Differentiation of BCS-TC2 human colon adenocarcinoma cells by sodium butyrate: increase in 5''-nucleotidase activity

Body mass index (BMI) is the most commonly used measure of obesity. Recently, some investigators have advocated direct measurement of adiposity rather than use of the BMI. This study was undertaken to determine the ability of BMI to predict body fat levels in three populations of West African heritage living in different environments. A total of 1,054 black men and women were examined in Nigeria, Jamaica, and the United States during 1994 and 1995. A standardized protocol was used to measure height, weight, waist and hip circumferences, and blood pressure at all sites; percentage of body fat was estimated using bioelectrical impedance analysis. Percentage of body fat and BMI were highly correlated within site- and sex-specific groups, and the resulting r2 ranged from 0.61 to 0.85. The relation was quadratic in all groups except Nigerian men, in whom it was linear. The regression coefficients were similar across sites, yet the mean body fat levels differed significantly (p < 0.001) as estimated by the intercept, making intersite comparison difficult. Compared with BMI, percentage of body fat was not a better predictor of blood pressure or waist or hip circumference.     

Expression and enzymic activity of ecto 5''-nucleotidase in the human male genital tract

The factor structure of the Millon Clinical Multiaxial Inventory (Millon, 1994; MCMI-III) was assessed among 444 African American inpatient substance abusers and constitutes the first factor analysis of the MCMI-III. We found 3 main factors: General Maladjustment, Paranoid Behavior/Thinking With Detached Emotionality, and Antisocial Acting Out. These factors were essentially similar to previous findings of factor studies with the MCMI and MCMI-II across diverse populations. This factor invariance should lend credibility to the revised test and spur additional research into its psychometric properties.     

Decreased resistance to gemcitabine (2',2'-difluorodeoxycitidine) of cytosine arabinoside-resistant myeloblastic murine and rat leukemia cell lines: role of altered activity and substrate specificity of deoxycytidine kinase

We determined the potential activity of 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) in 1-beta-D-arabinofuranosylcytidine (ara-C)-sensitive and-resistant leukemia cell lines. Both drugs are phosphorylated by deoxycytidine kinase (dCK); the triphosphates, dFdCTP and ara-CTP, respectively, are incorporated into DNA. In the murine leukemia cell line L1210, induction of resistance to ara-C resulted in the 2200-fold resistant subline L4A6. The Brown Norway rat myelocytic leukemia ara-C-sensitive cell line (BCLO) was >300-fold more sensitive to ara-C than its variant Bara-C. In L1210 cells, gemcitabine was 8-fold more active than ara-C; in L4A6, BCLO, and Bara-C cells, gemcitabine was 16-, 28-, and more than 3-fold more active than ara-C, respectively. A partial explanation for these differences may be the higher dCK activity in the parental cell lines L1210 and BCLO with gemcitabine compared to ara-C as a substrate. DCK activity was not or hardly detectable in the resistant L4A6 and Bara-C cell. In the rat leukemia cell lines, deoxycytidine (dCyd) phosphorylation activity showed an aberrant pattern, since the activity with dCyd was 1.5-fold higher in the Bara-C cell line compared with BCLO, possibly due to thymidine kinase 2. The wild-type L1210 cells accumulated at least 3-fold more ara-CTP and dFdCTP than the rat leukemia cell line BCLO. The ara-C-resistant variants L4A6 and Bara-C did not accumulate dFdCTP or ara-CTP. In conclusion, gemcitabine was more active than ara-C in all leukemia cell lines tested. The sensitivity of the wild-type cell lines correlates with the accumulation of dFdCTP and ara-CTP, but is independent of dCK. However, both resistant variants had decreased dCK activities, but were relatively more sensitive to dFdC than to ara-C.     

