Μg μL Research Articles

Analyses of pollen-tube growth and biological action of S-RNase in the style of self-compatible Japanese pear

The Japanese pear ‘Osa-Nijisseiki’ (S 2S 4 SM) (SM = stylar-part mutant) is a self-compatible bud mutant that originated from self-incompatible ‘Nijisseiki’ (S 2S 4). The S 4-allele of the pear is deleted and it shows unilateral incompatibility to cultivar with an S 2S 4 genotype. However, when pollen-tube growth was compared between cross-compatible [‘Osa-Nijisseiki’ × ‘Okusankichi’ (S 5S 7)], unilateral-compatible [‘Osa-Nijisseiki’ × ‘Kikusui’ (S 2S 4)], self-compatible (‘Osa-Nijisseiki’ × ‘Osa-Nijisseiki’), and unilateral-incompatible pollination (‘Kikusui’ × ‘Osa-Nijisseiki’), pollen-tube growth clearly showed the following order: cross-compatible > unilateral-compatible > self-compatible > unilateral-incompatible. This indicates that the ‘Osa-Nijisseiki’ style produces specific inhibitor(s) not only to S 2- and S 4-pollen but also “self-pollen”, because the phenotype of S 4 SM-pollen is the same as S 4-pollen. Stylar protein analysis demonstrated that ‘Osa-Nijisseiki’ produces S 2-RNase (RNase associated with S 2-allele) together with a small amount of S 4-RNase. The purified S 4-RNase possessed almost the same inhibitory action on the growth of S 4-pollen-tubes in vitro at 1 μg μl −1 as that from original ‘Nijisseiki’. These results suggest that the depressed growth of unilateral-compatible and self-pollen-tubes in ‘Osa-Nijisseiki’ is due to this biologically active S 4-RNase. Growth of self-pollen-tubes may also be depressed by inhibitor(s) specific to “self-pollen” unrelated to S-alleles.

Development of a CMOS-compatible PCR chip: comparison of design and system strategies

In the last decade research in chips for DNA amplification through the polymerase chain reaction (PCR) has been relatively abundant, but has taken very diverse approaches, leaving little common ground for a straightforward comparison of results. Here we report the development of a line of PCR chips that is fully compatible with complementary-metal-oxide-semiconductor (CMOS) technology and its revealing use as a general platform to test and compare a wide range of experimental parameters involved in PCR-chip design and operation. Peltier-heated and polysilicon thin-film driven PCR chips have been produced and directly compared in terms of efficiency, speed and power consumption, showing that thin-film systems run faster and more efficiently than Peltier-based ones, but yield inferior PCR products. Serpentine-like chamber designs have also been compared with standard rectangular designs and with the here reported rhomboidal chamber shape, showing that serpentine-like chambers do not have detrimental effects in PCR efficiency when using non-flow-through schemes, and that chamber design has a strong impact on sample insertion/extraction yields. With an accurate temperature control (±0.2 °C) we have optimized reaction kinetics to yield sound PCR amplifications of 25 µl mixtures in 20 min and with 24.4 s cycle times, confirming that a titrated amount of bovine albumin serum (BSA, 2.5 µg µl−1) is essential to counteract polymerase adsorption at chip walls. The reported use of a CMOS-compatible technological process paves the way for an easy adaption to foundry requirements and for a scalable integration of electro-optic detection and control circuitry.

Development and Validation of an HPTLC?Densitometric Method for Determination of ACE Inhibitors

A high-performance thin layer chromatographic method coupled with densitometric analysis has been developed for measurement of benazepril and cilazapril, both pure and in their commercial dosage forms. The active substances were extracted from tablets with methanol (mean recovery 102%) and chromatographed on silica gel 60 F254 HPTLC plates in horizontal chambers with ethyl acetate–acetone–acetic acid–water, 8:2:0.5:0.5 (v/v), as mobile phase. Chromatographic separation of these ACE inhibitors was followed by UV densitometric quantitation at 215 nm. Calibration plots were constructed in the range 0.4 to 2.0 μg μL−1 for benazepril (2.0–10.0 μg spot−1) and from 0.5 to 1.5 μg μL−1 for cilazapril (4.0–12.0 μg spot−1) with good correlation coefficients (r ≥ 0.990). The method was used to determine benazepril and cilazapril in pharmaceutical preparations with satisfactory precision (1.4% < RSD < 5.6%) and accuracy (1.7 < RE < 5.1).

