Disposition of ropinirole in animals and man.

1. The disposition and metabolic fate of ropinirole, a novel compound indicated for the symptomatic treatment of Parkinson's disease, was studied in the mouse, rat, cynomolgus monkey and man, following oral and intravenous administration of ropinirole hydrochloride. 2. In all species, nearly all of the p.o. administered dose (94%) was rapidly absorbed from the gastrointestinal tract following administration of 14C-ropinirole hydrochloride. In rat and monkey, the compound distributed rapidly beyond total body water and was shown to cross the blood-brain barrier. Blood clearance of the compound was high, approximately equal to one-half the hepatic blood flow in the monkey and similar to the hepatic blood flow in rat. Terminal phase elimination half-lives for the compound were relatively short (0.5 and 1.3 h in rat and monkey respectively), although there was evidence of a second elimination phase in the monkey with an elimination half-life of approximately 5-11 h. Plasma concentrations of ropinirole after the intravenous dose were not determined in the mouse and were below the lower limit of quantification in man (0.08 ng/ml) at the doses used in the studies described in this paper. 3. In both animals and man, ropinirole was extensively metabolized. In the rat, the major metabolic pathway was via hydroxylation of the aromatic ring to form 7-hydroxy ropinirole. In mouse, monkey and man, the major pathway was via N-depropylation. The N-despropyl metabolite was metabolized further to form 7-hydroxy and carboxylic acid derivatives. Metabolites formed in all species were generally metabolized further by glucuronidation. 7-Hydroxy ropinirole is the only metabolite of ropinirole previously shown to possess significant dopamine agonist activity in vivo. In all species, the major route of excretion of ropinirole-related material after oral or intravenous administration of the compound was renal (60-90% of dose).

Saturation mutagenesis of the beta subunit of the human granulocyte-macrophage colony-stimulating factor receptor shows clustering of constitutive mutations, activation of ERK MAP kinase and STAT pathways, and differential beta subunit tyrosine phosphorylation.

The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are heterodimeric complexes consisting of cytokine-specific alpha subunits and a common signal-transducing beta subunit (hbetac). We have previously demonstrated the oncogenic potential of this group of receptors by identifying constitutively activating point mutations in the extracellular and transmembrane domains of hbetac. We report here a comprehensive screen of the entire hbetac molecule that has led to the identification of additional constitutive point mutations by virtue of their ability to confer factor independence on murine FDC-P1 cells. These mutations were clustered exclusively in a central region of hbetac that encompasses the extracellular membrane-proximal domain, transmembrane domain, and membrane-proximal region of the cytoplasmic domain. Interestingly, most hbetac mutants exhibited cell type-specific constitutive activity, with only two transmembrane domain mutants able to confer factor independence on both murine FDC-P1 and BAF-B03 cells. Examination of the biochemical properties of these mutants in FDC-P1 cells indicated that MAP kinase (ERK1/2), STAT, and JAK2 signaling molecules were constitutively activated. In contrast, only some of the mutant beta subunits were constitutively tyrosine phosphorylated. Taken together, these results highlight key regions involved in hbetac activation, dissociate hbetac tyrosine phosphorylation from MAP kinase and STAT activation, and suggest the involvement of distinct mechanisms by which proliferative signals can be generated by hbetac.

Antipsychotic drug affinities at alpha2-adrenoceptor subtypes in post-mortem human brain.

Although there is substantial interest in the possible role of alpha2-adrenoceptors in the antipsychotic efficacy of clozapine, there has so far been no systematic investigation of antipsychotic drug affinities for alpha2-adrenoceptor subtypes in the human brain. We have assessed the ability of three classical and four 'atypical' antipsychotic drugs to displace binding of [3H]RX821002 to alpha2-adrenoceptors in human post-mortem brain tissue. All seven drugs displaced radioligand from an apparent single site in the frontal cortex, consistent with the sole presence of the alpha2A-subtype in this region. In the caudate nucleus, all drugs except risperidone differentiated two sites, of which one was equivalent to the cortical alpha2A-subtype and the second, accounting for approximately two-thirds of specific radioligand binding, showed higher affinity for the antipsychotics. This second site, on the basis of prazosin's relatively high affinity, is consistent with an alpha2B-adrenoceptor identity. The new antipsychotic quetiapine showed the greatest selectivity for this receptor site; both quetiapine and clozapine had affinities for the alpha2B site which were greater than their affinities for human D2 dopamine receptors. The possible role of this site in the mechanisms underlying aspects of antipsychotic drug atypicality is discussed.

