Effects of diazepam administration on melatonin synthesis in the rat pineal gland in vivo.

The effect of diazepam (DZP) on melatonin synthesis in rat pineal gland was investigated in vivo. Subcutaneous injection of DZP (3 mg/kg) 1 h before the start of darkness significantly suppressed nocturnal elevations of pineal N-acetylserotonin (NAS) and melatonin contents in rats, and caused a 2-h delay in reaching the maximum melatonin level in the dark phase. DZP treatment also markedly suppressed the dark-induced increase of pineal N-acetyltransferase activity, which catalyzes the rate-limiting step in melatonin synthesis, but had no effect on hydroxyindole-O-methyltransferase activity, which catalyzes the final step of melatonin formation. Pineal norepinephrine and dopamine contents, in contrast, were not altered by DZP injection. The distribution rate of DZP to the brain reached the highest level 30 min after a single injection, while that to the pineal gland was observed 5 h later (i.e., 4 h after the start of darkness). It is clear that the inhibitory effect of DZP on melatonin synthesis in rat pineal gland appears concomitantly with the increase in the distribution volume of DZP into this gland. These results suggest that the inhibitory effect of DZP on melatonin synthesis results from the drug's direct action on the rat pineal gland.     

Determination of melatonin and monoamines in rat pineal using reversed-phase ion-interaction chromatography with fluorescence detection

A method is reported for the ion-interaction, reversed-phase separation of 24 compounds (chiefly monoamines) arising from the metabolism of tyrosine and tryptophan. These compounds were separated as two groups. The first group comprised 3,4-dihydroxyphenylethylene glycol, tyrosine, 3-methoxy-4-hydroxyphenyl glycol, 5-hydroxytryptophan, norepinephrine, 3,4-dihydroxyphenylacetic acid, epinephrine, 5-hydroxyindole-3-acetic acid, homovanillic acid, 5-hydroxytryptophol, dopamine, tryptophan. N-acetylserotonin, N-acetyltryptophan, 5-methoxytryptophan and serotonin. The mobile phase consisted of a 6.8:93.2 (v/v) mixture of acetonitrile and an aqueous solution containing 0.16 M ammonium phosphate, 0.06 M citric acid, 0.15 mM disodium EDTA, 10 mM dibutylamine and 6 mM sodium 1-octanesulphonate at pH 4.50. The second group of compounds comprised 6-hydroxymelatonin, 5-methoxyindole-3-acetic acid, indole-3-acetic acid, 5-methoxytryptamine, tryptamine, 5-methoxytryptophol, melatonin and tryptophol. The mobile phase consisted of a 16:84 (v/v) mixture of acetonitrile and an aqueous solution containing 0.05 M ammonium phosphate, 0.05 M citric acid, 0.15 mM disodium EDTA, 25 mM dibutylamine and 5 mM sodium 1-octanesulphonate at pH 5.30. Detection was by fluorescence measurement (lambda ex = 280 nm, lambda em = 340 nm). The proposed method exhibited linear calibration over the biochemically significant concentration range, with detection limits in the 10-200 pg range. Excellent precision for peak areas and retention times was observed, even over a period of 24 h. The applicability of amperometric detection (at 0.72V) is also demonstrated. The method is applied to the determination of monoamines in individual rat pineals. Low nanogram levels of tyrosine, norepinephrine, 5-hydroxyindole-3-acetic acid, tryptophan, serotonin and 6-hydroxymelatonin, and picogram levels of 5-hydroxytryptophan, 5-hydroxytryptophol, 5-methoxyindole-3-acetic acid, indole 3-acetic acid, 5-methoxytryptophol and melatonin were indicated in most of the samples.     

Determination of six indolic compounds, including melatonin, in rat pineal using high-performance liquid chromatography with serial fluorimetric-electrochemical detection.

A reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, N-acetylserotonin, tryptophan, 5-hydroxytryptamine (serotonin) and melatonin in rat pineal using a buffered aqueous eluent containing acetonitrile and methanol as organic modifiers and an ion-pairing agent to assist in controlling the retention of compounds containing an amine group. Serial fluorimetric-electrochemical detection provided additional assurance of compound identity. Analyte preparation simply involved the sonication of pineals in dilute perchloric acid containing an antioxidant and a chelating agent, followed by centrifugation to clarify. The method simplifies the determination of this range of indolic compounds, which normally would require at least two separate runs with different eluents. Detection limits for melatonin were 60 and 135 pg for fluorimetric and electrochemical detection, respectively. (This represented the "worst case" considering the levels and detection limits of all compounds present.) Using flow programming and a flow-rate varying between 1 and 1.5 ml/min, the analysis time was 27.5 min, which made the determination of ten samples in a working day possible.     

Concurrent on-line sampling of melatonin in pineal microdialysates from conscious rat and its analysis by high-performance liquid chromatography with electrochemical detection

Dynamic changes of melatonin in microdialysates from the pineal gland of a freely moving rat were repeatedly determined by using on-line high-performance liquid chromatography with electrochemical detection. The detection limit for melatonin, ca. 5 pg, was well below that achieved with other systems. We observed a drastic increase of extracellular pineal melatonin during the transitional phase from the light period to the dark period. This application of microdialysis is a useful tool in the study of the physiological role of the mammalian pineal body.     

