Presence of 2-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline and 1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, novel endogenous amines, in parkinsonian and normal human brains

2-Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline and 1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline were identified for the first time as novel endogenous amines in parkinsonian and normal human brains by gas chromatography-mass spectrometry. It is of interest that these tetrahydroisoquinolines are analogues of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) which produces Parkinson's disease.     

A N-methyltransferase in human brain catalyses N-methylation of 1,2,3,4-tetrahydroisoquinoline into N-methyl-1,2,3,4-tetrahydroisoquinoline, a precursor of a dopaminergic neurotoxin, N-methylisoquinolinium ion

N-methylation of 1,2,3,4-tetrahydroisoquinoline (TIQ) present in human brain was found by a N-methyltransferase in human brain homogenate. Formation of N-methyl-1,2,3,4-tetrahydroisoquinoline (NMTIQ) from TIQ was quantitatively assayed by high-performance liquid chromatography with electrochemical detection. The reaction required S-adenosyl-L-methionine (SAM) as a methyl donor and in terms of SAM the value of the Michaelis constant, Km, and of the maximal velocity, Vmax, were 5.11 +/- 1.69 microM and 7.31 +/- 0.21 pmol/min/mg protein, respectively. The value of Km and Vmax in terms of TIQ were 20.9 +/- 5.5 microM and 7.98 +/- 1.21 pmol/min/mg protein, respectively. The optimal pH of the reaction was 8.25. A major part of the N-methyltransferase activity was found in the cytosolic fraction of human cortex. Enzymatic formation of NMTIQ indicates that in human brain this compound may be an intermediate of biosynthesis of a potent neurotoxin of dopamine metabolism, N-methylisoquinolinium ion, from naturally-occurring TIQ.     

A new polyamine derivative, a structural analog of spermine, with in vivo activity as an inhibitor of ethanol appetite

This report describes the design and synthesis of the synthetic polyamine DCD (N,N'-bis-(3-aminopropyl)cyclohexane-1,4-diamine, tetramethanesulfonate), a structural analog of spermine, and its in vivo activity as an inhibitor of alcohol consumption in a free-paradigm carried out on genetically high-ethanol-consuming UChB rats. After acute treatment with DCD (daily single dose, 20 mg/kg, p.o., 3 days), a 19% decrease in ethanol intake was obtained, without affecting the levels of food and water intake. After chronic treatment (daily single dose, 20mg/kg, p.o., 60 days) a decrease of up to 60% in ethanol intake with respect to the basal period was provoked; this effect was significantly maintained during the post-treatment period and, according to the data obtained from the determination of acetaldehyde levels in blood, was not related to a possible disulfiram-like effect. The design of this new compound was carried out using molecular modeling techniques, with the structures of natural polyamines (putrescine, spermidine, and spermine) and biosynthetically related diamines (1,3-diaminopropane; DAP) as templates. These polyamines have shown activity as inhibitors of ethanol appetite in the same experimental model.     

Streptidine, a metabolic derivative produced after administration of streptomycin in vivo, is vestibulotoxic in rats

Streptomycin is the treatment of choice in developing countries for patients suffering from tuberculosis or other infectious diseases. However, it produces incapacitating vestibular symptoms whose onset is delayed and gradual. This observation led to the notion that a streptomycin metabolic derivative and not the antibiotic itself is the damaging agent for the inner ear. To study further the existence of this ototoxic metabolite, chronic treatment with streptomycin or its putative derivative streptidine was carried out in young male Long Evans rats. The presence of streptomycin or streptidine in the blood of animals of either experimental group was assessed by high performance liquid chromatography and analysis of swimming behavior was used to evaluate vestibular damage. Features of the sensory epithelium and quantification of hair cells were attained in sections of the utricular organ of all groups by light microscopy. After 25, 35 and 45 days of treatment with streptomycin, a metabolite with the same chromatographic properties as the streptidine standard run in parallel was identified in the blood of rats. Concentrations of the metabolite were 2.26 microg/ml on the 25th day and around 8.0 microg/ml in both the 35th and the 45th day of treatment, while streptomycin was below its detection level at either period. In streptidine-treated rats, the concentration of this compound was 1.0, 1.84 and 4.94 microg/ml on the 25th, 35th and 45th treatment days, respectively. Treatment with either streptomycin or streptidine resulted in similar abnormal swimming patterns and histological alterations of the utricular epithelium. Loss of hair cells was roughly equivalent even though streptidine was administered in a dose 90% lower than streptomycin. The gradual appearance of streptidine as a metabolic derivative of the antibiotic in the blood of rats or the administration of this compound alone, causing similar functional and structural vestibular deterioration seen in streptomycin-treated animals, supports the notion that streptidine is a potential contributor to ototoxicity after prolonged antibiotic administration.     