Growth and carbohydrate status of coppice shoots of hybrid poplar following shoot pruning.

Fifteen, 1-year-old Populus maximowiczii Henry x P. nigra L. 'MN9' trees were decapitated and allowed to sprout. After 8 weeks, all had 6 to 10 coppice shoots. All shoots, except the tallest (dominant) shoot, were removed from five of the trees (pruned treatment), and shoot growth, gas exchange and carbohydrate status were compared in the pruned and unpruned trees. Although photosynthetic rate of recently mature leaves of pruned trees was approximately 50% greater than that of leaves on the dominant shoot of unpruned trees, and the dry weight of leaves of pruned trees was 37% greater than that of the leaves on the dominant shoot of unpruned trees, the shoot dry matter relative growth rate did not differ between treatments. Concentrations of water-soluble carbohydrates and starch in the uppper stem and leaves of the dominant shoot were similar in pruned and unpruned trees. However, relative to that of the dominant shoot in unpruned trees, the lower stem in pruned trees was depleted in both soluble carbohydrates and starch. Starch deposition, assessed as the quantity of (14)C-starch in tissues 24 h after a fully expanded source leaf was labeled with (14)CO(2), was 3.9 times greater in roots of pruned trees than in roots of unpruned trees. We conclude that early removal of all but the dominant shoot reduces the carbohydrate status of the roots and the lower portion of the stem by eliminating the excised shoots as a source of photosynthate.

Changes in protein synthesis during drought conditioning in roots of jack pine seedlings (Pinus banksiana Lamb.).

The impact of drought conditioning on the ability of eight-week-old jack pine (Pinus banksiana Lamb.) seedlings to withstand drought was assessed. Two progressive cycles of drought conditioning significantly increased the survival of seedlings subjected to a subsequent prolonged drought. The in vivo accumulation of several root membrane proteins during drought conditioning was correlated with an increase in seedling survival. A group of root proteins, ranging in molecular mass from 43 to 47 kDa, increased accumulation during one cycle of drought conditioning and to a lesser extent during two cycles of drought conditioning. The accumulation of several low molecular mass membrane and soluble proteins also increased during drought conditioning, suggesting that these proteins may play an important role in the enhancement of drought tolerance. In vitro translation studies showed a general increase in the abundance of protein products encoded by mRNAs from drought-conditioned seedlings. Although the majority of the in vitro translation products appeared in both control and drought-conditioned seedlings, one mRNA encoding a 15 kDA translated protein was more prominent during the second cycle of drought conditioning.

Carbohydrate mobilization following shoot defoliation and decapitation in hybrid poplar.

The effects of shoot defoliation, decapitation, and disbudding on carbon mobilization were investigated in rooted cuttings of Populus maximowiczii x nigra L. 'MN9'. Ten days after complete shoot defoliation or decapitation, the stem starch concentration of treated plants declined to one-half that of intact plants, and there were similar or greater reductions in the concentrations of glucose, fructose, sucrose, galactose, and shikimic acid. Partial shoot defoliation (50%) and complete disbudding had no effect on stem starch concentration, but stem sucrose concentration was reduced in all treatments. Sucrose depletion preceded and may have induced other changes in the carbon status of plants subjected to leaf or shoot removal. Four days after shoot decapitation, the sucrose concentration of roots of treated plants was reduced to 25% of that of intact plants. However, the concentrations of fructose and glucose increased in the roots of treated plants and was followed by the accumulation of shikimic acid, salicyl alcohol, unknown compound A and salicin. The possible role of increased concentrations of root organic solutes in the water relations and regrowth process of decapitated plants is discussed.

Influence of photoperiod and temperature on frost hardiness and free amino acid concentrations in black spruce seedlings.