Cytotoxic activity and mechanism of action of 5-Aza-2'-deoxycytidine in human CML cells

We investigated the cytotoxic activity and some aspects of the mode of action of 5-aza-2'-deoxycytidine (Aza-dC) in 21 primary cultures of leukemic cells freshly obtained from patients with chronic myeloid leukemia (CML) in blast crisis. The cytotoxic potency of Aza-dC was comparable or even greater than that of 1-beta-D-arabinofuranosylcytosine (Ara-C) in most cases, suggesting that this drug has potential in the therapy of blast crisis of CML. Drug incorporation into DNA was evaluated by exposing leukemic cells simultaneously to 3H-Aza-dC at the concentration of 0.1 micrograms/ml and 14C-thymidine (TdR) used as internal standard. Incorporation of Aza-dC into DNA was detectable in all cases. In 17 samples we evaluated the DNA integrity of leukemic cells exposed to Aza-dC using alkaline elution techniques. The drug caused a detectable amount of DNA alkali labile sites (ALS). DNA-ALS increased in cells exposed to Aza-dC concentrations from 0.1 to 1 microgram/ml but did not further increase at 10 micrograms/ml. A plateau in the levels of DNA-ALS was also seen in human K562 cells exposed to increasing concentrations of Aza-dC from 5 to 10 micrograms/ml, whereas in these cells Aza-dC incorporation into DNA increased with increasing Aza-dC concentrations. Therefore, DNA-ALS caused by Aza-dC are not simply the result of the chemical decomposition of azacytosine molecules incorporated into DNA, but are presumably the result of a saturable DNA repair mechanism (e.g., glycosylases) leading to formation of the apyrimidinic sites.     

Activation of protein kinase C-alpha and translocation of the myristoylated alanine-rich C-kinase substrate correlate with phorbol ester-enhanced noradrenaline release from SH-SY5Y human neuroblastoma cells

The aim of this study was to investigate the mechanism by which short-term pretreatment with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM) enhances noradrenaline (NA) release from the human neuroblastoma cell line SH-SY5Y. Subcellular fractionation and immunocytochemical studies demonstrated that an 8-min TPA treatment caused translocation of the alpha-subtype of protein kinase C (PKC) from the cytosol to the plasma membrane. In contrast, TPA altered the distribution of PKC-epsilon from cytosolic and membrane-associated to cytoskeleton- and membrane-associated. TPA had no effect on the cytosolic location of PKC-zeta. Subcellular fractionation studies also showed that the myristoylated alanine-rich C-kinase substrate (MARCKS), a major neuronal PKC substrate that has been implicated in the mechanism of neurotransmitter release, translocated from membranes to cytosol in response to an 8-min TPA treatment. Under these conditions the level of phosphorylation of MARCKS increased threefold. The ability of TPA to enhance NA release and to cause the translocation and phosphorylation of MARCKS was inhibited by the PKC inhibitor Ro 31-8220 (10 microM). Selective down-regulation of PKC subtypes by prolonged exposure to phorbol 12,13-dibutyrate (100 nM) attenuated the TPA-induced enhancement of NA release and the translocation of MARCKS over an interval similar to that of down-regulation of PKC-alpha (but not -epsilon or -zeta). Thus, we have demonstrated a strong correlation between the translocation of MARCKS and the enhancement of NA release from SH-SY5Y cells due to the TPA-induced activation of PKC-alpha.     

Synthesis and biological activity of a 1 alpha,25-dihydroxyvitamin D2 analog bearing an amide group in the side-chain

Synthesis of a 1 alpha,25-dihydroxyvitamin D2 analog (3), in which the double bond in the side-chain is replaced by an amide group, is described. Condensation of a carboxylic acid (8) with an amine (6) gave an amide (9), which in turn led to 3 via several steps. The analog (3) could not bind to the chick cytosol vitamin D receptor, which indicated the importance of the hydrophobic interaction of the C(22)-C(23) double bond in 1 alpha,25-dihydroxyvitamin D2 (2) with the vitamin D receptor.     