Effects of single-walled carbon nanotubes on the polymerase chain reaction

The effects of single-walled carbon nanotubes on the polymerase chain reaction(PCR) were investigated via quantitative PCR product measurements, scanningelectron microscopy (SEM), high-resolution transmission electron microscopy(TEM) and x-ray photoelectron spectroscopy (XPS). The results showed thatadding single-walled carbon nanotubes (SWCNTs) into the reaction liquidincreases the amount of PCR product at SWCNT concentrations below3 µg µl−1,but this effect is reversed at higher SWCNT concentrations.Similar effects were observed in PCR reactions with or withoutMg2+.Both SEM and HRTEM results showed that the DNA templates and Taq enzymes areattached to bundles of SWCNTs in PCR products. XPS results showed that the C 1sbinding energy in PCR products increased after reaction, with the emergence of two newpeaks beside the main peak compared with carbon nanotubes before reaction, suggestingthat there might be a chemical reaction between SWCNTs and PCR components. Inconclusion, SWCNTs may increase the PCR efficiency at a concentration range of less than3 µg µl−1 in the reaction liquid and have the potential to act as catalysts in a variety of biochemicalreactions.

Simultaneous densitometric determination of quinapril and hydrochlorothiazide in the combination tablets

A new, simple, and accurate TLC method, using normal- and reversed-phase techniques and densitometric detection, has been developed for measurement of quinapril and hydrochlorothiazide in combination tablets. UV detection at λ = 210 nm was used to quantify the analytes. The drugs were chromatographed on silica gel 60 F 254 HPTLC plates and on octadecilsilane (RP-18) TLC plates, in horizontal chambers, with ethyl acetate-acetone-acetic acid, 8 + 2 + 0.5 ( v/v ) and methanol-0.07 m phosphate buffer, pH 2.5, 6 + 4 ( v/v ), respectively, as mobile phases. The active substances were extracted from tablets with methanol (96% < mean recovery < 104%). Calibration curves were constructed in the range 0.4 to 2.4 μg μL −1 for quinapril and 0.25 to 1.5 μg μL −1 for hydrochlorothiazide, with good correlation ( r ≥ 0.998). The precision ( RSD < 4.4%) and accuracy (2.91 < RE < 3.92) were satisfactory for TLC-densitometric determination of quinapril in combination with hydrochlorothiazide in commercial tablets.

Application of videodensitometric and classical densitometric HPTLC for quantification of fluoroquinolone derivatives in pure and tablet forms

A new, simple, and rapid high-performance thin layer chromatographic method coupled with densitometry has been developed and validated for the determination of enoxacin, ofloxacin, and pefloxacin in tablets. HPTLC analysis was performed on silica gel 60F 254 plates in horizontal chambers with dichloromethane-methanol-25% NH 3 , 7 + 5 + 1.5, as mobile phase. Detection and quantification were performed by videodensitometry at λ = 254 nm and by classical densitometry at the wavelength of maximum absorption of the drugs determined. The active substances were extracted from tablets with a 0.05% solution of ammonia in methanol (enoxacin, ofloxacin) or with methanol (pefloxacin). Calibration plots were constructed in the range 0.1 to 0.6 μg μL −1 and were linear for all analytes with good correlation coefficients ( r from 0.992 to 0.9998). The precision of the proposed chromatographic methods, expressed as RSD , were between 7.56 and 10.08. The mean recoveries of the three antibiotics from tablets ranged from 97...