We investigated the effects of a 4-week exposure to an 8-h or 18-h photoperiod at 5 or 25 degrees C on the development of hardiness to -20 degrees C and the accumulation of proline (Pro), arginine (Arg) and tryptophan (Trp) in shoots of black spruce (Picea mariana (Mill.) B.S.P.) seedlings. The greatest degree of hardening to -20 degrees C occurred in seedlings exposed to an 8-h photoperiod at 25 degrees C, and some hardening occurred in seedlings exposed to 5 degrees C in either an 8-h or 18-h photoperiod. Proline accumulated in shoots in response to 5 degrees C and either an 8-h or 18-h photoperiod, whereas Trp accumulated in response to an 8-h photoperiod at either temperature, and Arg only accumulated in shoots in the 5 degrees C + 8-h photoperiod treatment. Only the accumulation of Trp was significantly related to the degree of hardiness to -20 degrees C.

The truncation that generated the v-cbl oncogene reveals an ability for nuclear transport, DNA binding and acute transformation.

The v-cbl oncogene is the transforming gene of the murine Cas NS-1 retrovirus which induces pre-B cell lymphomas and myeloid leukaemias. Sequencing of c-cbl has revealed that v-cbl was generated by a large truncation that removed 60% of the C-terminus of the corresponding protein. In this study we prepared antibodies to cbl and found that c-cbl encodes a 120 kDa protein which is localized in the cytoplasm with a cytosolic and cytoskeletal distribution. Immunofluorescence studies show a striking pattern of brightly staining vesicles in mitotic cells similar to that observed with cytokeratin antibodies. In contrast to p120c-cbl, which is exclusively cytoplasmic, the p100gag-v-cbl encoded by Cas NS-1 is localized in both the cytoplasm and the nucleus. This redistribution to the nucleus correlates with the ability of cbl to induce acute transformation. Furthermore the truncated protein encoded by v-cbl can bind DNA, unlike the full-length protein. These results suggest that the C-terminus of cbl is involved in the retention of p120c-cbl in the cytoplasm and the inhibition of DNA binding. The findings also suggest that a truncated protein encoded by c-cbl exists in the nucleus of normal cells.

A rearrangement of the c-cbl proto-oncogene in HUT78 T-lymphoma cells results in a truncated protein.

The murine Cas NS-1 retrovirus carries the v-cbl oncogene and induces pro-B, pre-B and myeloid tumors in mice inoculated at birth. The human homolog of v-cbl is located on chromosome 11q23.3, which is in the region of translocation breakpoints in a range of acute leukemias. The sequencing of the human and murine c-cbl proto-oncogenes has revealed v-cbl as a markedly truncated form of c-cbl that encompasses 40% of the amino terminus. This truncation deletes a proline- and serine/threonine-rich domain and a putative leucine zipper. In this study we describe the nature of a truncated form of c-cbl present in the cutaneous T-cell lymphoma line HUT78. In these cells a genetic rearrangement involving unknown sequences and Alu repeat sequences results in the insertion of a premature stop codon that removes 259 amino acids from the carboxy terminus. This results in the translation of a 72 kDa protein, compared with the 120 kDa protein encoded by full-length c-cbl, which lacks a portion of the proline-rich domain and the entire leucine zipper.

The sequences of the human and mouse c-cbl proto-oncogenes show v-cbl was generated by a large truncation encompassing a proline-rich domain and a leucine zipper-like motif.

The murine Cas NS-1 retrovirus carries the v-cbl oncogene and induces pre-B cell lymphomas and myeloid leukemias. The cellular homolog of v-cbl has been identified in mouse and human DNA, and was recently mapped to mouse chromosome 9 and human chromosome 11q23. To determine the coding sequences of the human and mouse c-cbl proto-oncogenes cDNA clones were isolated from libraries prepared from the human T cell leukemia lines CCRF-CEM and HUT 78, and the mouse pre-B cell line 70Z/3. DNA sequencing revealed an open reading frame encoding 906 amino acids in the human cDNAs and 896 amino acids in 70Z/3. The sequence showed that v-cbl is a markedly truncated form of murine c-cbl containing 355 N-terminal amino acids. The nucleotide sequence of v-cbl is identical to murine c-cbl in this region, and the human sequence has only five amino acid changes in the v-cbl portion. The most notable features of the sequence which was lost in the generation of v-cbl is a C-terminal leucine zipper and a stretch of 208 amino acids containing 23% proline and 19% serine/threonine residues. This proline-rich sequence has similarities to the transcriptional activation domains of some transcription factors, and v-cbl's transforming potential may be due to the loss of this region and the leucine zipper.