Phospholipase D activity of human amnion cells stimulated with phorbol ester and bradykinin

Twenty-four-hour intraesophageal pH monitoring is presently considered the most reliable diagnostic test for gastroesophageal reflux. Prolonged esophageal pH measurements can be obtained in hospitalized patients with a stationary technique and in ambulant outpatients by means of a portable device; however, there have been no studies that have examined whether the two approaches provide a similar diagnostic accuracy. We performed a prospective study to compare stationary and ambulatory pH-metry in the diagnosis of gastroesophageal reflux. Seventy-seven control subjects and 178 patients with proven gastroesophageal reflux disease were randomized to either ambulant or static pH-metry, which was performed with standard pH electrodes, sensors, and recorders. Reflux events (intraesophageal pH < 4.0) analyzed were: number of episodes; total, upright, and supine reflux time; number of episodes lasting > 5 min; and duration of the longest episode. A composite score of all reflux events according to DeMeester was also calculated. The limits of normality were defined as the 95th percentiles of the control groups. Both controls and patients assigned to either pH monitoring method were comparable. Of 255 studies attempted, 243 (95%) were completed successfully. The results showed similar median values of reflux events for the two control groups and for the two patients groups. Percent total reflux time provided a good separation between normal and abnormal reflux, with a sensitivity of 0.92 for static pH-metry and 0.68 for the ambulant procedure (respective 95th percentiles, 3.4 and 4.6).(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism and metabolic actions of 6-methylpurine and 2-fluoroadenine in human cells

Activation of purine nucleoside analogs by Escherichia coli purine nucleoside phosphorylase (PNP) is being evaluated as a suicide gene therapy strategy for the treatment of cancer. Because the mechanisms of action of two toxic purine bases, 6-methylpurine (MeP) and 2-fluoroadenine (F-Ade), that are generated by this approach are poorly understood, mechanistic studies were initiated to learn how these compounds differ from agents that are being used currently. The concentration of F-Ade, MeP, or 5-fluorouracil required to inhibit CEM cell growth by 50% after a 4-hr incubation was 0.15, 9, or 120 microM, respectively. F-Ade and MeP were also toxic to quiescent MRC-5, CEM, and Balb 3T3 cells. Treatment of CEM, MRC-5, or Balb 3T3 cells with either F-Ade or MeP resulted in the inhibition of protein, RNA, and DNA syntheses. CEM cells converted F-Ade and MeP to F-ATP and MeP-ribonucleoside triphosphate (MeP-R-TP), respectively. The half-life for disappearance of HeP-ribonucleoside triphosphate from CEM cells was approximately 48 hr, whereas the half-lives of F-ATP and ATP were approximately 5 hr. Both MeP and F-Ade were incorporated into the RNA and DNA of CEM cells. These studies indicated that the mechanisms of action of F-Ade and MeP were quite different from those of other anticancer agents, and suggested that the generation of these agents in tumor cells by E. coli PNP could result in significant advantages over those generated by either herpes simplex virus thymidine kinase or E. coli cytosine deaminase. These advantages include a novel mechanism of action resulting in toxicity to nonproliferating and proliferating tumor cells and the high potency of these agents during short-term treatment.     

Ribonuclease L, a 2-5A-dependent enzyme: purification to homogeneity and assays for 2-5A binding and catalytic activity