Statin treatment increases formation of carbon monoxide and bilirubin in mice: a novel mechanism of in vivo antioxidant protection

Heme oxygenase (HO) has a central role in cellular antioxidant defences and vascular protection, and it may mediate pleiotropic actions of drugs used in cardiovascular therapy. We investigated whether long-term use of statins upregulates HO activity and increases carbon monoxide (CO) and bilirubin levels in vivo. Adult FvB mice were given atorvastatin or rosuvastatin (5 mg/kg) daily by i.p. injections for 1, 2, or 3 weeks. HO activity, tissue CO, bilirubin, and antioxidant levels, total plasma bilirubin, and carboxyhemoglobin (COHb) were measured. Fold changes in heart HO activity significantly increased after 1, 2, and 3 weeks of atorvastatin (1.24 +/- 0.06 (p < or = 0.05); 1.29 +/- 0.26 (p < or = 0.03); 1.33 +/- 0.08 (p < 0.01), respectively) and 2 and 3 weeks of rosuvastatin (1.23 +/- 0.20 (p < or = 0.03); 1.63 +/- 0.42 (p < 0.01), respectively). Heart tissue CO and COHb levels also increased after 3 weeks with atorvastatin (1.30 +/- 0.24 (p < or = 0.05); 1.92 +/- 0.17 (p < or = 0.001), respectively) and rosuvastatin (1.47 +/- 0.13 (p < or = 0.004); 1.63 +/- 0.12 (p < or = 0.001), respectively). Significant increases in heart antioxidant levels were observed after statin treatment and corroborated by heart bilirubin content elevations. Antioxidant level increases were abolished by treatment with an HO inhibitor. These findings suggest that the induction of HO and the production of its products, CO and bilirubin, may be a mechanism by which statins exert antioxidant actions and confer cardioprotection in vivo.     

Identification of a novel binding site for calmodulin in ammodytoxin A, a neurotoxic group IIA phospholipase A2.

The molecular mechanism of the presynaptic neurotoxicity of snake venom phospholipases A2 (PLA2s) is not yet fully elucidated. Recently, new high-affinity binding proteins for PLA2 toxins have been discovered, including the important intracellular Ca2+ sensor, calmodulin (CaM). In the present study, the mode of interaction of group IIA PLA2s with the Ca2+-bound form of CaM was investigated by mutational analysis of ammodytoxin A (AtxA) from the long-nosed viper (Vipera ammodytes ammodytes). Several residues in the C-terminal part of AtxA were found to be important in this interaction, particularly those in the region 115-119. In support of this finding, introduction of Y115, I116, R118 and N119, present in AtxA, into a weakly neurotoxic PLA2 from Russell's viper (Daboia russellii russellii) increased by sevenfold its binding affinity for CaM. Furthermore, two out of four peptides deduced from different regions of AtxA were able to compete with the toxin in binding to CaM. The nonapeptide showing the strongest inhibition was that comprising the AtxA region 115-119. This stretch contributes to a distinct hydrophobic patch within the region 107-125 in the C-terminal part of the molecule. This lacks any substantial helical structure and is surrounded by several basic residues, which may form a novel binding motif for CaM on the molecular surface of the PLA2 toxin.     