Metabolism of temelastine (SK&F 93944) in hepatocytes from rat, dog, cynomolgus monkey and man.

A species comparison of the metabolic pathways of temelastine has been made using hepatocyte preparations from rat, dog, cynomolgus monkey, and man. Metabolites and unchanged temelastine were separated by HPLC and were compared with authentic standards by retention. The characteristic UV spectra of SK&F 93944 and its metabolites aided in the preliminary identification of metabolites in hepatocyte incubates, subsequently confirmed by liquid chromatography/mass spectrometry (LC/MS). The metabolic profile of temelastine is complex, both in vivo and in vitro, but all of the metabolites identified unambiguously from in vivo studies have also been demonstrated in vitro. Moreover, the time-dependent nature of the metabolic profile has been investigated in rat hepatocytes. Marked differences in the rate of production, extent of accumulation, and distribution between cells and culture medium have been observed for specific metabolites. Species differences in the metabolism of temelastine by rat, dog, cynomolgus monkey, and human hepatocytes have been observed. In particular, SK&F 94224 (a hydroxylated metabolite of temelastine) was not detected in human hepatocyte incubations at appreciable concentrations, but was present in varying amounts in the other species and especially in incubations from dog hepatocytes. Temelastine N-glucuronide was not detected in the rat hepatocyte system but was present to a modest or significant extent in hepatocyte incubations from dog, cynomolgus monkey, and man.

The analysis and characterization of isomeric metabolites of temelastine by the combined use of thermospray liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry.

This paper describes the development and application of a combined thermospray liquid chromatographic/mass spectrometric and liquid chromatographic/tandem mass spectrometric method for distinguishing between five isomeric metabolites of Temelastine, comprising four hydroxylated metabolites and one N-oxide. The method allows the unambiguous characterization of all of the isomers either on their own or in the presence of each other. Clear results were obtained for the characterization of these metabolites in biological samples. Temelastine showed extensive phase I and phase II metabolism and the methodology was used to study the aglycone structures of the glucuronide conjugates derived from the hydroxylated metabolites. Photo-diode array ultraviolet spectroscopy was used as a complementary technique to help elucidate the site of glucuronidation in these species.

The structural identification of drug metabolites using thermospray liquid chromatography/mass spectrometry.

The structural identification of drug metabolites has been carried out using thermospray liquid chromatography/mass spectrometry (LC/MS). It has allowed the direct analysis of biological samples, in this case in vitro hepatocyte incubations, with the minimum of sample preparation. The technique also provided molecular weight information on several conjugates including glucuronides, a glutathione conjugate and one unidentified conjugate. A number of minor metabolites were also successfully identified using this method. The examples discussed in this paper illustrate the value of LC/MS in identifying unknown drug metabolites covering a wide polarity range in a complex biological mixture. However, this would not have been possible, if the interface had been unable to handle gradient separations.

Rapid structural analysis of the in vitro and in vivo metabolism of SK & F 95448 by the combined use of thermospray liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry.

The thermospray liquid chromatography/mass spectrometry (LC/MS) interface has had a major impact on the direct analysis of the metabolic fate of xenobiotics in complex biological media. This paper outlines the rapidity and power of the LC/MS approach, and shows how detailed structural information can be obtained without recourse to individual compound isolation. This provides a great saving in time and effort. The additional specificity of liquid chromatography/tandem mass spectrometry is highlighted in identifying the sites of metabolic transformation. The ability to handle biological samples with little or no clean-up using wide high-performance liquid chromatographic gradients is a key feature of the success of this methodology.

Application of thermospray liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry for the identification of cynomolgus monkey and human metabolites of SK & F 101468, a dopamine D2 receptor agonist.