Effects of melatonin on reproductive and accessory reproductive organs in male rats.

Effects of daily injections of melatonin on male rat reproductive and accessory reproductive organs were studied. The weight of the prostate was decreased by treatment with a high dose of melatonin (8.0 mg/kg, s.c., injection time; 17:00) once daily for 30 d. The weights of the other accessory reproductive organs and testes were not influenced by melatonin. Lower doses (0.8, 2.4, 4.8 mg/kg), had no effect. After the successive treatment with melatonin (8.0 mg/kg), the testosterone levels in testes and in serum and the conversion rate of [3H]testosterone to [3H]dihydrotestosterone in the prostate were not influenced. The activity of acid phosphatase and the uptake of [3H]testosterone by the prostate, in contrast, were significantly decreased after the successive treatment with melatonin. These data suggest that melatonin may have a direct inhibitory action on male rat prostate, though only at a high dose.     

克林沙星酰胺衍生物的简易合成及其体内外生物活性

通过特色羧酸与克林沙星吡咯环氨基直接反应,得到19个未见文献报道的克林沙星酰胺衍生物,产物收率52.8%～97.5%.目标分子结构得到1H NMR,13C NMR,HR MS的确证.在琼脂扩散法初筛的基础上进行MIC活性验证,进而对活性较好的化合物开展急性毒性试验以及化合物稳定性实验,由此发现了抗菌活性更强、毒性更低、...     

Variation of melatonin and serotonin content in rat pineal gland with sex and oestrous phase difference determined by high-performance liquid chromatography with fluorimetric detection

Simultaneous determination of melatonin and serotonin in rat pineal gland is described using reversed-phase high-performance liquid chromatography with fluorimetric detection. These indoles were analysed isocratically within 15 min. In this work, veratric acid (3,4-dimethoxybenzoic acid), which has fluorescence characteristics (lambda ex = 290 nm, lambda em = 350 nm) around the wavelength of native fluorescence of melatonin (lambda ex = 285 nm, lambda em = 345 nm), was used as an internal standard. This method was applied to the determination of melatonin and serotonin in male and female rat pineal gland. No significant differences between the two groups were observed in the pineal melatonin and serotonin contents. The pineal melatonin and serotonin contents were compared with the oestrous and the di-oestrous phases of female rats. They were not widely different from each other.     

Gram-scale synthesis of a glucopyranosylidene-spiro-thiohydantoin and its effect on hepatic glycogen metabolism studied in vitro and in vivo

A high yielding, simple synthesis is described starting from d-glucose to produce gram quantities of a glucopyranosylidene-spiro-thiohydantoin. This compound efficiently inhibited the activity of rat liver glycogen phosphorylase a; moreover, it also activated phosphorylase phosphatase which, in turn, decreased the amount of glycogen phosphorylase a. Both effects result in the inhibition of glycogen mobilization and the formation of glucose from glycogen.     

Studies on chemical protectors against radiation. XXXV. Effects of radioprotective Chinese traditional medicines on radiation-induced lipid peroxidation in vivo and in vitro.

The fluctuation of lipid peroxidation (LP) in 9 tissues was investigated in mice for 7 d after whole-body X-irradiation with a lethal dose of bone marrow death. LP increased significantly in bone marrow, thymus, spleen and liver following irradiation, and slightly in brain and testis, but not in blood plasma, submaxillary gland or kidney. The effects of 7 radioprotective Chinese traditional medicines (CTMs) and cysteamine (MEA) on the radiation-induced LP in 4 tissues were studied by i.p. injection before or after irradiation and their LP content in tissues was measured 2 d after irradiation. Most CTMs showed significant inhibition of radiation-induced LP in bone marrow and liver, especially when injected prior to irradiation. Some CTMs also showed such inhibition in spleen. MEA only inhibited the increase of LP in liver when injected before irradiation, but enhanced the increase of LP in spleen. None of these radioprotectors including MEA was recognized to inhibit radiation-induced LP in thymus. The in vitro experiments were carried out using mouse liver microsomal suspensions (MS). The MS were prepared from normal (non-irradiated) mice. Each of the 8 radioprotectors was added to MS before or after irradiation and then post-irradiation-incubated at 37 degrees C. All markedly inhibited radiation-induced LP if added before irradiation, but were slightly less effective if added after.     

In vivo microdialysis/liquid chromatography/tandem mass spectrometry for the on-line monitoring of melatonin in rat

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been coupled to in vivo microdialysis for on-line monitoring of melatonin in a freely moving rat for a period of 15 hours. A microdialysis probe was surgically implanted into the jugular vein of the rat, and deionized water was used as the perfusion medium at a flow rate of 1.0 microL/min. Microdialysis samples were collected in an on-line injector with sample injection every 30 minutes. Melatonin was dosed by intraperitoneal (i.p.) injection and then monitored by microdialysis/LC/MS/MS. The whole experiment, including the microdialysis sampling and sample injection into the LC/MS system, was fully automated. Metabolites of melatonin were identified off-line by LC/MSn experiments. Two metabolites were identified as 6-hydroxymelatonin and cyclic 2-hydroxymelatonin, consistent with ones found previously in the literature.     