We have already presented a two-dimensional cell motility assay using a highly metastatic variant (L-10) of human rectal adenocarcinoma cell line RCM-1 as a motility model of tumour cells of epithelial origin. In this model, L-10 cells showed locomotion as a coherent sheet when stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA), and we called this type of movement "cohort migration". Electron and immunoelectron microscopic study of the migrating cell sheets demonstrated localized release from cell-cell adhesion only at the lower portion of the cells with loss of E-cadherin immunoreactivity, and this change was associated with increased tyrosine phosphorylation of the E-cadherin-catenin complex, including beta-catenin. Cell-extracellular matrix (ECM) interactions involved in this TPA-induced cohort migration and their effect on tyrosine phosphorylation of the E-cadherin-catenin complex have now been investigated. L-10 cell cohort migration was almost completely inhibited by addition of Arg-Gly-Asp (RGD) peptide into the medium, and thus RGD dependent. Cohort migration was stimulated on type I and IV collagens, fibronectin (FN)- and laminin-coated substratum, but was inhibited by RGD only on FN-coated surface. By using immunofluorescent techniques, FN was demonstrated preferentially around migrating cells, and a protein synthesis inhibitor, cycloheximide, inhibited the migration by about 75%. FN produced by L-10 cells were found to be mostly EDA+ FN when analysed by RT-PCR. Moreover, anti-FN antibody, but not anti-vitronectin antibody, inhibited the TPA-induced cohort migration almost completely. Thus, it was likely that L-10 cells produced FN themselves and moved on the FN substrate in an RGD-dependent manner. However, stimulation of migration by type I collagen coating and inhibition by RGD treatment did not affect the tyrosine phosphorylation of the E-cadherin-catenin complex induced by TPA, indicating that cell-cell interactions were adjusted to suit cell migration, irrespective of the condition of cell-ECM adhesion, during TPA-induced cohort migration.     

Alniditan, a new 5-hydroxytryptamine1D agonist and migraine-abortive agent: ligand-binding properties of human 5-hydroxytryptamine1D alpha, human 5-hydroxytryptamine1D beta, and calf 5-hydroxytryptamine1D receptors investigated with [3H]5-hydroxytryptamine and [3H]alniditan

Alniditan is a new migraine-abortive agent. It is a benzopyran derivative and therefore structurally unrelated to sumatriptan and other indole-derivatives and to ergoline derivatives. The action of sumatriptan is thought to be mediated by 5-hydroxytryptamine (5-HT)1D-type receptors. We investigated the receptor-binding profile in vitro of alniditan compared with sumatriptan and dihydroergotamine for 28 neurotransmitter receptor subtypes, several receptors for peptides and lipid-derived factors, ion channel-binding sites, and monoamine transporters. Alniditan revealed nanomolar affinity for calf substantia nigra 5-HT1D and for cloned h5-HT1D alpha, h5-HT1D beta and h5-HT1A receptors (Ki = 0.8, 0.4, 1.1, and 3.8 nM, respectively). Alniditan was more potent than sumatriptan at 5-HT1D-type and 5-HT1A receptors. Alniditan showed moderate-to-low or no affinity for other investigated receptors; sumatriptan showed additional binding to 5-HT1F receptors. Dihydroergotamine had a much broader profile with high affinity for several 5-HT, adrenergic and dopaminergic receptors. In signal transduction assays using cells expressing recombinant h5-HT1D alpha, h5-HT1D beta, or h5-HT1A receptors, alniditan (like 5-HT) was a full agonist for inhibition of stimulated adenylyl cyclase (IC50 = 1.1, 1.3, and 74 nM, respectively, for alniditan). Therefore, in functional assays, the potency of alniditan was much higher at 5-HT1D receptors than at 5-HT1A receptors. We further compared the properties of [3H]alniditan, as a new radioligand for 5-HT1D-type receptors, with those of [3H]5-HT in membrane preparations of calf substantia nigra, C6 glioma cells expressing h5-HT1D alpha, and L929 cells expressing h5-HT1D beta receptors. [3H]Alniditan revealed very rapid association and dissociation binding kinetics and showed slightly higher affinity (Kd = 1-2 nM) than [3H]5-HT. We investigated 25 compounds for inhibition of [3H]alniditan and [3H]5-HT binding in the three membrane preparations; Ki values of the radioligands were largely similar, although some subtle differences appeared. Most compounds did not differentiate between 5-HT1D alpha and 5-HT1D beta receptors, except methysergide, ritanserin, ocaperidone, risperidone, and ketanserin, which showed 10-60-fold higher affinity for the 5-HT1D alpha receptor. The Ki values of the compounds obtained with 5-HT1D receptors in calf substantia nigra indicated that these receptors are of the 5-HT1D beta-type. We demonstrated that alniditan is a potent agonist at h5-HT1D alpha and h5-HT1D beta receptors; its properties probably underlie its cranial vasoconstrictive and antimigraine properties.     

