Photo-control of nitric oxide synthase activity using a caged isoform specific inhibitor

Nitric oxide (NO) plays a critical role in a number of physiological processes and is produced in mammalian cells by nitric oxide synthase (NOS) isozymes. Because of the diverse functions of NO, pharmaceutical interventions which seek to abrogate adverse effects of excess NOS activity must not interfere with the normal regulation of NO levels in the body. A method has been developed for the control of NOS enzyme activity using the localized photochemical release of a caged isoform-specific NOS inhibitor. The caged form of an iNOS inhibitor has been synthesized and tested for photosensitivity and potency. UV and multiphoton uncaging were verified using a hemoglobin-based assay. IC(50) values were determined for the inhibitor (70+/-11 nM), the caged inhibitor (1098+/-172 nM), the UV uncaged inhibitor (67+/-26 nM) and the multiphoton uncaged inhibitor (73+/-11 nM). UV irradiation of the caged inhibitor resulted in a 86% reduction in iNOS activity after 5 min. Multiphoton uncaging had an apparent first order time constant of 0.007+/-0.001 min(-1). A therapeutic range exists, with molar excess of inhibitor to enzyme from 3- to 7-fold, over which the full dynamic range of the inhibition can be exploited.     

Bichromophoric fluorescent photolabile protecting group for alcohols and carboxylic acids

Novel bichromophoric fluorescent photolabile protecting group, (5-dansyloxy-3-hydroxynaphthalen-2-yl)methyl (DNS-NQMP), allows for the independent photochemical release and fluorescent imaging of caged substrates. Irradiation of DNS-NQMP-caged alcohols and carboxylic acids with 300 or 350 nm light results in fast (k(release) ~ 10(5) s(-1)), efficient (Φ = 0.2), and quantitative release of the substrates. This uncaging chemistry is compatible with aqueous media and DNS-NQMP-protected hydroxy compounds are hydrolytically stable at neutral pH. Upon excitation with 400 nm light, caged compounds show intense green emission (λ(max) = 559 nm) with 21% fluorescence quantum yield. Fluorescent readout conducted using 400 nm or longer wavelengths does not cause substrate release. The DNS-NQMP chromophore retains its fluorescent properties after photo-uncaging reaction.     

The Reaction Rates of NO with Horseradish Peroxidase Compounds I and II

In this study the reactions between nitric oxide (NO) and horseradish peroxidase (HRP) compounds I and II were investigated. The reaction between compound I and NO has biphasic kinetics with a clearly dominant initial fast phase and an apparent second-order rate constant of (7.0 +/- 0.3) x 10(5) M(-1) s(-1) for the fast phase. The reaction of compound II and NO was found to have an apparent second-order rate constant of k(app) = (1.3 +/- 0.1) x 10(6) M(-1) s(-1) or (7.4 +/- 0.7) x 10(5) M(-1) s(-1) when measured at 409 nm (the isosbestic point between HRP and HRP-NO) and 419 nm (lambda(max) of compound II and HRP-NO), respectively. Interestingly, the reaction of compound II with NO is unusually high relative to that of compound I, which is usually the much faster reaction. Since horseradish peroxidase is prototypical of mammalian peroxidases with respect to the oxidation of small substrates, these results may have important implications regarding the lifetime and biochemistry of NO in vivo after inflammation where both NO and H(2)O(2) generation are increased several fold.     

Activity of ovarian nitric oxide synthase (NOs) during ovulatory process in the rat: relationship with prostaglandins (PGs) production.

