The allelopathic potential ofCoridothymus capitatus L. (Labiatae). Preliminary studies on the roles of the shrub in the inhibition of annuals germination and/or to promote allelopathically active actinomycetes

Summary Suppression of annuals at various intensities was observed around some shrubs ofCoridothymus capitatus growing on kurkar formation in the coastal hills of Israel. The phenomenon was clearly observed as annuals-free belts of 15–20 cm around ‘aggressive’ shrubs. Quantitatively, density of annuals decreased by 16 fold in the annual-free belts as compared to a distance of 60–80 cm from the canopies of the shrubs. Their dry matter was decreased by 5.4 fold around the shrubs. Suppression rate of emergence of planted seeds of annuals (Plantago psyllium andErucaria hispanica) early in the season was 45% higher around ‘aggressive’C. capitatus than that around ‘non-aggressive’ ones. In the laboratory, seed germination of the annuals was strongly suppressed by diffusates and volatiles from shoots, as well as from their water extracts and their essential oils. Incubation of fresh shoots ofC. capitatus in soil collected from around ‘non-aggressive’ shrubs, for 7 days, increased population levels of actinomycetes by 9.6 fold and by 36.7 fold when soil was collected from around ‘aggressive’ shrubs. Isolates of some soil-borne actinomycetes inhibited germination of the test plantsLactuca sativa andAnastatica hierochuntica on agar plates (4–98%). The preliminary results indicate a possible synergistic inhibitory effect induced by essential oils of the aromatic shrub and the phytototic activity of actinomycetes.     

Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides,unassisted by transfection reagents

For the past 15–20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called ‘gymnosis’) that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.     

Towards clarification of the biological role of microcystins, a family of cyanobacterial toxins

Microcystins constitute a serious threat to the quality of drinking water worldwide. These protein phosphatase inhibitors are formed by various cyanobacterial species, including Microcystis sp. Microcystins are produced by a complex microcystin synthetase, composed of peptide synthetases and polyketide synthases, encoded by the mcyA-J gene cluster. Recent phylogenetic analysis suggested that the microcystin synthetase predated the metazoan lineage, thus dismissing the possibility that microcystins emerged as a means of defence against grazing, and their original biological role is not clear. We show that lysis of Microcystis cells, either mechanically or because of various stress conditions, induced massive accumulation of McyB and enhanced the production of microcystins in the remaining Microcystis cells. A rise in McyB content was also observed following exposure to microcystin or the protease inhibitors micropeptin and microginin, also produced by Microcystis. The extent of the stimulation by cell extract was strongly affected by the age of the treated Microcystis culture. Older cultures, or those recently diluted from stock cultures, hardly responded to the components in the cell extract. We propose that lysis of a fraction of the Microcystis population is sensed by the rest of the cells because of the release of non-ribosomal peptides. The remaining cells respond by raising their ability to produce microcystins thereby enhancing their fitness in their ecological niche, because of their toxicity.     

Selective hydroboration of dieneamines. Formation of hydroxyalkylphenothiazines as MDR modulators

N-dienylphenothiazines synthesized from tetrazolo[1,5-a]pyridinium salts by treatment with phenothiazine were subjected to catalytic hydrogenation to yield N-butylphenothiazines, whereas transformation of these dienes with borane dimethyl sulfide (BH(3) × Me(2)S) resulted in selective hydroboration of one double bond and full reduction of the other double bond to give 2-hydroxybutylphenothiazines. Position of the hydroxyl group was supported by NMR spectroscopy and verified by X-ray analysis. Comparison of MDR modulatory activity of the new derivatives revealed that the hydroxybutyl compounds are promising candidates for development of novel MDR inhibitors.     