Metabolism of methoxyphenamine and 2-methoxyamphetamine in P4502D6-transfected cells and cell preparations

1. Control and P4502D6-transfected human B-lymphoblastoid cell lines (cHol and h2D6v2 respectively) were used to study 2D6-mediated metabolism of methoxyphenamine (MPA) and 2-methoxyamphetamine (2MA). The main metabolites were products of O-dealkylation and aromatic hydroxylation at the 5-position. In addition, N-desmethyl-methoxyphenamine (NDMP) was also identified as a minor metabolite of MPA in extracts of these cells, confirming previous reports of 2D6-mediated N-demethylation of MPA. 2. An additional ring-hydroxylated metabolite of MPA and 2MA has been tentatively identified as the corresponding 3-hydroxy-2-methoxy derivative. 3. MPA metabolism in whole cells was time dependent, with approximately 30% of the MPA metabolized after 72 h. A 35% conversion of MPA was achieved on average with cell lysates. Only 18% 2MA was metabolized. By contrast, control cells (cHol) showed no evidence of any MPA or 2MA metabolites even after 96-h incubation. 4. Continuous presence of haemin/dimethylsulphoxide (DMSO) throughout the 4-day incubation with MPA resulted in a shift in the metabolite profile towards the production of NDMP at the expense of the other products. 5. In summary, h2D6v2 cells, lysates and microsomes can form all metabolites of MPA and can be used in drug interaction studies.     

A carbocyclic nucleoside analogue is a TNF-alpha inhibitor with immunosuppressive action: role of prostaglandin E2 and protein kinase C and comparison with pentoxifylline

PURPOSE: To compare the activity of topical 0.05% and 0.01% mequitazine versus vehicle in the prevention of allergic conjunctivitis induced by a conjunctival provocation test with allergens. METHODS: Forty subjects with a history of grass pollen allergic conjunctivitis were enrolled in this comparative, randomized, double-masked study. Fifteen minutes before the conjunctival provocation test, subjects received one instillation alone of 0.05% mequitazine eyedrops in one eye and in the fellow eye either one drop of 0.01% dose or vehicle, at the same dosage. Then a specific conjunctival provocation test (CPT) was performed with the allergen threshold concentration previously defined. The therapeutic efficacy was assessed by the decrease in a symptomatic composite score representing the allergic reaction. RESULTS: Topical mequitazine at the dose of 0.05% significantly decreases the intensity of the allergic reaction compared to vehicle. Moreover, the group treated with this dose needed a higher allergen concentration to trigger ocular signs. The 0.01% dose appears efficient only on itching, compared to vehicle. CONCLUSION: The results of this study support the efficacy and the onset of action of 0.05% mequitazine eyedrops versus 0.01% or vehicle in the prevention of allergic conjunctivitis induced by a conjunctival provocation test.     

RNase L inhibitor is induced during human immunodeficiency virus type 1 infection and down regulates the 2-5A/RNase L pathway in human T cells