Ghrelin,a novel growth hormone-releasing peptide,in the treatment of chronic heart failure

Ghrelin is a novel growth hormone (GH)-releasing peptide, isolated from the stomach, which has been identified as an endogenous ligand for growth-hormone secretagogues receptor (GHS-R). This peptide also causes a positive energy balance by stimulating food intake and inducing adiposity through growth hormone-independent mechanisms. In addition, ghrelin has some cardiovascular effects, as indicated by the presence of its receptor in blood vessels and the cardiac ventricles. In vitro, ghrelin inhibits apoptosis of cardiomyocytes and endothelial cells. In humans, infusion of ghrelin decreases systemic vascular resistance and increases cardiac output in patients with heart failure. Repeated administration of ghrelin improves cardiac structure and function and attenuates the development of cardiac cachexia in rats with heart failure. These results suggest that ghrelin has cardiovascular effects and regulates energy metabolism through GH-dependent and -independent mechanisms. Thus, administration of ghrelin may be a new therapeutic strategy for the treatment of severe chronic heart failure (CHF).     

Complex formation of cytochrome C with a calixarene carboxylic acid derivative: a novel solubilization method for biomolecules in organic media

A calixarene carboxylic acid derivative has been found to form a complex with the cationic protein cytochrome c. The solubilized cytochrome c was stable and showed peroxidase activity in chloroform. The calix[6]arene and the calix[8]arene achieved quantitative extraction of the protein. The calix[6]arene, whose cavity is well-fitted to a protonated amino group, exhibited a selectivity to lysine-rich proteins due to the recognition of the epsilon-amino groups in lysine residues on the surface of the protein. This is the first report showing protein extraction by calixarenes. The solubilized cytochrome c could catalyze an oxidative reaction in organic solvents. This host compound functions as a novel solubilization tool for biomolecules and a separation tool for lysine-rich proteins.     

The stimulatory effect of chronic lithium treatment on basal thyrotropin secretion in rats: In vivo antagonism by methylparaben

Chronic treatment of rats with lithium chloride was examined in order to determine its effect on hypothalamic monoamine and metabolite content, basal thyrotropin (TSH) secretion and thyroid function. The hypothalamic concentrations of noradrenaline (NA), dopamine (DA) and its metabolites, dihydroxyphenylacetic acid. (DOPAC) and homovanillic acid (HVA) in the lithium treated rats remained unaltered when compared to control levels. NA turnover and the NA metabolite, 3-methoxy-4-hydroxyphenylglycol (total MHPG), were significantly lower (p<0.01), whereas both serotonin (5-HT) and its metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), were significantly higher (p<0.01 and p<0.02, respectively) in the lithium treated rat hypothalami than in controls. Chronic lithium treatment significantly elevated basal TSH levels (p<0.05). This effect was antagonized by methylp-hydroxybenzoate (methylparaben, p<0.01), which did not itself affect basal TSH levels. Free serum T₃ and T₄ levels were not significantly affected by chronic lithium treatment, although T₄ tended to be slightly lower than control levels. The monoamine changes observed in the hypothalamus of lithium treated rats did not appear to account for the elevated TSH levels observed in these rats since NA activity which is generally regarded as stimulatory was decreased and 5-HT which has an inhibitory effect on TSH secretion, was increased. The elevated TSH levels may have been due to a reduced negative feedback inhibition of TSH release by the mildly reduced circulating T₄ levels caused by chronic lithium treatment. A further possibility is that the pituitary cGMP (and hence TSH) response to TRH may have been enhanced by chronic lithium treatment and methylparaben may have antagonized this effect.     