Nitric oxide (NO) is synthesized by the rat ovary and a role in the follicular development, the ovulation, and the luteal formation has been postulated. The aims this study were to determine the activity of nitric oxide synthase (NOs) enzyme during the ovulatory process and to demonstrate the existence of a relationship between the ovarian NO production and the synthesis of prostaglandins (PGs) involved in the follicular rupture. Prepuberal rats treated with PMSG/hCG to induce ovulation were used. The NOs activity, measured by [(14)C]citrulline formation, showed an increase after PMSG administration and reached a maximum at 10 h after hCG injection. NOs activity remained high up to 24 h post ovulation. At 10 h after the hCG injection, the activity of Ca(2+)-dependent NOs (constitutive NOs) was similar to that seen at 0 h, and the activity of Ca(2+)-independent NOs (inducible NOs) increased from 14.4 to 51% of total activity. The in vitro ovarian production of PGE and PGF(2alpha) was inhibited by L-NAME and stimulated by 3-morpho-linosydnonimine (SIN-1), a NO donor. The in vivo production of ovarian prostaglandins was also inhibited by the intrabursal administration of two NOs inhibitors, N(G)-nitro-L-arginine methyl ester (L-NAME) and N(G)-monomethyl-L-arginine (L-NMMA). Our results suggest that the inducible NOs (iNOs) is the main isoform involved in the ovulatory process and that the NO produced stimulates the synthesis of both PGE and PGF(2alpha) from the cyclooxygenase pathway, to enhance the process of follicle rupture.     

Tryptophan-derived ultraviolet filter compounds covalently bound to lens proteins are photosensitizers of oxidative damage

The human eye is chronically exposed to light of wavelengths >300 nm. In the young human lens, light of wavelength 300-400 nm is predominantly absorbed by the free Trp derivatives kynurenine (Kyn), 3-hydroxykynurenine (3OHKyn), and 3-hydroxykynurenine-O-beta-D-glucoside (3OHKynG). These ultraviolet (UV) filter compounds are poor photosensitizers. With age, the levels of the free UV filters in the lens decreases and those of protein-bound UV filters increases. The photochemical behavior of these protein-bound UV filters and their role in UV damage are poorly elucidated and are examined here. UVA illumination of protein-bound UV filters generated peroxides (principally H2O2) in a metabolite-, photolysis-time-, and wavelength-dependent manner. Unmodified proteins, free Trp metabolites, and Trp metabolites that do not bind to lens proteins gave low peroxide yields. Protein-bound 3OHKyn (principally at Cys residues) yielded more peroxide than comparable Kyn and 3OHKynG adducts. Studies using D2O and sodium azide implicated 1O2 as a key intermediate. Illumination of the protein-bound adducts also yielded protein-bound Tyr oxidation products (DOPA, di-tyrosine) and protein cross-links via alternative mechanisms. These data indicate that the covalent modification of lens proteins by Kyn derivatives yields photosensitizers that may enhance oxidation in older lenses and contribute to age-related nuclear cataract.     

Flash photolysis of caged nitric oxide inhibits proximal tubular fluid reabsorption in free-flow nephron

A nonobstructing optical method was developed to measure proximal tubular fluid reabsorption in rat nephron at 0.25 Hz. The effects of uncaging luminal nitric oxide (NO) on proximal tubular reabsorption were investigated with this method. Proximal fluid reabsorption rate was calculated as the difference of tubular flow measured simultaneously at two locations (0.8-1.8 mm apart) along a convoluted proximal tubule. Tubular flow was estimated on the basis of the propagating velocity of fluorescent dextran pulses in the lumen. Changes in local tubular flow induced by intratubular perfusion were detected simultaneously along the proximal tubule, indicating that local tubular flow can be monitored in multiple sites along a tubule. The estimated tubular reabsorption rate was 5.52 +/- 0.38 nl.min(-1).mm(-1) (n = 20). Flash photolysis of luminal caged NO (potassium nitrosylpentachlororuthenate) was induced with a 30-Hz UV nitrogen-pulsed laser. Release of NO from caged NO into the proximal tubule was confirmed by monitoring intracellular NO concentration using a cell-permeant NO-sensitive fluorescent dye (DAF-FM). Emission of DAF-FM was proportional to the number of laser pulses used for uncaging. Photolysis of luminal caged NO induced a dose-dependent inhibition of proximal tubular reabsorption without activating tubuloglomerular feedback, whereas uncaging of intracellular cGMP in the proximal tubule decreased tubular flow. Coupling of this novel method to measure reabsorption with photolysis of caged signaling molecules provides a new paradigm to study tubular reabsorption with ambient tubular flow.     