The interferon-regulated 2-5A/RNase L pathway plays a major role in the antiviral and antiproliferative activities of these cytokines. Several viruses, however, have evolved strategies to escape the antiviral activity of the 2-5A/RNase L pathway. In this context, we have cloned a cDNA coding for the RNase L inhibitor (RLI), a protein that specifically inhibits RNase L and whose regulated expression in picornavirus-infected cells down regulates the activity of the 2-5A/RNase L pathway. We show here that RLI increases during the course of human immunodeficiency virus type 1 (HIV-1) infection, which may be related to the downregulation of RNase L activity that has been described to occur in HIV-infected cells. In order to establish a possible causal relationship between these observations, we have stably transfected H9 cells with RLI sense or antisense cDNA-expressing vectors. The overexpression of RLI causes a decrease in RNase L activity and a twofold enhancement of HIV production. This increase in HIV replication correlates with an increase in HIV RNA and proteins. In contrast, reduction of RLI levels in RLI antisense cDNA-expressing clones reverses the inhibition of RNase L activity associated with HIV multiplication and leads to a threefold decrease in the viral load. This anti-HIV activity correlated with a decrease in HIV RNA and proteins. These findings demonstrate that the level of RLI, via its modulation of RNase L activity, can severely impair HIV replication and suggest the involvement of RLI in the inhibition of the 2-5A/RNase L system observed during HIV infection.     

Interaction of cyclin-dependent kinase 2 and the Lyn tyrosine kinase in cells treated with 1-beta-D-arabinofuranosylcytosine

The cyclin dependent kinase 2 (Cdk2) is required for initiation and progression of DNA replication. Activation of Cdk2 involves binding to cyclin E or cyclin A and dephosphorylation of Tyr15. The present studies demonstrate that treatment of U-937 cells with 1-beta-D-arabinofuranosylcytosine (ara-C) is associated with tyrosine phosphorylation of Cdk2 and inhibition of Cdk2 activity. The results also demonstrate that Cdk2 directly associates with the Src-like tyrosine kinase Lyn as a consequence of ara-C-treatment. Confocal microscopy studies show that Lyn is detectable in the nucleus and that it colocalises with Cdk2. Subcellular fractionation and coimmunoprecipitation studies further demonstrate nuclear binding of Lyn and Cdk2. We also show that Lyn phosphorylates Tyr15 of Cdk2 and that incubation of Lyn with Cdk2 results in inhibition of Cdk2 activity. These findings suggest that the association of Lyn and Cdk2 in ara-C-treated cells may contribute to regulation of Cdk2-dependent cell cycle checkpoints.     

Metabolism of 17 beta-hydroxy-2alpha, 3alpha-cyclopropano-5alpha-androstane in the rabbit

The neutral urinary excretion products of 17beta-hydroxy-2alpha, 3alpha-cyclopropano-5alpha-androstane from the rabbit, dosed orally, were investigated. Column chromatography yielded five crystalline metabolites which were identified by GLC and spectroscopic measurements. Three of these substances were hydroxylated in the 4alpha-position and one in the 6alpha-position with the cyclopropane ring intact. The fifth substance, 17beta-hydroxy-3beta-methyl-5alpha-androstan-2-one, can be derived from initial hydroxylation of the cyclopropane ring at C-2 followed by ring opening. The dosed substance and triol material was shown to be present by GLC and m.s. measurements. GLC determinations show that hydroxylation has occurred at C-4-C-6-C-2.     

Identification and sequence of human PKY, a putative kinase with increased expression in multidrug-resistant cells, with homology to yeast protein kinase Yak1

The authors report a clinical case of endometriosis the abdomen rectum muscle, in woman 28 years old, after a cesarean section delivery. On the basis of literature on the topic, the following are taken into consideration, the incidence, the pathogenesis, the clinical characteristics of this kind of pathology and the aspects which might facilitate the diagnostic approach and correct therapeutic to be given or follow. Parietal endometriosis is an extremely rare disease with incidence in feminine population of 0.03-1%. The pathogenesis is still ill-known. Lack of the classical symptoms and the unusual site can make diagnosis difficult. Pathognomonics but not always present are the presence of tumescence palpable of the abdominal wall near or proximity of preceding surgical scar, the cyclic character of painful symptomatology, the augmentation of volume and the bleeding in period menstrual or premenstrual. The ultrasonography, the computerized axial tomography, the nuclear magnetic resonance can facilitate the preoperative diagnosis but they do not always furnish reports of certainty. The aspirate-needle in ultrasonography control can furnish one of orientation diagnosis. The diagnosis of certainty is founded on the histologic examination after biopsy or excision. The treatment of the abdominal wall endometriosis is surgically essential. The excision of tumescence, easy usually, it is the only means to obtain the definitive recovery. The medical therapy postoperative is adjuvant in the treatment of unrecognized pelvic centres of endometriosis.     