In vivo characterization of a novel GnRH (gonadotropin-releasing hormone) antagonist, LXT-101, in normal male rats

LXT-101 is a newly developed GnRH (gonadotropin-releasing hormone) analogue. In this study, the in vivo pharmacological profile in intact male rats and binding characters of LXT-101 were illustrated, and regulation of mRNA of hormone receptors related to the pituitary-gonadal axis during and after administration was observed to reveal its molecular mechanism of potent effect and reversibility. After single subcutaneous injections, LXT-101 produced a dose- and time-dependent suppression of serum testosterone level. Multiple administrations and osmotic pump implantation revealed that the time of onset and dose needed to maintain the effect of chemical castration decreased as the frequency of injection increased and gave direct proof that depot formulation could significantly improve the duration of antagonist delivery and pharmacological activities compared to the injectable formulation. And LXT-101 showed excellent character of regulating the pituitary-gonadal axis quickly and reversibly. Competitive binding assay showed that LXT-101 could specifically bind a pituitary GnRH receptor with high affinity. These results indicated that LXT-101 is fit for sustained-release formulation and it might possibly be developed as an ideal candidate for treating sex hormone-sensitive tumors and other disorders.     

A Novel and Neurotoxic Tetrahydroisoquinoline Derivative In Vivo: Formation of 1,3-Dimethyl-1,2,3,4-Tetrahydroisoquinoline, a Condensation Product of Amphetamines, in Brains of Rats Under Chronic Ethanol Treatment

Serotonin binding protein (SBP) is a vesicular protein found in neurectoderm-derived cells that store 5-hydroxytryptamine (5-HT, serotonin), such as central and peripheral serotonergic neurons and paraneurons (parafollicular cells of the thyroid). 5-HT is stored as a complex with SBP in vivo. Two forms of the protein are found. These differ in molecular mass: one is 45 kDa and the other 56 kDa. It has been suggested that the 56-kDa form of SBP may be the precursor of the 45-kDa form. To study the relationship between these two proteins, we have used a covalently bound radiolabeled probe to analyze their binding domains. A photoaffinity reagent, N-(4-azido-2-nitrophenyl)-5-hydroxytryptamine (NAP-5-HT), was synthesized and characterized by nuclear magnetic resonance spectroscopy, mass spectra, and UV-visible absorption spectra. A 1 M excess of NAP-5-HT inhibited the binding of [3H]5-HT to SBP by 50%. NAP[3H]5-HT was also synthesized and attached to both high- and low-affinity binding sites on both forms of SBP. The high-affinity constants for 45-kDa and 56-kDa proteins were 0.8 nM and 0.02 nM, respectively, whereas the low-affinity constants were 0.3 microM and 0.15 microM. When the high-affinity site of partially purified SBP was photoaffinity-labeled with the reagent, two covalently labeled proteins (45 kDa and 56 kDa) were found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition of the labeling of both proteins by 50% was observed in the presence of a 15-fold molar excess of 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-thyroid cancer properties of a novel isoflavone derivative, 7-(O)-carboxymethyl daidzein conjugated to N-t-Boc-hexylenediamine in vitro and in vivo

The incidence of thyroid cancer is up to 3 folds higher in women than in men, suggesting that estrogenic effects may be involved in the pathogenesis of this malignancy. Here, we explore whether or not human thyroid cancer cell growth can be curbed by a novel isoflavone derivative generated in our laboratory, the N-t-Boc-hexylenediamine derivative of 7-(O)-carboxymethyl daidzein (cD-tboc). With the exception of the follicular cancer cell line WRO, estrogen receptor (ER)α mRNA was only marginally expressed in cell lines derived from papillary (NPA), follicular (MRO), anaplastic thyroid carcinoma (ARO) such that the expression of estrogen receptor (ER) βmRNA was more abundant than that of ERα mRNA in these cell types. Estradiol-17β (E2; 0.03-300nmol/l) per se increased proliferation in all four cell-types. The ERβ-specific agonist DPN increased [(3)H]-thymidine incorporation in all four thyroid cancer cell lines, whereas the ERα-specific agonist PPT increased growth only in NPA and WRO. By contrast, cD-tboc, derived from the weak estrogen daidzein, did not cause cell growth and dose-dependently diminished cell growth in all four cell lines via apoptosis and not necrosis, as detected by the release of histone-DNA fragments. The cytotoxic growth inhibitory effect of cD-tboc in these cells was modulated by E2 and the general caspase inhibitor Z-VAD-FMK, and the magnitude of this salvage was cell type-and dose-dependent. When nude mice carrying ARO thyroid xenografts were treated with cD-tboc, tumor volume decreased significantly, and no apparent toxicity was observed. These results suggest that cD-tboc may be a promising agent for therapy of thyroid carcinoma either alone or in combination with existing cytotoxic drugs.     