Flash photolysis using a light emitting diode: an efficient, compact, and affordable solution

Flash photolysis has become an essential technique for dynamic investigations of living cells and tissues. This approach offers several advantages for instantly changing the concentration of bioactive compounds outside and inside living cells with high spatial resolution. Light sources for photolysis need to deliver pulses of high intensity light in the near UV range (300-380 nm), to photoactivate a sufficient amount of molecules in a short time. UV lasers are often required as the light source, making flash photolysis a costly approach. Here we describe the use of a high power 365 nm light emitting diode (UV LED) coupled to an optical fiber to precisely deliver the light to the sample. The ability of the UV LED light source to photoactivate several caged compounds (CMNB-fluorescein, MNI-glutamate, NP-EGTA, DMNPE-ATP) as well as to evoke the associated cellular Ca(2+) responses is demonstrated in both neurons and astrocytes. This report shows that UV LEDs are an efficient light source for flash photolysis and represent an alternative to UV lasers for many applications. A compact, powerful, and low-cost system is described in detail.     

Nitric oxide inhibits LPS-induced tumor necrosis factor synthesis in vitro and in vivo

The effect of nitric oxide (NO) on LPS-stimulated TNF-α synthesis has been studied in vitro and in vivo. The synthesis of TNF-α in J774 macrophages stimulated with LPS (0.l μg/ml) was increased in concentration-related fashion by NO synthase inhibitor L-NMMA (3-30-300 μM) and reduced by either L-arginine (3-30-300 μM) or the NO donor SIN-1 (1-10-1OOμM). The level of TNF-α in the serum of LPS-challenged rats (6mg/kg/i.p.) was increased in animals pre-treated s.c. with L-NMMA (10 and 50mg/kg) and reduced in those given L- arginine (100 and 300mg/kg). These results show a negative feedback mechanism exhibited by NO on TNF-α synthesis suggesting an important regulatory link between NO and TNF-α in pathological processes.     

A spectrophotometric method for the direct detection and quantitation of nitric oxide, nitrite, and nitrate in cell culture media

A method for the spectrophotometric determination of nitric oxide, nitrite, and nitrate in tissue culture media is presented. The method is based on the nitric oxide-mediated nitrosative modification of sulfanilic acid that reacts with N-(1-naphthyl)ethylenediamine dihydrochloride forming an orange-colored product absorbing at 496 nm. Nitric oxide levels were determined in culture media from this absorbance measurement using chemiluminescence standardization. Extinction coefficients of 5400 and 6600 M(-1) cm(-1) were determined for the nitric oxide product in assay solutions containing 0.1 or 100 mM KPO4 buffer (pH 7.4), respectively, with a limit of detection of 1 microM. Acidification of these reactions (pH 2.4) generated a pink-colored product absorbing at 540 nm allowing for quantitation of total nitric oxide/nitrite levels using extinction coefficients of 38,000 and 36,900 M(-1) cm(-1), for the assay solutions described. The limit of detection of this assay was approximately 300 nM. Using the 100 mM KPO4 buffer system, nitrate levels were determined following reduction to nitrite using a copper-coated cadmium reagent with an extinction coefficient of 29,500 M(-1) cm(-1) and a detection limit of 0.5 microM. The utility of these assays was demonstrated in the standardization of nitric oxide-saturated cell culture media, and the release of nitric oxide by the NONOate compound DEA/NO.     