Cloning of human Stat5B. Reconstitution of interleukin-2-induced Stat5A and Stat5B DNA binding activity in COS-7 cells

We have isolated a second human Stat5 cDNA, Stat5B, and demonstrated that the genes encoding both Stat5A and Stat5B are located at chromosome 17q11.2. Both genes were constitutively transcribed in peripheral blood lymphocytes. By using specific antisera, we demonstrated that both Stat5A and Stat5B are activated by interleukin-2 (IL-2) in peripheral blood lymphocytes, natural killer-like YT leukemia cells, and human T cell lymphotropic virus type I-transformed MT-2 T cells. In COS-7 cells, which constitutively express the Janus family tyrosine kinase Jak1, reconstitution of IL-2-induced Stat5A and Stat5B DNA binding activities was dependent on the coexpression of Jak3 along with the IL-2 receptor beta chain and the common cytokine receptor gamma-chain. This IL-2-induced Stat5 activation was dependent on the presence of either of two tyrosines (Tyr-392 or Tyr-510) in the IL-2 receptor beta chain, indicating that either of these two tyrosines can serve as a docking site. Moreover, we demonstrated that human Stat5 activation is also dependent on Tyr-694 in Stat5A and Tyr-699 in Stat5B, indicating that these tyrosines are required for dimerization. The COS-7 reconstitution system described herein provides a valuable assay for further elucidation of the IL-2-activated JAK-STAT pathway.     

Petunia p34cdc2 protein kinase activity in G2/M cells obtained with a reversible cell cycle inhibitor, mimosine

Protoplasts isolated from petunia leaf mesophyll are non-cycling cells mostly with 2C content. Cells regenerating from protoplast culture enter mitosis after 48 h. This experimental model is used to relate p34cdc2 kinase activity to cell cycle phase. Our results show that the histone H1 phosphorylation, and hence p34cdc2 kinase activity, peaks with G2+early M cell cycle phase. However, a trace kinase activity was already present when most cells were entering S phase. To obtain a maximum of cells in G1+S phases, the protoplast culture was treated with the rare amino acid, mimosine. Mimosine blocked plant cells derived from protoplast culture both at G1 and in early and mid S phase. Despite the increased G1+S level, p34cdc2 kinase activity did not increase. This suggests that the trace activity appearing when the majority of cells are entering S does not correspond to any putative p34cdc2 activation at G1/S transition but to the activation of the minor 4C population initially present in the leaf: the hypothesis remains that p34cdc2 kinase activity is solely related to G2+M phase in petunia.     

High-performance liquid chromatographic determination of the enantiomers of the benzoquinolinone LY191704, a human type 1 5 alpha-reductase inhibitor, in plasma

A stereoselective reversed-phase liquid chromatographic method for the determination of compounds LY300502 and LY300503 (enantiomers of LY191704) in rat and dog plasma was developed. The assay involved extraction of the compounds using a strong cation-exchange solid-phase extraction column, from which the compounds are eluted with 1% of 1 M HCI in methanol. The enantiomers were separated on a Daicel Chiralcel OD-R column. The mobile phase consisted of water-acetonitrile-methanol (50:40:10, v/v) at a flow-rate of 0.3 ml/min. UV detection was achieved at 220 nm. The disposition of the enantiomers of LY191704 in rats and dogs was found to be stereoselective and species specific.