Alstrostines in Rubiaceae: Alstrostine A from Chassalia curviflora var. ophioxyloides and a novel derivative,rudgeifoline from Rudgea cornifolia

Monoterpenoid indole alkaloids have frequently been isolated from the species-rich Psychotria alliance (Rubiaceae), a complex group including several tribes and genera. In our aim of understanding the chemical diversification within this remarkably heterogeneous group, members of two genera of the tribe Palicoureeae have been studied. Alstrostine A was isolated from Chassalia curviflora var. ophioxyloides, and a novel derivative, rudgeifoline from Rudgea cornifolia, respectively. Alstrostines, an unusual class of alkaloids comprising one tryptamine and two iridoid units, have recently been discovered in Alstonia rostrata (Apocynaceae). The presence of alstrostines in two rubiaceous species is remarkable but not unexpected as both families share similar biosynthetic pathways and are capable of synthesizing related alkaloids.     

Synthesis of beta-glucanases during sporulation in Saccharomyces cerevisiae: formation of a new, sporulation-specific 1,3-beta-glucanase.

A biphasic synthesis of 1,3-beta-glucanase occurred when cells of Saccharomyces cerevisiae AP-1 (a/alpha) were incubated in sporulation medium. The capacity to degrade laminarin increased very slowly during the first 7 h but at a much faster rate thereafter. Changes occurring during the first period were not sporulation specific since the moderate increase in activity against laminarin was insensitive to glutamine and hydroxyurea and also took place in the nonsporulating strain S. cerevisiae AP-1 (alpha/alpha). However, the changes taking place after 7 h must be included in the group of sporulation-specific events since they were inhibited by glucose, glutamine, and hydroxyurea and did not occur in the nonsporulating diploid. Consequently, only when the cells had been incubated for at least 7 h in sporulation medium did full induction of activity against laminarin take place upon shift to a medium which favored vegetative growth. Changes in the relative proportions of the vegetative glucanases, namely, endo- and exo-1,3-beta-glucanase, and the formation of a new sporulation-specific 1,3-beta-glucanase account for the observed events and are the consequence of the expression of the sporulation program.     

A comparison between effects of chronic ethanol consumption, ethanol withdrawal and fasting in ethanol-fed rats on the free cytosolic NADP+/NADPH ratio and NADPH-regenerating enzyme activities in the liver

Effects of chronic ethanol consumption, withdrawal and fasting on the free cytosolic NADP+/NADPH ratio and NADPH-regenerating enzyme activities of rat liver were studied. Ethanol consumption was shown to decrease the NADP+/NADPH ratio in non-fasted rats, and both ethanol withdrawal and fasting in ethanol-fed animals appeared to increase the ratio to the normal or higher level. Any treatment of rats caused the complex interaction on hepatic NADPH-regenerating enzyme activities, none of the enzyme activity correlating with the free cytosolic NADP+/NADPH ratio. Relationship between free cytosolic NADP+/NADPH ratio and lipogenic capacity of withdrawn rat liver is discussed, and a hypothesis for development of the fatty liver is suggested.     

