Effect of zinc-chelating dipeptides on osteoblastic MC3T3-E1 cells: Activation of aminoacyl-tRNA synthetase

The effect of zinc-chelating dipeptides on osteoblastic MC3T3-E1 cells was investigated. As zinc compounds, we used zinc sulfate, AHZ, di(N-acetyl-β-alanyl-l-histidinato)zinc (AAHZ), and di(histidino)zinc (HZ). Cells were cultured for 72 h in the presence of zinc compounds (10⁻⁸–10⁻⁵M). The effect of AHZ (10⁻⁷ and 10⁻⁶M) to increase protein and deoxyribonucleic acid (DNA) contents in the cells was the greatest in comparison with those of other zinc compounds. Zinc sulfate and HZ at 10⁻⁷M did not have an effect on the cellular protein content. AHZ (10⁻⁶M) had a potent effect on cell proliferation, although zinc sulfate (10⁻⁶M) had no effect. β-Alanyl-l-histidine (10⁻⁶ and 10⁻⁵M) did not have an appreciable effect on the cells. Those effects of AHZ (10⁻⁶M) on osteoblastic cells were completely abolished by the presence of cycloheximide (10⁻⁶M). AHZ (10⁻⁸–10⁻⁵M) directly activated [³H]leucyl-tRNA synthetase in the cell homogenate, whereas the effect of zinc sulfate was seen at 10⁻⁶ and 10⁻⁵M. The present study suggests that the chemical form of zinc-chelating β-alanyl-l-histidine (AHZ) can reveal a potent anabolic effect on osteoblastic cells, and that AHZ directly stimulates protein synthesis.     

The effects of FMRFamide, serotonin, and acetylcholine on the isolated crop-gizzard of the earthworm, Lumbricus terrestris

We examined the effects of FMRFamide, serotonin, and acetylcholine on the isolated crop-gizzard of the earthworm, Lumbricus terrestris. FMRFamide caused a decrease in contraction amplitude with a threshold between 10⁻⁹ and 10⁻⁸ M and a biphasic change in contraction rate. The contraction rate increased with a threshold between 10⁻⁸ and 10⁻⁷ M and decreased with a threshold between 10⁻⁶ and 10⁻⁵ M. Serotonin decreased the contraction rate with a threshold between 10⁻⁸ and 10⁻⁷ M and the amplitude with a threshold between 10⁻⁸ and 10⁻⁷ M. Acetylcholine increased the contraction rate with a threshold between 10⁻⁸ and 10⁻⁷ M and caused a biphasic change in contraction amplitude. Amplitude rose with a threshold between 10⁻⁹ and 10⁻⁸ M and decreased with a threshold between 10⁻⁶ and 10⁻⁵ M. These results suggest that all three neurotransmitters may play a role in controlling the movement of the digestive tract in L. terrestris.     

Entropy decrease associated to solute compartmentalization in the cell

We have deduced equations to quantify the entropy associated to the compartmentalization of components in eukaryotic cells as a function of cell and compartment volumes, and of the concentration of solutes. On the basis of known and plausible values of volume and solute concentrations and the deduced equations, we estimate that the contribution of compartmentalization to the decrease of entropy is approximately −14.4 × 10⁻¹⁴ J K⁻¹ cell⁻¹ (−0.7 J K⁻¹ L⁻¹) in the case of Saccharomyces cerevisiae, a typical eukaryotic cell, and approximately −49.6 × 10⁻¹⁴ J K⁻¹ cell⁻¹ (−1.0 J K⁻¹ L⁻¹) in the more complex Chlamydomonas reinhardtii. When compared with other potential contributing factors, such as the informational entropy of DNA and the conformational entropy of proteins, compartmentalization appears as an essential development that significantly decreased the entropy of living cells during biological evolution.     