Identification of human plasma metabolites exhibiting time-of-day variation using an untargeted liquid chromatography-mass spectrometry metabolomic approach

Although daily rhythms regulate multiple aspects of human physiology, rhythmic control of the metabolome remains poorly understood. The primary objective of this proof-of-concept study was identification of metabolites in human plasma that exhibit significant 24-h variation. This was assessed via an untargeted metabolomic approach using liquid chromatography-mass spectrometry (LC-MS). Eight lean, healthy, and unmedicated men, mean age 53.6 (SD ± 6.0) yrs, maintained a fixed sleep/wake schedule and dietary regime for 1 wk at home prior to an adaptation night and followed by a 25-h experimental session in the laboratory where the light/dark cycle, sleep/wake, posture, and calorific intake were strictly controlled. Plasma samples from each individual at selected time points were prepared using liquid-phase extraction followed by reverse-phase LC coupled to quadrupole time-of-flight MS analysis in positive ionization mode. Time-of-day variation in the metabolites was screened for using orthogonal partial least square discrimination between selected time points of 10:00 vs. 22:00 h, 16:00 vs. 04:00 h, and 07:00 (d 1) vs. 16:00 h, as well as repeated-measures analysis of variance with time as an independent variable. Subsequently, cosinor analysis was performed on all the sampled time points across the 24-h day to assess for significant daily variation. In this study, analytical variability, assessed using known internal standards, was low with coefficients of variation <10%. A total of 1069 metabolite features were detected and 203 (19%) showed significant time-of-day variation. Of these, 34 metabolites were identified using a combination of accurate mass, tandem MS, and online database searches. These metabolites include corticosteroids, bilirubin, amino acids, acylcarnitines, and phospholipids; of note, the magnitude of the 24-h variation of these identified metabolites was large, with the mean ratio of oscillation range over MESOR (24-h time series mean) of 65% (95% confidence interval [CI]: 49-81%). Importantly, several of these human plasma metabolites, including specific acylcarnitines and phospholipids, were hitherto not known to be 24-h variant. These findings represent an important baseline and will be useful in guiding the design and interpretation of future metabolite-based studies.     

Utilizing ESEEM spectroscopy to locate the position of specific regions of membrane-active peptides within model membranes

Membrane-active peptides participate in many cellular processes, and therefore knowledge of their mode of interaction with phospholipids is essential for understanding their biological function. Here we present a new methodology based on electron spin-echo envelope modulation to probe, at a relatively high resolution, the location of membrane-bound lytic peptides and to study their effect on the water concentration profile of the membrane. As a first example, we determined the location of the N-terminus of two membrane-active amphipathic peptides, the 26-mer bee venom melittin and a de novo designed 15-mer D,L-amino acid amphipathic peptide (5D-L9K6C), both of which are antimicrobial and bind and act similarly on negatively charged membranes. A nitroxide spin label was introduced to the N-terminus of the peptides and measurements were performed either in H2O solutions with deuterated model membranes or in D2O solutions with nondeuterated model membranes. The lipids used were dipalmitoyl phosphatidylcholine (DPPC) and phosphatidylglycerol (PG), (DPPC/PG (7:3 w/w)), egg phosphatidylcholine (PC) and PG (PC/PG (7:3 w/w)), and phosphatidylethanolamine (PE) and PG (PE/PG, 7:3w/w). The modulation induced by the 2H nuclei was determined and compared with a series of controls that produced a reference "ruler". Actual estimated distances were obtained from a quantitative analysis of the modulation depth based on a simple model of an electron spin situated at a certain distance from the bottom of a layer with homogeneously distributed deuterium nuclei. The N-terminus of both peptides was found to be in the solvent layer in both the DPPC/PG and PC/PG membranes. For PE/PG, a further displacement into the solvent was observed. The addition of the peptides was found to change the water distribution in the membrane, making it "flatter" and increasing the penetration depth into the hydrophobic region.     