The macrocyclic effect and vasodilation response based on the photoinduced nitric oxide release from trans-[RuCl(tetraazamacrocycle)NO](2+)

Irradiation of trans-[RuCl(cyclam)(NO)](2+), cyclam is 1,4,8,11-tetraazacyclotetradecane, at pHs 1-7.4, with near UV light results in the release of NO and formation of trans-[Ru(III)Cl(OH)(cyclam)](+) with pH dependent quantum yields (from approximately 0.01 to 0.16 mol Einstein(-1)) lower than that for trans-[RuCl([15]aneN(4))(NO)](2+), [15]aneN(4) is 1,4,8,12-tetaazacyclopentadecane, (0.61 mol Einstein(-1)). After irradiation with 355 nm light, the trans-[RuCl([15]aneN(4))(NO)](2+) induces relaxation of the aortic ring, whereas the trans-[RuCl(cyclam)(NO)](2+) complex does not. The relaxation observed with trans-[RuCl([15]aneN(4))(NO)](2+) is consistent with a larger quantum yield of release of NO from this complex.     

Synthesis and biological evaluation of a novel photo-activated histone deacetylase inhibitor

Hydroxamic acid-based histone deacetylase inhibitors (HDACi) are a class of epigenetic agents with potentially broad therapeutic application to several disease states including post angioplasty mediated neointimal hyperplasia (NIH). Precise spatiotemporal control over the release of HDACi at the target blood vessel site is required for the safe and successful therapeutic use of HDACi in the setting of drug eluting balloon catheter (DEBc) angioplasty treatment of NIH. We aimed to develop and characterise a novel photoactive HDACi, as a potential coating agent for DEBc.Metacept-3 1 was caged with a photo-labile protecting group (PPG) to synthesise a novel UV365nm active HDACi, caged metacept-3 15. Conversion of caged metacept-3 15 to active/native metacept-3 1 by UV365nm was achievable in significant quantities and at UV365nm power levels in the milliwatt (mW) range.In vitro evaluation of the biological activity of pre and post UV365nm activation of caged metacept-3 15 identified significant HDACi activity in samples exposed to short duration, mW range UV365nm. Toxicity studies performed in human umbilical vein endothelial cells (HUVEC’s) identified significantly reduced toxicity of caged metacept-3 15 pre UV365nm exposure compared with native metacept-3 1 and paclitaxel (PTX).Taken together these findings identify a novel photo-activated HDACi, caged metacept-3 15, with pharmacokinetic activation characteristics and biological properties which may make it suitable for evaluation as a novel coating for targeted DEBc angioplasty interventions.     

P(3)-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP for the rapid activation of the Na(+),K(+)-ATPase

P(3)-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP (pHP-caged ATP) has been investigated for its application as a phototrigger for the rapid activation of electrogenic ion pumps. The yield of ATP after irradiation with a XeCl excimer laser (lambda = 308 nm) was determined at pH 6.0-7.5. For comparison, the photolytic yields of P(3)-[1-(2-nitrophenyl)]ethyl ATP (NPE-caged ATP) and P(3)-[1, 2-diphenyl-2-oxo]ethyl ATP (desyl-caged ATP) were also measured. It was shown that at lambda = 308 nm pHP-caged ATP is superior to the other caged ATP derivatives investigated in terms of yield of ATP after irradiation. Using time-resolved single-wavelength IR spectroscopy, we determined a lower limit of 10(6) s(-1) for the rate constant of release of ATP from pHP-caged ATP at pH 7.0. Like NPE-caged ATP, pHP-caged ATP and desyl-caged ATP bind to the Na(+), K(+)-ATPase and act as competitive inhibitors of ATPase function. Using pHP-caged ATP, we investigated the charge translocation kinetics of the Na(+),K(+)-ATPase at pH 6.2-7.4. The kinetic parameters obtained from the electrical measurements are compared to those obtained with a technique that does not require caged ATP, namely parallel stopped-flow experiments using the voltage-sensitive dye RH421. It is shown that the two techniques yield identical results, provided the inhibitory properties of the caged compound are taken into account. Our results demonstrate that under physiological (pH 7.0) and slightly basic (pH 7.5) or acidic (pH 6. 0) conditions, pHP-caged ATP is a rapid, effective, and biocompatible phototrigger for ATP-driven biological systems.     