Interaction of anthelmintic benzimidazoles and benzimidazole derivatives with bovine brain tubulin

The binding and inhibitory properties of 11 benzimidazoles for bovine brain tubulin were investigated. The effects of the benzimidazoles on the initial rates of microtubule polymerization were determined by a turbidimetric assay. The median inhibitory concentrations (I₅₀) for nocodazole, oxibendazole, parbendazole, mebendazole and fenbendazole ranged from 1.97 · 10⁻⁶ to 6.32 · 10⁻⁶ M. Benomyl, cambendazole and carbendazim had I₅₀ values from 5.83 · 10⁻⁵ to 9.01 · 10⁻⁵ M. Thiabendazole had an I₅₀ value of 5.49 · 10⁻⁴ M. Inhibitor constants (K_i) were determined by the colchicine binding assay. Oxibendazole, fenbendazole, and cambendazole had K_i values of 3.20 · 10⁻⁵, 1.73 · 10⁻⁵ and 1.10 · 10⁻⁴ M, respectively. Oxibendazole and fenbendazole were competitive inhibitors of colchicine. In contrast, cambendazole was a noncompetitive inhibitor of colchicine. The ability of these benzimidazoles to inhibit microtubule polymerization and the mode of action for the anthelmintic benzimidazoles is discussed.     

Effects of aqueous hop (Humulus Lupulus L.) extract on vascular reactivity in rats: Mechanisms and influence of gender and hormonal status

Phytoestrogens, naturally occurring plant compounds having oestrogenic and/or anti-oestrogenic activity, are present in many human foodstuffs including hop. Moderate intakes of isoflavonoid phytoestrogens have been associated with a reduction in cardiovascular diseases incidence. So, it is possible that hop (Humulus Lupulus L.) might similarly contribute to the reported health-beneficial effects of moderate beer consumption. Thus, the purpose of this study was to investigate in vitro effects of aqueous hop extract on thoracic vascular reactivity in Sprague Dawley male and female rats. Endothelium-intact thoracic arterial rings from male rats (MALE, n=8), sham-ovariectomized (Sham OVX) female (n=8) and ovariectomized (OVX) female rats (n=8) were used. We assessed the relaxation induced by aqueous hop extract (10⁻⁹, 10⁻² g/l) in aortic rings precontracted with norepinephrine (10⁻⁷ M), in the absence or in the presence of l-NAME (10⁻⁴ M), indomethacin (10⁻⁵ M), thapsigargin (10⁻⁴ M), iberiotoxin (3.10⁻⁸ M), apamin (3.10⁻⁸ M) and TEA (3.10⁻⁴ M). Aqueous hop extract induced relaxation of endothelium-intact thoracic arterial rings in MALE and Sham OVX rats, whereas a weak effect was observed in OVX rats. This vasorelaxation was strongly inhibited in presence of l-NAME, indomethacin and thapsigargin. These data indicated that aqueous hop extract-induced vasodilation, in male and intact female rats, is mediated by NOS activation, cyclooxygenase products and Ca²⁺ pathways. Moreover, our results suggested that effect of hop in enhancing vascular reactivity was independent of gender but strongly related to hormonal status.     

Dopamine-stimulated cyclic AMP and binding of [H]dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [³H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10⁻⁸ M and was half-maximal at 7.9±3.4·10⁻⁷M. The increase at 1·10⁻⁵ M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10⁻⁹ M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC₅₀ value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10⁻⁵ M dopamine was 2.3±0.9·10⁻⁶M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [³H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The K_d value for high affinity binding sites was 0.43±0.1·10⁻⁷M and 4.7±1.6·10⁻⁷M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2±0.4·10^/t-7M noradrenaline, 2·10^/t-7 M epinine, 4.1±1.8·10^/t-6M fluphenazine, 8.0±1.6·10^/t-6M haloperidol, 4.2±1.2·10⁻⁶Mcis-flupenthixol, 2.7±0.4·10⁻⁵Mtrans-flupenthixol, >1·10⁻⁵M apomorphine, sulpiride, naloxone and isoproterenol.     