Roles of 5-HT 1A receptors in the dorsal raphe and dorsal hippocampus in anxiety assessed by the behavioral effects of 8-OH-DPAT and S 15535 in a modified Geller–Seifter conflict model

8-OH-DPAT [8-hydroxy-2-(di- N-propylamino)tetralin], a 5-HT 1A receptor agonist, and S 15535 (4-benzodioxan-5-yl)1-(indan-2-yl)piperazine, a partial agonist at 5-HT 1A receptors, were administered into the dorsal raphe nucleus and dorsal hippocampus and their behavioral effects were assessed in a modified Geller–Seifter conflict model. Injected into the dorsal raphe nucleus 8-OH-DPAT, 1 μg but not 0.04 or 0.2 μg 0.5 μl −1, and S 15535, 2.5 μg but not 0.1 or 0.5 μg 0.5 μl −1, significantly increased punished responding with no effect on rates of unpunished or time-out responding. WAY 100635, a selective 5-HT 1A receptor antagonist, injected subcutaneously at 0.3 mg kg −1 30 min before 1 μg 8-OH-DPAT or 2.5 μg S 15535 in the dorsal raphe, completely antagonized their effects on punished responding. At doses ranging from 1 to 10 μg μl −1 injected into the CA1 region of the dorsal hippocampus neither 8-OH-DPAT nor S 15535 modified punished responding or the rates of time-out. At the highest doses, 8-OH-DPAT significantly reduced unpunished responding whereas S 15535 had the opposite effect. The results suggest that stimulation of 5-HT 1A receptors in the dorsal raphe nucleus has anxiolytic-like effects whereas stimulation of postsynaptic receptors in the dorsal hippocampus has no anxiolytic or anxiogenic effects, at least judging from changes in rates of punished responding. These results are compatible with the hypothesis that 5-HT 1A receptor agonists and partial agonists attenuate anxiety by reducing serotonergic transmission in brain areas innervated by the dorsal raphe nucleus.

Antisense oligodeoxynucleotides against the muscarinic m 2, but not m 4, receptor supports its role as autoreceptors in the rat hippocampus

Antisense oligodeoxynucleotides against muscarinic m 2 and m 4 receptors were used to investigate the role of these receptor subtypes as negative autoreceptors in the regulation of acetylcholine (ACh) release in the rat hippocampus. Following the continuous infusion of antisenses into the third ventricle (1 μg μl −1 h −1, 3 days), 3 H -AF-DX 384/muscarinic M 2-like binding was significantly decreased in the medial septum by the antisense against the m 2 receptor whereas M 2-like binding in the dorsal striatum was decreased by the antisense against the m 4 receptor. In contrast, 3 H -pirenzepine/muscarinic M 1-like binding was unaffected by either antisense treatment in any of the brain areas investigated. When perfused into the hippocampus via a dialysis probe, the purported muscarinic M 2 receptor antagonist AF-DX 384 (100 nM) increased hippocampal ACh release in freely moving rats. This effect of AF-DX 384 was significantly attenuated by the m 2, but not the m 4, receptor antisense treatment. Hippocampal choline acetyltransferase activity was not affected by either antisense treatments. Taken together, these results suggest that the molecularly defined muscarinic m 2 receptor regulates hippocampal ACh release by acting as a negative autoreceptor. In contrast, the molecularly defined m 4 receptor is unlikely to be directly involved in the negative regulation of ACh release in the rat hippocampus. Therefore, inhibiting muscarinic m 2 receptor function may be an alternative approach to regulate the release of ACh in neurodegenerative diseases associated with impaired cholinergic functions.

Essential oil composition of Cistus albidus leaves

The essential oils of Cistus albidus leaves growing wild on calcareous and siliceous soils in Provence (South of France) were analysed by GC and GC-MS. The major component, for both oils, is α-zingiberene (116.5–45.2 μg μl −1), and differences in essential oil composition between the Cistus populations from the two soil types, as well as the chemotaxonomic aspect of α-zingiberene accumulation, are discussed. The composition of essential oils of Cistus albidus leaves from calcareous and siliceous soils was determined. Both contained α-zingiberene as the main constituent, and the differences in composition between the Cistus populations from the two soil types are discussed.