Cooperativity in the dissociation of nitric oxide from hemoglobin.

The dissociation of nitric oxide from hemoglobin, from isolated subunits of hemoglobin, and from myoglobin has been studied using dithionite to remove free nitric oxide. The reduction of nitric oxide by dithionite has a rate of 1.4 X 10(3) M-1 S-1 at 20 degrees in 0.05 M phosphate, pH 7.0, which is small compared with the rate of recombination of hemoglobin with nitric oxide (25 X 10(6) M-1 S-1 (Cassoly, R., and Gibson, Q. H. (1975) J. Mol. Biol. 91, 301-313). The rate of NO combination with chains and myoglobin was found to be 24 X 10(6) M-1 S-1 and 17 X 10(6) M-1 S-1, respectively. Hence, the observed progress curve of the dissociation of nitric oxide is dependent upon the dithionite concentration and the total heme concentration. Addition of excess carbon monoxide to the dissociation mixture reduces the free heme yielding a single exponential process for chains and for myoglobin which is dithionite and heme concentration independent over a wide range of concentrations. The rates of dissociation of nitric oxide from alpha chains, from beta chains, and from myoglobin are 4.6 X 10(-5) S-1, 2.2 X 10(-5) S-1, and 1.2 X 10(4) S-1, respectively, both in the presence and in the absence of carbon monoxide at 20 degrees in 0.05 M phosphate, pH 7.0. Analogous heme and dithionite concentration dependence is found for the dissociation of nitric oxide from tetrameric hemoglobin. The reaction is cooperative, the intrinsic rate constants for the dissociation of the 1st and 4th molecules of NO differing about 100-fold. With hemoglobin, replacement of NO by CO at neutral pH is biphasic in phosphate buffers. The rate of the slow phase is 1 X 10(-5) S-1 and is independent of pH. The amplitude of the fast phase increases with lowering of pH. By analogy with the treatment of the HbCO + NO reaction given by Salhany et al. (Salhany, J.M., Ogawa, S., and Shulman, R.G. (1975) Biochemistry 14, 2180-2190), the fast phase is attributed to the dissociation of NO from T state molecules and the slow phase to dissociation from R state molecules. Analysis of the data gives a pH-independent value of 0.01 for the allosteric constant c (c = Kr/Kt where Kr and Kt are the dissociation constants for NO from the R and T states, respectively) and pH-dependent values of L (2.5 X 10(7) at pH 7 in 0.05 M phosphate buffer). The value of c is considerably greater than that for O2 and CO. Studies of the difference spectrum induced in the Soret region by inositol hexaphosphate are also reported. This spectrum does not arise directly from the change of conformation between R and T states. The results show that if the equilibrium binding curve for NO could be determined experimentally, it would show cooperativity with Hill's n at 50% saturation of about 1.6.     

The first supramolecular orthovanadate receptor -- structural mimics of vanadium haloperoxidase

Tris(2-guanidinium-ethyl)amine (1) was prepared as a supramolecular receptor of hydrogen orthovanadate (HVO(4)(2-)) to mimic the active site of vanadium haloperoxidase (V-HPO). Both (1)H and (51)V NMR titration indicated 1:1 complex (5) formation between (1) with HVO(4)(2-) with a binding constant of 1.1 x 10(3) M(-1). Similar as V-HPO, a UV band at 307 nm was observed upon binding of HVO(4)(2-) to (1). According to DFT calculations UV transitions >300 nm observed for both the enzyme and its mimic are due to V-N interactions.     