Biodeterioration of some herbal raw materials by storage fungi and aflatoxin and assessment of Cymbopogon flexuosus essential oil and its components as antifungal

Deterioration of raw materials of six medicinal plants viz. Terminalia arjuna, Acorus calamus, Rauvolfia serpentina, Holarrhena antidysenterica, Withania somnifera and Boerhaavia diffusa was examined. Some of the contaminated raw materials were found to be deteriorated by toxigenic strains of Aspergillus flavus and contain aflatoxin B₁ (41.0–95.4 μg kg⁻¹) which is above the permissible limit. Essential oil of Cymbopogon flexuosus and its components was found efficient in checking fungal growth and aflatoxin production. C. flexuosus essential oil absolutely inhibited the growth of A. flavus and aflatoxin B₁ production at 1.3 μl ml⁻¹ and 1.0 μl ml⁻¹ respectively. The individual oil components were more efficacious than the Cymbopogon oil as such which emphasizes masking of their efficacy when combined together. Eugenol exhibited potent antifungal and aflatoxin inhibitory activity at 0.3 μl ml⁻¹ and 0.1 μl ml⁻¹ respectively. Eugenol was found superior over some prevalent synthetic antimicrobials and exhibited broad fungitoxic spectrum against some biodeteriorating moulds. Prospects of exploitation of the oil and its components as acceptable plant based antimicrobials in qualitative as well as quantitative control of biodeterioration of herbal raw materials have been discussed.     

Graft copolymerization of N-vinylformamide onto sodium carboxymethylcellulose and study of its swelling, metal ion sorption and flocculation behaviour

The present paper reports the graft copolymerization of N-vinylformamide onto sodium carboxymethylcellulose by free radical polymerization using potassium peroxymonosulphate/thiourea redox system in an inert atmosphere. The reaction conditions for maximum grafting have been optimized by varying the reaction variables, including the concentration of N-vinylformamide (12.0 × 10⁻²–28.0 × 10⁻² mol dm⁻³), potassium peroxymonosulphate (4.0 × 10⁻³–12.0 × 10⁻³ mol dm⁻³), thiourea (1.2 × 10⁻³–4.4 × 10⁻³ mol dm⁻³), sulphuric acid (2.0 × 10⁻³–10.0 × 10⁻³ mol dm⁻³), sodium carboxymethylcellulose (0.2–1.8 g dm⁻³) along with time duration (60–180 min) and temperature (25–45° C). Water swelling capacity, metal ion sorption and flocculation studies of synthesized graft copolymer have been performed with respect to the parent polymer. The graft copolymer has been characterized by FTIR spectroscopy and thermogravimetric analysis.     

Crack velocity and the fracture of bone

The fracture mechanics parameters associated with the fracture of transversely oriented bovine femur compact tension specimens have been determined for a slowly propagating and stable crack, as a function of cross head speed. It was found that an increase in cross head speed from 1.7–33 × 10⁻⁶ m sec⁻¹ produced an increase in the crack velocity from 2.1–27 × 10⁻⁵ m sec⁻¹ and an associated increase in the critical strain energy release rate (G_c) from 920 to 2780 J m⁻² and in the critical stress intensity factor (K_c) from 2.4 to .     

Small-scale turbulence affects the division rate and morphology of two red-tide dinoflagellates

The effects of small-scale turbulence on two species of dinoflagellates were examined in cultures where the turbulent forces came randomly from all directions and were intermittent both spatially and temporally; much like small-scale turbulence in the ocean. With Lingulodinium polyedrum (Stein) Dodge (syn. Gonyaulax polyedra), division rate increased linearly (from 0.35 to 0.5 per day) and the mean cross-sectional area (CSA) decreased linearly (from 1100 to 750 μm²) as a function of the logarithmic increase in turbulence energy dissipation rate (). These effects were noted when values increased between 10⁻⁸ and 10⁻⁴ m² s⁻³. However, when increased to 10⁻³ m² s⁻³, division rate sharply decreased and mean CSA increased. Over the same range of , Alexandrium catenella (Wheedon and Kofoid) Balech had its division rate decrease linearly (from 0.6 to 0.45 per day) and its CSA increase linearly (from 560 to 650 μm²) as a function of the logarithmic increase in . Even at the highest examined (10⁻³ m² s⁻³), which may be unrealistically high for their ambits, both L. polyedra and A. catenella still had fairly high division rates, 0.2 and 0.45 per day, respectively. Turbulence strongly affected chain formation in A. catenella. In non-turbulent cultures, the mode was single cells (80–90% of the population), but at of 10⁻⁵ to 10⁻⁴ m² s⁻³, the mode was 8 cells per chain. At the highest (10⁻³ m² s⁻³), the mode decreased to 4 cells per chain. The vertical distributions of A. catenella populations in relation to hydrographic flow fields were studied in the summers of 1997 and 1998 in East Sound, Washington, USA (latitude 48°39′N, 122°53′W). In both summers, high concentrations of A. catenella were found as a subsurface bloom in a narrow depth interval (2 m), where both current shear and turbulence intensity were at a minimum. Other researchers have shown that A. catenella orients its swimming in shear flows, and that swimming speed increases with chain length. These responses, when combined with our observations, support a hypothesis that A. catenella actively concentrates at depths with low turbulence and shear.     