Insecticidal effects of organotin(IV) compounds on Plutella xylostella (Linnaeus) larvae I: Topical application toxicity and antifeedant effect

AbstractStructure–activity relationship studies were conducted with early fourth‐instar larvae of a highly resistant strain of the diamondback moth, Plutella xylostella (Linnaeus) on (1) toxicity by topical appliction of 43 organotin compounds, and (2) the antifeedant effect of a selected number (17) of these compounds on treated Brassica chinensis (Chinese cabbage) leaves.The toxicity data revealed that the triorganotins (R3SnX) were, without exception, more toxic than the commercial sample of malathion (84% active ingredient) used in the tests. Among the diorganotins, phenylcyclopentyltin oxide proved to be as active as malathion. Within the triorganotin series, the tricyclohexyltins were generally more toxic than the triphenyltins, the most active tricyclohexyltin compound being (c‐C6H11)3Sn(2‐pyridinethiolato N‐oxide) (LC50 0.03 μg μl−1), which was almost 500‐fold more active than malathion. The most active compound in the triphenyltin class was O, S‐bis(triphenyltin)mercaptoacetate (LC50 0.30 μg μl−1). Variations in the anionic X group resulted only in marginal changes in activity in the (c‐C6H11)3Sn series, but significant changes in activity were obtained with the Ph3Sn compounds, especially the ring‐substituted phenoxyacetates, (4‐ZC6H4)OCH2(O)COSnPh3. In the mixed triorganotin compounds an increase in activity was observed when one of the phenyl groups in Ph3SnOH was replaced by the p‐chlorophenyl group.In the antifeedant tests, the tricyclohexyltins were found to be generally more effective than the triphenyltins. In most cases, antifeedant activity paralleled the toxicity by topical application trends in the (c‐C6H11)3Sn series, but in the Ph3Sn series an inverse trend was observed. The diorganotin compound (c‐C5H9)PhSnO exerted a relatively pronounced antifeedant activity which was comparable with that of a number of triphenyltin derivatives.It was established from histological studies of the mid‐gut cross‐sections of the treated larvae that, in most cases, the organotins affected the columnar cells physiologically; an exception was noted for Ph3SnOC(O)C6H4COOH‐4 which, like malathion, caused severe morphological damage to the cell membrane.

Separation and determination of betaine in an oriental medicine by liquid chromatography

Recent interest in assaying betaine in plant tissue stimulated work on the complete separation of this substance from numerous impurities. A suitable separation of betaine was achieved by liquid chromatography on an amino-bonded silica gel. Pretreatment of samples of an aqueous extract of Jikoppi, an oriental medicine, was primarily based on a method developed by Vialle et al. in which an extract of Jikoppi was passed through a Dowex 50W-X8 (H +) column. The betaine retained in the column was eluted with 4% ammonia solution. The solution was then purified by passing it through column containing a mixed bed of Amberlite IRC-50 [H +] and Dowex 1-X4 (OH −). The combined effluent and washings from the purification column were evaporated into a syrup. A 15-mg portion of glycine was added to this condensed product as an internal standard. A laboratory-packed Kaseisorb LC NH 2 super (5 μm) column satisfactorily separated betaine and glycine from impurities using acetonitrile-water (75 + 25) containing 0.1% of Brij-35 with a detection wavelength of 210 nm. Linearity of the calibration based on the peak-area ratio was demonstrated over the entire working range of 0.25–18 μg μl −1. The relative 95% fiducial limits of betaine were determined to be 1.3–1.6% for the 0.5–12-μg range and 0.5–1.2% for the 14-18-μg range ( n=5). The recovery of betaine through the whole analytical procedure was 93% ( n=5). Betaine contents in six Jikoppi powders were also determined.

Fluorometric determination of gallium with 2-hydroxy-1-naphthaldehyde-semicarbazone; Application to aluminium metal.

A fluorometric method for the determination of trace amounts of Ga with 2-hydroxy-1-naphthaldehyde-semicarbazone (2-HNS) was applied to the analysis of aluminium metal and its alloy. To a sample solution containing less than 5.0 μg of gallium, were added 0.1% sodium fluoride solution (Ga 05.0 μg : 50 μl, Ga 01.0μg: 20 μl. Ga 00.2μg: 5 μg μl), 2 ml of 2-HNS solution in DMF (Ga 05.0 μg : 0.05%, Ga 01.0 μg : 0.025%, Ga 00.2μg: 0.01%) and 1 ml of 2 w/v% ammonium acetate solution. The pH of the solution was adjusted to 3.0+0.1 with hydrochloric acid or ammonia water, and the solution was diluted to 25 ml with water. The fluorescence intensity was measured at 450 nm (λex: 395 nm) against the standard quinine sulfate solution (10, 2 or 0.5 μg/ml). Gallium was separated from interfering elements by extracting with isopropylether, and the method was then found to be applicable to the determination of gallium above 0.1 ppm in practical samples.