Induction of G2/M phase arrest and apoptosis by a novel enediyne derivative, THDA, in chronic myeloid leukemia (K562) cells

We studied the effect of 2-(6-(2-thieanisyl)-3(Z)-hexen-1,5-diynyl)aniline(THDA), a newly developed anti-cancer agent, on cell proliferation, cell cycle progression, and induction of apoptosis in K562 cells. THDA was found to inhibit the growth of K562 cells in a time-and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest and apoptosis in K562 cells following 24 h exposure to THDA. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 were increased in a time-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that THDA did not change the steady-state levels of cyclin B1, cyclin D3 and Cdc25C, but decreased the protein levels of Cdk1, Cdk2 and cyclin A. THDA also caused a marked increase in apoptosis, which was associated with activation of caspase-3 and proteolytic cleavage of poly (ADP-ribose) polymerase. These molecular alterations provide an insight into THDA-caused growth inhibition, G2/M arrest and apoptotic death of K562 cells.     

Anti-tumor effects of B-2, a novel 2,3-disubstituted 8-arylamino-3H-imidazo[4,5-g]quinazoline derivative, on the human lung adenocarcinoma A549 cell line in vitro and in vivo

This study evaluated the selective effects of 2-isopropyl-3-butyl-8-(4-fluorophenylamino)-3H-imidazo[4,5-g]quinazoline (B-2), a member of a series of quinazolines, on the cell survival and growth of the non-small cell lung cancer (NSCLC) cell line A549 in vitro and in vivo. Cytotoxicity assay, hollow fiber assay and cell xenograft model experiment revealed that B-2 showed selective effects on A549 cell survival or growth in a dose-dependent manner. At a dose of 100mg/kg, B-2 showed stronger efficacy on tumor growth inhibition in nude mice than Iressa. Exposure of A549 cells to B-2 caused inhibition of EGFR-dependent ERK-MAPK activation. In addition, inhibition of cell cycle progression and induction of mitochondria-dependent apoptosis might contribute to present the multitarget pathway of non-small cell lung cancer treatment of B-2.     

In vitro and in vivo characterization of a novel semaphorin 3A inhibitor,SM-216289 or xanthofulvin

SM-216289 (xanthofulvin) isolated from the fermentation broth of a fungal strain, Penicillium sp. SPF-3059, was identified as a strong semaphorin 3A (Sema3A) inhibitor. Sema3A-induced growth cone collapse of dorsal root ganglion neurons in vitro was completely abolished in the presence of SM-216289 at levels less than 2 mum (IC50 = 0.16 mum). When dorsal root ganglion explants were co-cultured with Sema3A-producing COS7 cells in a collagen gel matrix, SM-216289 enabled neurites to grow toward the COS7 cells. SM-216289 diminished the binding of Sema3A to its receptor neuropilin-1 in vitro, suggesting a direct interference of receptor-ligand association. Moreover, our data suggest that SM-216289 interacted with Sema3A directly and blocked the binding of Sema3A to its receptor. We examined the efficacy of SM-216289 in vivo using a rat olfactory nerve axotomy model, in which strong Sema3A induction has been reported around regenerating axons. The regeneration of olfactory nerves was significantly accelerated by a local administration of SM-216289 in the lesion site, suggesting the involvement of Sema3A in neural regeneration as an inhibitory factor. SM-216289 is an excellent molecular probe to investigate the function of Sema3A, in vitro and in vivo, and may be useful for the treatment of traumatic neural injuries.     

In vivo ethanol elimination in man, monkey and rat: a lack of relationship between the ethanol metabolism and the hepatic activities of alcohol and aldehyde dehydrogenases

The in vivo ethanol elimination in human subjects, monkeys and rats was investigated after an oral ethanol dosage. After 0.4 g. ethanol/kg of body weight, ethanol elimination was much slower in human subjects than in monkeys. In order to detect a rise in monkey plasma ethanol concentrations as early as observed in human subjects, ethanol had to be administered at a dose of 3 g/kg body weight. Ethanol metabolism in rats was also much faster than in human subjects. However, human liver showed higher alcohol dehydrogenase activity and higher low Km aldehyde dehydrogenase activity than rat liver. Thus, our data suggest a lack of relationship between hepatic ethanol-metabolizing activities and the in vivo ethanol elimination rate.