The acetylcholine receptor: Isolation of a brain nicotinic receptor and its preliminary characterization in lipid bilayer membranes

The technique of affinity chromatography with the curarizing neurotoxins of Naja naja venom has been employed to extract nicotinic acetylcholine receptors from the brain tissues of mouse and hog. Both carbochol and hexamethonium were used as linear or step gradients to elute the receptor and its properties were investigated in lipid bilayer membranes. Of particular interest is the observation that discrete quanta of conductance could be observed across an NaCl gradient of 1.0:0.1 M. By switching the voltage-clamp across the bilayer between a positive and negative 80 mV, the separate Na⁺ and Cl⁻ conductances of these quanta could be estimated and the following conductances of the smallest discrete quanta were observed: 3.7 · 10⁻¹¹ Ω⁻¹ (Na⁺) and 5.9 · 10⁻¹¹ Ω⁻¹ (Cl⁻) for mouse brain receptors; 3.8 · 10⁻¹¹ Ω⁻¹ (Na⁺) and 4.7 · 10⁻¹¹ Ω⁻¹ (Cl⁻) for hog brain receptors. Large aggregates of receptors appeared to activate and deactivate as multiples of a basic conductance size, although there is evidence that they may not represent the actual gating of ion channels. A “background noise” that is not within the temporal capability of the recording system is also present at an intensity that seems to parallel the number of activated receptors, and in view of recent electrophysiological evidence that the relaxation lifetime of the open channel state is of a millisecond duration, it may be that this “noise” actually represent the channel gating.     

Antagonistic effect of calcium, zinc and selenium against cadmium induced chromosomal aberrations and micronuclei in root cells of Hordeum vulgare

The antagonistic effect of calcium (Ca²⁺), zinc (Zn²⁺) and selenium (Se⁴⁺) at different concentrations (10⁻²–10⁻⁶ M) against cadmium (Cd²⁺) induced genotoxic effects in root cells of Hordeum vulgare were studied. The results showed that 10⁻³–10⁻⁵ M could induce chromosomal aberrations and micronuclei formation. But in the treatment with 10⁻²–10⁻⁶ M of Ca²⁺, Zn²⁺ and Se⁴⁺ together with Cd²⁺ (10⁻³–10⁻⁵ M), respectively, the frequencies of chromosomal aberrations and micronuclei effectively decreased after 48 h of treatment. The treatment with 10⁻⁴–10⁻⁶ M of Ca²⁺ together with 10⁻⁴–10⁻⁵ M Cd²⁺, 10⁻⁶ M of Zn²⁺ together with 10⁻⁵ M Cd²⁺ and 10⁻⁶ M of Se⁴⁺ together with 10⁻⁵ M Cd²⁺ suggested rather obvious antagonistic effects. The order of the antagonisms of Ca²⁺, Se⁴⁺ and Zn²⁺ against Cd²⁺ toxicity was Ca²⁺>Se⁴⁺>Zn²⁺. The degree of antagonisms of Ca²⁺, Se⁴⁺ and Zn²⁺ against Cd²⁺ related to their concentration ratio.     