Influence of ancillary ligand L in the nitric oxide photorelease by the [Ru(L)(tpy)NO] complex and its vasodilator activity based on visible light irradiation

The photochemical and pharmacological studies of the novel [Ru(L)(tpy)NO]³⁺ L = bpy (2,2′-bipyridine), NH · NHq (quinonediimine) and NH₂.NH₂cat (o-phenylenediamine) were investigated in aqueous medium. The synthesized nitrosyl ruthenium complexes showed nitric oxide (NO) release under light irradiation at 355 nm for [Ru(L)(tpy)NO]³⁺ complex with quantum yield of 0.14 ± 0.02, 0.47 ± 0.03 and 0.46 ± 0.02 mol Einstein⁻¹ for L = bpy, NH · NHq and NH₂ · NH₂cat, respectively, and 0.0065 ± 0.001 mol Einstein⁻¹ for light irradiation at 532 nm for [Ru(NH · NHq)(tpy)NO]³⁺ complex. The photochemical pathway at 355 nm light irradiation was described as a multi-step mechanism, although at 532 nm it was better attributed to a photo-induced electron transfer. The vasorelaxation induced by NO release produced by light irradiation in visible region from physiological solution of [Ru(NH · NHq)(tpy)NO]³⁺ complex was evaluated and compared with sodium nitroprusside (SNP). The results showed very similar vasodilator power between both species.     

Chronic methionine load-induced hyperhomocysteinemia impairs the relaxation induced by bradykinin in the isolated rat carotid

This study investigates the effects of chronic methionine intake on bradykinin (BK)-relaxation. Vascular reactivity experiments were performed on carotid rings from male Wistar rats. Treatment with methionine (0.1, 1 or 2 g kg⁻¹ per day) for 8 and 16 weeks, but not for 2 and 4 weeks, reduced the relaxation induced by BK. Indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and SQ29548, a selective thromboxane A₂ (TXA₂)/prostaglandin H₂ (PGH₂) receptor antagonist prevented the reduction in BK-relaxation observed in the carotid from methionine-treated rats. Conversely, AH6809, a selective prostaglandin F_2α (PGF_2α) receptor antagonist did not alter BK-relaxation in the carotid from methionine-treated rats. The nitric oxide synthase (NOS) inhibitors L-NAME, L-NNA and 7-nitroindazole reduced the relaxation induced by BK in carotids from control and methionine-treated rats. In summary, we found that chronic methionine intake impairs the endothelium-dependent relaxation induced by BK and this effect is due to an increased production of endothelial vasoconstrictor prostanoids (possibly TXA₂) that counteracts the relaxant action displayed by the peptide.     

Thermal biology, torpor use and activity patterns of a small diurnal marsupial from a tropical desert: sexual differences

Many small desert dasyurids employ torpor almost daily during winter, because cold nights and low food availability impose high energetic costs. However, in Western Australia the arid zone extends into tropical, coastal regions, where winter temperature conditions are far less severe. We studied the thermal biology and activity patterns of free-ranging kaluta (~27 g), a dasyurid restricted to these tropical spinifex deserts, during the Austral winter (June–July) and in addition quantified activity patterns in captivity. Unlike most dasyurids, wild and captive kalutas were almost exclusively diurnal and retreated into underground burrows during the night. Despite being active during the warmer part of the day, kalutas entered torpor daily. However, torpor patterns differed remarkably between males and females. While females spent most of the night torpid at body temperatures (T _b) as low as 21°C, close to soil temperature, males entered multiple short and shallow bouts (T _b > 25°C) during the night. Males also maintained higher T _bs during the early morning when active, occupied larger home ranges and covered greater distances while foraging than females. Hence, males appear to expend more energy than the similar-sized females both while foraging and during the rest phase. We propose that physiological as well as behavioural preparations for the September mating season that culminate in a complete male die-off might already impose energetic costs on males during winter.     

Microwave-assisted synthesis and structure–activity relationships of neuroactive pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine derivatives

We described herein the optimization of the synthetic methodology exploited to obtain the pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine sedative prototype 1a and novel analogues designed by successive molecular simplifications. By applying microwave irradiation during the hetero Diels-Alder key-step to obtain the heterotricyclic scaffold, under solvent-free conditions, we were able to obtain the desired compounds in drastically shorter times and better yields. Additionally, in vivo evaluation of the sedative effects of these heterocyclic derivatives showed that 1a and the novel structurally-related analogue 1e were the most efficient compounds to impair the locomotor activity in mice at the dose of 10 μmol/kg.