacco mosaic virus in vitro . lichenan did not interfere with cell-free protein synthesis programmed with naked RNA or virus particles cotranslationally disassembled. Thus, inhibition of virus replication by lichenan is probably not attributed to direct interaction of lichenan and viral structural components, but rather to reduced sensitivity of the host tissue. Treatment of a suspension of cultured cells of Nicotiana tabacum with lichenan was accompanied by a slight increase in phenylalanine ammonia-lyase (PAL) activity. However, lichenan applied to leaf tissue did not significantly stimulate PAL-activity, therefore, antiviral action apparently did not depend on the induction of the phenylpropanoid pathway. According to experiments with lichenan and digitonin (used as a callose elicitor) restriction of viruses to initially-infected cells by means of callose deposition also appears to be an improbable mechanism of viral inhibition. In cell suspensions treated with lichenan, slight changes in the extracellular concentration of free K⁺ were observed that did not reflect a marked K⁺ leakage, as caused by digitonin. This finding requires further examination. According to the present results, it is likely that lichenan affects the early virus-cell interactions that occur after virus disassembly.     

Presynaptic Mechanism of Action of 4-Aminopyridine: Changes in Intracellular Free Ca2+ Concentration and Its Relationship to B-50 (GAP-43) Phosphorylation

Abstract: Recently we have shown that 4-aminopyridine (4-AP), a drug known to enhance transmitter release, stimulates the phosphorylation of the protein kinase C substrate B-50 (GAP-43) in rat brain synaptosomes and that this effect is dependent on the presence of extracellular Ca²⁺. Hence, we were interested in the relationship between changes induced by 4-AP in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and B-50 phosphorylation in synaptosomes. 4-AP (100 μ M ) elevates the [Ca²⁺]_i (as determined with fura-2) to approximately the same extent as depolarization with 30 m M K⁺ (from an initial resting level of 240 n M to ∼480 n M after treatment). However, the underlying mechanisms appear to be different: In the presence of 4-AP, depolarization with K⁺ still evoked an increase in [Ca²⁺]_i, which was additive to the elevation caused by 4-AP. Several Ca²⁺ channel antagonists (CdCl₂, LaCl₃, and diphenylhydantoin) inhibited the increase in B-50 phosphorylation by 4-AP. It is interesting that the increase in [Ca²⁺]_i and the increase in B-50 phosphorylation by 4-AP were attenuated by tetrodotoxin, a finding pointing to a possible involvement of Na⁺ channels in this action. These results suggest that 4-AP (indirectly) stimulates both Ca²⁺ influx and B-50 phosphorylation through voltage-dependent channels by a mechanism dependent on Na⁺ channel activity.     

An Endogenous Aminoenkephalinase Inhibitor: Purification and Characterization of Arg0-Met5-Enkephalin from Bovine Striatum

Abstract: Arg⁰-Met⁵-enkephalin (Arg⁰-MEK) was isolated from bovine striatum and purified to homogeneity. The peptide was extracted with trichloroacetic acid, followed by column chromatography successively on Bio-Sil C₈, semipreparative HPLC Radial-Pak C₁₈, fast protein liquid chromatography (FPLC) Mono S, HPLC Ultrasphere-ODS, Supelco C₁₈, Lichromsorb C₁₈, and μBondapak C₁₈. The peptide content was followed by radioimmunoassay with an antibody against synthetic Met-enkephalin. For each of the six HPLC and FPLC systems, the elution time of the immunoreactive fractions coincided exactly with that of synthetic Arg⁰-MEK. The purified peptide showed a highly homogeneous profile in three different analytical HPLC systems. Its retention time and three-dimensional UV spectrum were identical to those of the synthetic Arg⁰-MEK. The structure of the purified material was identified by microsequencing as the hexapeptide Arg-Tyr-Gly-Gly-Phe-Met. Ninety percent of the purified peptide was in oxidized form containing equimolar amounts of Met-( R )- and Met-( S )-sulfoxide. The reduced Arg⁰-MEK inhibited aminoenkephalinase with a K _i of 2.2 µ M , and its sulfoxide analogue inhibited it with a K _i of 8.9 µ M . The reduced or oxidized peptide suppressed the electrically induced contraction of rat vas deferens with an ED₅₀ of 5 µ M , and the effect could be reversed by equimolar naloxone. Our data indicate that Arg⁰-MEK is an immediate Met-enkephalin precursor and an endogenous inhibitor.