Synthesis, characterization and applications of graft copolymer (Chitosan-g-N,N-dimethylacrylamide)

An unreported graft copolymer of N,N-dimethylacrylamide (DMA) with chitosan has been synthesized under nitrogen atmosphere using peroxymonosulphate/mandelic acid redox pair. The effect of reaction conditions on grafting parameters i.e. grafting ratio, efficiency, conversion, add on and homopolymer has been studied. Experimental results show that maximum grafting has been obtained at 1.0 g dm⁻³ concentration of chitosan, 30 × 10⁻² mol dm⁻³ concentration of N,N-dimethylacrylamide and 7.0 × 10⁻³ mol dm⁻³ concentration of hydrogen ion. It has also been observed that grafting ratio, add on, conversion and efficiency increase upto 3.2 × 10⁻³ mol dm⁻³ of mandelic acid, 12.0 × 10⁻³ mol dm⁻³ of potassium peroxymonosulphate, 150 min of time and 40 °C of temperature. Grafted polymer has been characterized by FTIR spectroscopy and thermogravimetric analysis. Water swelling capacity of chitosan-g-N,N-dimethylacrylamide has been determined. It has been observed that the graft copolymer is thermally more stable than parent backbone.     

Pressure jump relaxation kinetics of frog skin open circuit voltage and short circuit current

The relaxation kinetics of frog skin open circuit voltage, V_oc, and short circuit current I_sc, was studied by analyzing the effects of subjecting the tissue to sudden increments of hydrostatic pressure. Both V_oc and I_sc are perturbed by the pressure jump. Changes in V_oc can be resolved into three components: a rapid decrease (phase I), a second, additional decrease with time constant 2.2 s (phase II), and finally a very slow increase found only in some preparations. The amplitudes of phases I and II are linear in the range of pressures studied (<350 atm) and have respective pressure coefficients of −1.2 · 10⁻⁴atm⁻¹ and −3.7 · 10⁻⁴atm⁻¹.Under short circuit conditions phases I and II persist. The pressure coefficients of the amplitudes of phase I and II, −4.3 · 10⁻⁴atm⁻¹ and −5.0 · 10⁻⁴atm⁻¹, respectively, are larger than those of V_oc, but the time constant of phase II, 2.2 s, is the same. The sum of the amplitudes of phases I and II is directly proportional to I_sc when it is inhibited with ouabain. It is argued that in both electrical states pressure perturbs the same transport mechanism giving rise to phases I and II of V_oc and I_sc.The magnitude of the pressure coefficients of these processes implies that they arise from chemical reactions, rather than from simple, physical solution properties. Comparison of the pressure jump kinetics with the previous spectral analysis of the electrical fluctuations of frog skin suggests a common origin for both sets of phenomena.     

Differential effects of prazosin and rauwolsin on the release of prostaglandins I2 and E2 from the perfused mesenteric arterial bed of the rabbit following nerve stimulation

Sympathetic nerve stimulation of the perfused mesenteric arterial bed of the rabbit, , increase the secretion of prostaglandin (PG)I₂ and PGE₂. Prazosin (4.8 × 10⁻⁶), and α₁ adrenergic receptor antagonist, inhibited this inrease in release of PGI₂ but not of PGE₂ whereas rauwolsin (10⁻⁷ M), an α₂ adrenergic receptor antagonist, inhibited the increase in release of PGE₂ but not of PGI₂. Prazosin (10⁻⁶ M) completely blocked the vasoconstrictor response to nerve stimulation, and to norepinephrine and phenylephrine administration, suggesting there to be little of an α₂ adrenergic receptor component in this response. It is concluded that the increase in PGI₂ release follows the activation of α₁ adrenergic receptors and is therefore post-junctional in origin, whereas the increase in PGE₂ release follows the activation of α₂ adrenergic receptors and may be pre- and/or post-junctional in origin.Indomethacin (2.8 × 10⁻⁷, 5.6 × 10⁻⁷ and 1.12 × 10^{−6 M} did not affect the vasoconstrictor responses to nerve stimulation at 10 Hz, whereas rauwolsin (10⁻⁷ M) in the presence of indomethacin substantially increased them. These results indicate that PGE₂ does not regulate norepinephrine release following nerve stimulation at 10 Hz to rabbit mesenteric arteries, and that the inhibition of norepinephrine release following stimulation of α₂ pre-junctional receptors is independent of PG involvement.     