A combination of thermospray liquid chromatography-mass spectrometry (LC-MS) and LC MS MS has allowed the structural elucidation of a number of metabolites of 4-[2-(dipropylamino)ethyl]-1,3-dihydro-2H-indol-2-one (SK & F 101468) in monkey urine. By using LC-MS-MS with the third quadrupole (Q3) set up in multiple ion detection (MID) mode, a number of metabolites were subsequently detected in the human urine and plasma samples despite very low dosing regimes. This was achieved with minimal sample preparation, e.g. for the urine sample centrifugation was the only preparative step, in order to remove particulate matter, prior to analysis. The good signal-to-noise ratio obtained for the human samples, using LC MS MS with Q3 set up for MID, raised the possibility of a LC-MS-MS quantitative assay. As a result, the detection limit of this method for SK&F 101468 when dissolved in methanol was determined to be in the region of 20 pg on column.

Human lymphocytes aged in vivo have reduced levels of methylation in transcriptionally active and inactive DNA.

The amount of 5-methylated cytosine (5 mC) in the sequence CmCGG has been measured in DNA extracted from uncultured peripheral blood human lymphocytes obtained from 24 young (mean age 25) and 22 old (mean age 75) individuals. When compared with the young group the old group had significantly reduced levels of 5 mC in total genomic DNA, in transcriptionally active DNA, and in genomic DNA from which transcriptionally active sequences had been removed. In both the young and old groups transcriptionally active DNA contained 10% less 5 mC than the residual 'inactive' DNA. These results indicate that loss of genomic DNA methylation may be involved in aging in vivo and underscore the association of gene regulation with the distribution of methylation in DNA.

The metabolism of 5-(4-acetamidophenyl)pyrazin-2(1H)-one in rat, dog and cynomolgus monkey.

1. The metabolism and disposition of 14C-acetamidophenyl pyrazinone has been studied in rat, dog and cynomolgus monkey. The compound was well absorbed and rapidly excreted in urine and faeces by all three species. 2. Distribution of 14C-pyrazinone was rapid and extensive with the exception of the central nervous system where concentrations were at, or below, the limit of detection. 3. Whereas, in in vitro studies, metabolites (but not the parent compound) weakly inhibited some activities of the cytochrome P-450 system, there was evidence from in vivo studies in the rat that the compound and/or its metabolite(s) are weak selective inducers of cytochrome P-450. 4. Metabolite patterns were similar in all three species. The major route of metabolism was glucuronidation at the oxygen of the pyrazinone ring. Other metabolites originated from metabolism by gut microflora with subsequent hepatic metabolism.

Drought tolerance, growth partitioning and vigor in eucalypt seedlings and rooted cuttings.

To clarify the physiological basis of productivity differences among rooted cuttings and seedlings of eucalypt species, relationships between morphology and water relations were examined in 4-month-old seedlings of Eucalyptus grandis W. Hill ex Maiden, E. urophylla S.T. Blake and E. cloeziana F. Muell. and in 4-month-old rooted cuttings of three E. grandis cultivars. Four-month-old seedlings had greater dry weights, lower leaf area/root dry weight (LA/RDW) ratios and lower shoot/root dry weight (S/R) ratios than 4-month-old rooted cuttings. For all cultivars of E. grandis, tall rooted cuttings, as defined by height at age 4 weeks, had greater dry weights by age 4 months and lower LA/RDW and S/R ratios than short rooted cuttings. There were differences in height growth, dry matter productivity and relative shoot and root development among cuttings of different E. grandis cultivars, but these differences were not as great as the differences between short and tall grades of the same cultivar and between seedlings and cuttings. Consistent with the differences in LA/RDW and S/R ratios, seedlings had higher daytime water potentials (Psi(x)) than cuttings, and tall cuttings had higher daytime values of Psi(x) than short cuttings. Differences in Psi(x) were also related to stomatal conductance (g(wv)), which was up to 300% greater in short cuttings than in tall cuttings. Among seedlings, those of E. cloeziana, which had the smallest dry weight at age 4 months, had the highest g(wv), whereas those of E. grandis, which had the greatest dry weight at age 4 months, had the lowest g(wv). Unlike seedlings and the tall cuttings, short cuttings lost turgor when subjected to drought. The differences observed in susceptibility to water stress may account in part for the associated differences in dry matter production. Xylem pressure potential and relative water deficit at zero turgor did not differ significantly among the types of plants studied, which suggests that differences in growth rates were not the result of differences in dehydration tolerance.