Fluorometric determination of gallium with 2-hydroxy-4-methylbenzaldehyde-semicarbazone; Application to aluminium metal.

A fluorometric method for the determination of trace amounts of Ga with 2-hydroxy-1-naphthaldehyde-semicarbazone (2-HNS) was applied to the analysis of aluminium metal and its alloy. To a sample solution containing less than 5.0 μg of gallium, were added 0.1% sodium fluoride solution (Ga 05.0 μg : 50 μl, Ga 01.0μg: 20 μl. Ga 00.2μg: 5 μg μl), 2 ml of 2-HNS solution in DMF (Ga 05.0 μg : 0.05%, Ga 01.0 μg : 0.025%, Ga 00.2μg: 0.01%) and 1 ml of 2 w/v% ammonium acetate solution. The pH of the solution was adjusted to 3.0+0.1 with hydrochloric acid or ammonia water, and the solution was diluted to 25 ml with water. The fluorescence intensity was measured at 450 nm (λex: 395 nm) against the standard quinine sulfate solution (10, 2 or 0.5 μg/ml). Gallium was separated from interfering elements by extracting with isopropylether, and the method was then found to be applicable to the determination of gallium above 0.1 ppm in practical samples.

Neurotensin perfusion of rat hypothalamus: Dissociation of dopamine release from body temperature change

To determine whether endogenous dopamine is involved in the impairment of body temperature induced by neurotensin, the local activity of [ 14C]dopamine in the hypothalamus of the unanesthetized rat was examined. A push-pull guide tube was implanted permanently above an intended site of perfusion within the anterior hypothalamic. pre-optic area or other region of the diencephalon. After the endogenous stores of dopamine at a specific site were labelled by microinjcction of 0.02–0.05 μCi of [ 14C]dopamine, an artificial cerebrospinal fluid was perfused at the site at a rate of 20 μl min and at successive 5 min intervals. Perfusion of neurotensin in concentrations of 0.05 or 0.1 μg μl in the dorsomedial hypothalamus, lateral hypothalamus, arcuate nucleus or diagonal band of Broca evoked a calcium-dependent efflux of [ 14C]dopamine. The release of dopamine induced hy neurotensin was functionally specific since it was: (1) not mimicked by the relatively inactive neurotensin analogue. [ d-Arg 9]neurotensin: (2) dependent on the morphological locus of the push-pull perfusion: and (3) not accompanied hy an efflux of [ 3H]norepinephrine when the site was double-labelled. Although neurotensin perfused in the anterior hypothalamic, pre-optic area caused a consistent decline in temperature, in most cases the temperature change did not correlate with an enhanced release of dopamine. Moreover, the release of dopamine. but not the temperature change, was abolished when neurotensin was perfused in a calcium-free medium. These results show that it is unlikely that the thermolytic action of neurotensin. at least within the hypothalamus, is mediated by the presynaptic release of dopamine.

Thermoregulatory effects of carbachol in the posterior hypothalamus of the pigeon

Thermoregulatory responses to intrahypothalamic injections of carbamylcholine (CCh) were recorded from unanaesthetized pigeons exposed to 20 and 38°C. The drug (0.25–1.0 μg μl −1) was administered into the posterior hypothalamus. 1. 2.At 20°C, CCh in most cases either produced a cessation of shivering and induced hypothermia, or, produced a biphasic response; a slight increase in body temperature was followed by hypothermia. In one pigeon, the injections intensified shivering and an elevation in body temperature was seen. 2. 3.Under ambient heat load (38°C), CCh depressed the thermally induced panting and caused body temperature to rise. A slight elevation in heart rate was also recorded. 3. 4.The results suggest that a cholinergic mechanism also in the posterior hypothalamus may be involved in the temperature regulation of the pigeon. Interaction between cholinergic and serotonergic systems seems possible, but remains to be elucidated.