Determination of free radical reaction products and metabolites of salicylic acid using capillary electrophoresis and micellar electrokinetic chromatography

Hydroxylated radical products of salicylic acid are often used as a relative measurement in free radical research. Several analytical methods exist to determine the amount of 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid. In this study we use capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) in order to determine these free radical products. The CZE experiment was optimized with a CZE simulation program in order to achieve an optimal pH. Calibration curves were recorded in the range 10⁻⁶–10⁻⁴ M and the detection limit was determined. For both CZE and MECC it was 2·10⁻⁷ M. Both methods resulted in a reproducible analysis of salicylate and its hydroxylated free radical products in 6 min.     

Comparison of anion-exchange and ion-modified reversed-phase liquid chromatography for the determination of S-sulfocysteine

A dual Hg–Au amalgam electrode is used to detect S-sulfocysteine (SSC) in this study. There exist two main components in the acetonitrile (ACN) rat brain extracts, namely, Cl⁻ and GSSG (oxidized glutathione), that are active in our detection system (GSH is not extracted in ACN). Two strong anion-exchange columns from different companies were used to separate the samples under different conditions, but SSC and Cl⁻ were not separated at the optimum detection pH of 5.2. The signal from Cl⁻ was greatly decreased by lowering the potential at the downstream electrode, though it cannot be completely eliminated. While a silver cartridge removed Cl⁻ from micromoles to several millimoles without any negative effect on the SSC signal in aqueous standards, a large negative peak which interferes with SSC detection was unfortunately introduced when a silver cartridge was applied to brain tissue samples. However, SSC and Cl⁻ in the samples are successfully separated by ion-modified reversed-phase LC in acetate buffer at the optimum detection pH (5.2). The separation conditions are 20 mM acetic acid, 2% methanol, 0.5 mM cetyltrimethylammonium p-toluene sulfonate (CTMA) (pH 5.2). Most importantly, the sensitivity of SSC under the optimum separation conditions is not sacrificed. The detection limit is 8 nM (20 μl injected).     

Mutagenicity of bisphenol A and its suppression by interferon-α in human RSa cells

Bisphenol A is used as a monomer in the production of polycarbonate plastic products. The widespread use of bisphenol A has raised concerns about its effects in humans. Since there is little information on the mutagenic potential of the chemical, the mutagenicity of bisphenol A was tested using human RSa cells, which has been utilized for identification of novel mutagens. In genomic DNA from cells treated with bisphenol A at concentrations ranging from 1×10⁻⁷ to 1×10⁻⁵ M, base substitution mutations at K-ras codon 12 were detected using PCR and differential dot-blot hybridization with mutant probes. Mutations were also detected using the method of peptide nucleic acid (PNA)-mediated PCR clamping. The latter method enabled us to detect the mutation in bisphenol A-treated cells at a dose (1×10⁻⁸ M) equivalent to that typically found in the environment. Induction of ouabain-resistant (Oua^R) phenotypic mutation was also found in cells treated with 1×10⁻⁷ and 1×10⁻⁵ M of bisphenol A. The induction of K-ras codon 12 mutations and Oua^R mutations was suppressed by pretreating RSa cells with human interferon (HuIFN)-α prior to bisphenol A treatment. The cells treated with bisphenol A at the concentration of 1×10⁻⁶ M elicited unscheduled DNA synthesis (UDS). These findings suggested that bisphenol A has mutagenicity in RSa cells as well as mutagens that have been tested in these cells, and furthermore, that a combination of the PNA-mediated PCR clamping method with the human RSa cell line may be used as an assay system for screening the mutagenic chemicals at very low doses.     