Effects of vindoline on rat cytochrome P450 isoform activities in vitro

A specific ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC–Q-TOF–MS/MS) method has been described for the simultaneous determination of the metabolites of tacrine, bupropion, diclofenac, dextromethorphan and midazolam, which are the five probe drugs of the five cytochrome P450 (CYP450) isoforms CYP1A2, CYP2B, CYP2C11, CYP2D1 and CYP3A4. The inhibition degree was determined by calculating the IC₅₀. The chromatographic separation was performed on a C₁₈ column with a mobile phase consisting of 0.1% formic acid and acetonitrile. The mass spectrometric analysis was conducted in positive electrospray ionization mode. The IC₅₀ values of CYP1A2, CYP2B, CYP2C11, CYP2D1 and CYP3A were 113.4, 83.78, 22.50, 9.081 and 52.76 μmol L⁻¹, respectively. The in vitro results demonstrated that vindoline could inhibit CYP2D1 activity in rats, and weak inhibitory effect on CYP2C11 and CYP3A, but had no obvious effects on CYP1A2 and CYP2B.     

Effects of the tumor promoter TPA on the induction of DNA synthesis in normal and RSV-transformed rat fibroblasts.

Induction of DNA synthesis by the tumor promoter tetradecanoyl phorbol acetate (TPA) was studied in a line of cultured rat fibroblasts (Rat-1) and their Rous sarcoma virus-transformed derivative (Rat-1 (RSV)). Following serum deprivation for 54 h to achieve quiescence, semiconservative DNA replication was measured by incubation of cells in BrdUrd and FdUrd after serum stimulation in the presence or absence of TPA. Optimal concentrations of TPA (0.1--0.5 microgram/ml) in serum-free medium induced a small increase (10--15%) in the amount of DNA made over a 30-h period in both Rat-1 and Rat-1 (RSV) cells. When Rat-1 cells were stimulated by a 4-h serum pulse, 30% of the DNA was replicated by 30 h. If the serum pulse was followed by TPA addition, 70% DNA replication was observed. If the serum pulse was preceded by TPA addition, the onset of DNA synthesis was delayed by several houses, but stimulation of DNA synthesis occurred. In contrast, the Rat-1 (RSV) cells did not show an increased in DNA synthesis induced by TPA in similar protocols, but the serum-induced onset of DNA synthesis was delayed by several hours in the presence of TPA. Therefore, TPA acts as a co-inducer of DNA synthesis in the Rat-1 but not in the Rat-1 (RSV) cells. The parent alcohol, phorbol, was inactive in Rat-1 cells, but delayed the onset of DNA synthesis in the Rat-1 (RSV) cells. We conclude that the co-inducing and delaying activities of TPA on DNA synthesis appear to be distinct and to act a different points in the G1 phase of the cell cycle.     

Synthesis, characterisation and in vitro anti-angiogenic potential of dendron VEGF blockers

To overcome the lack of in vivo stability of certain peptides used in cancer treatment and to increase their retention time in the extracellular matrix of the target tissue, the anti-angiogenic WHLPFKC sequence is synthesised at the uppermost branching generation of a poly(ε-lysine) dendron. The root of these dendrons is designed to interact preferentially with macromolecules of the extracellular matrix, whilst the uppermost branching generation of the dendron increased the exposed density of the bioactive peptide. Bioactivity testing of the blockers is performed on HUVECs. The results show that the dendron tethered with VEGF blockers was still able to inhibit proliferation and angiogenesis. Their relatively larger structure did not prevent the interaction with VEGF.     

Effects of urea induced protein conformational changes on ion exchange chromatographic behavior

Urea is widely employed to facilitate protein separations in ion exchange chromatography at various scales. In this work, five model proteins were used to examine the chromatographic effects of protein conformational changes induced by urea in ion exchange chromatography. Linear gradient experiments were carried out at various urea concentrations and the protein secondary and tertiary structures were evaluated by far UV CD and fluorescence measurements, respectively. The results indicated that chromatographic retention times were well correlated with structural changes and that they were more sensitive to tertiary structural change. Steric Mass Action (SMA) isotherm parameters were also examined and the results indicated that urea induced protein conformational changes could affect both the characteristic charge and equilibrium constants in these systems. Dynamic light scattering analysis of changes in protein size due to urea-induced unfolding indicated that the size of the protein was not correlated with SMA parameter changes. These results indicate that while urea-induced structural changes can have a marked effect on protein chromatographic behavior in IEX, this behavior can be quite complicated and protein specific. These differences in protein behavior may provide insight into how these partially unfolded proteins are interacting with the resin material.