Effects of oxidizing and reducing agents on ovine pulmonary artery responses to nitric oxide donors, sodium nitroprusside and 3-morpholino-sydnonimine

Nitrovasodilators-sodium nitroprusside (SNP; 10(-9)-10(-4) M) and 3-morpholino-sydnonimine (SIN-1; 10(-9)-10(-4) M) produced concentration-dependent relaxation of the fourth generation sheep pulmonary artery, preconstricted with 5-hydroxytryptamine (1 microM). Oxidizing agents [oxidized glutathione (GSSG, 1 mM) and CuSO4 (5 and 20 microM)] and reducing agents [dithiothreitol (DTT, 0.1 mM), ascorbic acid (1 mM) and reduced glutathione (GSH, 1 mM)] caused opposite effects on nitric oxide (NO)-induced vasodilation in the artery. Ascorbic acid and GSH potentiated the NO responses, while GSSG and CuSO4 inhibited relaxation caused by the nitrovasodilators. DTT, however, reduced the relaxant potency and efficacy of SNP and SIN-1. Pretreatment of the pulmonary artery strips with DTT (0.1 mM) inhibited SNP (10 microM)-induced Na(+)-K(+)-ATPase activity, while ascorbic acid (1 mM) and GSH (1 mM) had no effect either on basal or SNP (10 microM)-stimulated 86Rb uptake, an index of Na(+)-K(+)-ATPase activity, in ovine pulmonary artery. The results suggest that reducing agents like ascorbic acid may have beneficial effect in improving the vascular function under oxidative stress.     

S-nitrosoglutathione incorporated in poly(ethylene glycol) matrix: potential use for topical nitric oxide delivery.

Incorporation of nitric oxide (NO) donors in non-toxic polymeric matrices can be a useful strategy for allowing topical NO delivery. We have incorporated the NO-donor S-nitrosoglutathione (GSNO) into a liquid poly(ethylene glycol) (PEG)/H2O matrix through the S-nitrosation of GSH by a NO/O2 gas mixture. Kinetic measurements of GSNO decomposition associated with NO release were performed at 25, 35, and 45 degrees C in the dark and under irradiation with UV/Vis light, lambda>480 nm and lambda=333 nm. NO release from the liquid matrix to the gas phase was confirmed by mass spectrometry. The PEG/H2O matrix stabilizes GSNO leading to expressive reductions in the initial rates of thermal and photochemical NO release, compared to aqueous GSNO solution. This matrix effect is assigned to diffusional constrains imposed on the escape of the NO and GS radicals formed in the solvent cage. This effect allows the storage of PEG-GSNO formulations for extended periods (more than 65 days at freezer) with negligible decomposition. PEG-GSNO formulation seems therefore to be applicable in topical NO delivery and GSNO displays potential as a percutaneous absorption enhancer. Moreover, the rate of NO release can be locally increased by irradiation with visible light.     

UV-Induced destruction of light-harvesting complexes from purple bacterium Chromatium minutissimum

We studied UV-induced photodestruction of the native forms of bacteriochlorophyll a (Bchl a) from chromatophores and light harvesting complexes (LHC) of the sulphur photosynthetic bacterium Chromatium minutissimum. Irradiation of chromato- phores with 365-nm light (Soret band) or 280-nm light (absorption region of aromatic amino acids) led to the destruction of all long-wavelength forms of Bchl a. The quantum yields of photodestruction produced by the 280-nm light was higher than that produced by the 365-nm light. For the spectral forms of Bchl a absorbing at 850 nm and 890 nm, the difference was about one order of magnitude, and for the form absorbing at 800 nm the difference was almost two orders of magnitude. Similar UV sensitivity was observed for the Bchl a forms from isolated LHC. As a rule, the quantum yields of photodestruction induced by UV irradiation at 280 nm were about 100-1000 times higher (approximately 10(-3)-10(-4)) than that upon red light irradiation (approximately 10(-6)-10(-7)). We found that irradiation of chromatophores at 280 nm resulted in a crosslink between the core and peripheral LHC.     