Mitochondrial Membrane Potential Changes in Osteoblasts Treated with Parathyroid Hormone and Estradiol

This study assessed mitochondrial membrane potential changes in cultured osteoblasts treated with hormones known to regulate osteoblasts. A fluorescent carbocyanine dye, 5,5′, 6,6′-tetrachloro-1,1′, 3,3′-tetraethylbenzimidazolocarbocyanine iodide, also called JC-1, was used as a probe. JC-1 emits photons at 585 nm (orange–red) when the membrane potential in mitochondria is highly negative, but when the potential becomes reduced emission occurs at 527 nm (green). Osteoblasts were rinsed in serum-free medium for 5 min, then loaded with 1 × 10⁻⁶MJC-1 for 10 min. The distribution and intensity of JC-1 fluorescence were evaluated with a laser-scanning confocal microscope system. Hormone treatments included parathyroid hormone (PTH; 10⁻⁸M), 17β-estradiol (10⁻⁸M), and thyroxine (T₄; 10⁻⁸M). The potassium ionophore valinomycin (10⁻⁶M) was used as a control since it is known to disrupt the electrochemical gradient of mitochondria without interfering with the pH gradient. Valinomycin caused a profound, rapid increase (22.5% above untreated values) in the green/red ratio, which indicated a lowering of the mitochondrial membrane potential in all samples evaluated. PTH caused a less pronounced, but significant (7–14%), reduction in membrane potential in all cells examined. PTH is known to affect osteoblasts in a number of ways and is inhibitory to mitochondrial respiration; the results confirm this effect. For estradiol, half of the cells responded at a significant level, with a membrane potential reduction of 6 to 13% being recorded; the other half did not respond. Thyroxine did not alter mitochondrial membrane potential. Responses were detectable within 20 s for valinomycin, but occurred at a slower rate, over 200 to 300 s, following PTH and estradiol treatment. Responses to PTH and estradiol could be due to mitochondrial uptake of cytosolic Ca²⁺.     

Assay of R-apomorphine, S-apomorphine, apocodeine, isoapocodeine and their glucuronide and sulfate conjugates in plasma and urine of patients with Parkinson's disease

Analytical methods are described for the selective, rapid and sensitive determination of R- and S-apomorphine, apocodeine and isoapocodeine and the glucuronic acid and sulfate conjugates in plasma and urine. The methods involve liquid-liquid extraction followed by high-performance liquid chromatography with electrochemical detection. The glucuronide and sulfate conjugates are determined after enzymatic hydrolysis. For the assay of R- and S-apomorphine a 10 μm Chiralcel OD-R column is used and the voltage of the detector is set at 0.7 V. The mobile phase is a mixture of aqueous phase (pH 4.0)-acetonitrile (65:35, v/v). At a flow-rate of 0.9 ml min⁻¹ the total run time is ca. 15 min. The detection limits are 0.3 and 0.6 ng ml⁻¹ for R- and S- apomorphine, respectively (signal-to-noise ratio 3). The intra- and inter-assay variations are <5% in the concentration range of 2.5-25 ng ml⁻¹ for plasma samples, and <4% in the concentration range of 40-400 ng ml⁻¹ for urine samples. For the assay of apomorphine, apocodeine and isoapocodeine, a 5 μm C₁₈ column was used and the voltage of the detector set at 0.825 V. Ion-pairing chromatography was used. The mobile phase is a mixture of aqueous phase (pH 3.0)-acetonitrile (75:25, v/v). At a flow-rate of 0.8 ml min⁻¹ the total run time is ca. 14 min. The detection limits of this assay are 1.0 ng ml⁻¹ for apomorphine and 2.5 ng ml⁻¹ for both apocodeine and isoapocodeine (signal-to-noise ratio 3). The inter-assay variations are 5% in the concentration range of 5-40 ng ml⁻¹ for plasma samples and 7% in the concentration range of 50-500 ng ml⁻¹ for urine samples. The glucuronic acid and sulfate conjugates of the various compounds are hydrolysed by incubation of the samples with β-glucuronidase and sulfatase type H-1, respectively. Hydrolysis was complete after 5 h of incubation. No measurable degradation of apomorphine, apocodeine and isoapocodeine occurred during the incubation. A pharmacokinetic study of apomorphine, following the intravenous infusion of 30 μg kg⁻¹ for 15 min in a patient with Parkinson's disease, demonstrates the utility of the methods: both the pharmacokinetic parameters of the parent drug and the appearance of apomorphine plus metabolites in urine could be determined.