A protecting group for carboxylic acids that can be photolyzed by visible light

We report on a photolabile protecting (caging) group that is new for carboxylic acids. Unlike previously used caging groups for carboxylic acids, it can be photolyzed rapidly and efficiently in the visible wavelength region. The caging group 7-N,N-diethyl aminocoumarin (DECM) was used to cage the gamma-carboxyl group of glutamic acid, which is also a neurotransmitter. The caged compound has a major absorption band with a maximum at 390 nm (epsilon(390) = 13651 M(-)(1) cm(-)(1)). Experiments are performed at 400 nm (epsilon(400) = 12232 M(-)(1) cm(-)(1)) and longer wavelengths. DECM-caged glutamate is water soluble and stable at pH 7.4 and 22 degrees C. It photolyzes rapidly in aqueous solution to release glutamic acid within 3 micros with a quantum yield of 0.11 +/- 0.008 in the visible region. In whole-cell current-recording experiments, using HEK-293 cells expressing glutamate receptors and visible light for photolysis, DECM-caged glutamate and its photolytic byproducts were found to be biologically inert. Neurotransmitter receptors that are activated by various carboxyl-group-containing compounds play a central role in signal transmission between approximately 10(12) neurons of the nervous system. Caged neurotransmitters have become an essential tool in transient kinetic investigations of the mechanism of action of neurotransmitter receptors. Previously uncaging the compounds suitable for transient kinetic investigations required ultraviolet light and expensive lasers, and, therefore, special precautions. The availability of caged neurotransmitters suitable for transient kinetic investigations that can be photolyzed by visible light allows the use of simple-to-use, readily available inexpensive light sources, thereby opening up this important field to an increasing number of investigators.     

Polyamines induce rapid biosynthesis of nitric oxide (NO) in Arabidopsis thaliana seedlings

In this study, we examined the regulation by putrescine, spermidine and spermine of nitric oxide (NO) biosynthesis in Arabidopsis thaliana seedlings. Using a fluorimetric method employing the cell-impermeable NO-binding dye diaminorhodamine-4M (DAR-4M), we observed that the polyamines (PAs) spermidine and spermine greatly increased NO release in the seedlings, whereas arginine and putrescine had little or no effect. Spermine, the most active PA, stimulated NO release with no apparent lag phase. The response was quenched by addition of 2-aminoethyl-2-thiopseudourea (AET), an inhibitor of the animal nitric oxide synthase (NOS) and plant NO biosynthesis, and by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-1-oxy-3-oxide (PTIO), an NO scavenger. By fluorescence microscopy, using the cell-permeable NO-binding dye diaminorhodamine-4M acetoxymethyl ester (DAR-4M AM), we observed that PAs induced NO biosynthesis in specific tissues in Arabidopsis seedlings. Spermine and spermidine increased NO biosynthesis in the elongation zone of the Arabidopsis root tip and in primary leaves, especially in the veins and trichomes, while in cotyledons little or no effect of PAs beyond the endogenous levels of NO-induced fluorescence was observed. We conclude that PAs induce NO biosynthesis in plants.     

Parallel double helical DNA. 1. Evidence for the existence of a spiral A-T-paired base

The dependence of UV and CD spectra of oligonucleotide 3'-d(ApTpApTpApTpApTpApTp)-O(CH2)6O-5'-(pApTpApTpApTpApTp ApT) (eicosamer) in aqueous solution at pH 7 in the presence of 0.5 M NaCl on temperature and concentration was studied. It was shown that the eicosamer in concentrations below 5.10(-4) M forms a parallel stranded hairpin. From the thermal denaturation profile the thermodynamic parameters of parallel hairpin formation were determined. The values of delta H0, delta S0 and Tm were -90 +/- 8 kJ/mol, -300 +/- 20 J.mol-1.K-1 and 40.5 degrees C, respectively. The CD spectra of the parallel helix differ from those of B-form DNA by reduction of extreme magnitude at approximately 265 nm and appearance of a negative effect at approximately 285 nm.