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We investigated and compared hydrophilic retention mechanism (HILIC) of ten compounds (imidazoline and serotonin receptor ligands) in the thin-layer chromatographic (TLC) system and the high-performance liquid chromatographic (HPLC) system. The TLC and HPLC stationary phases were the chemically bonded amino (NH2) phases. In the TLC mode, the binary mobile phases were composed of acetonitrile (as the main component) and water or methanol (as the modifier). In the HPLC mode, the binary mobile phase was composed of acetonitrile (as the main component) and methanol (as the modifier). For each compound, we determined this region for which linear relationship between the retention and volume fraction of the mobile phase modifier was observed, and the retention behaviour was described by the linear solvent strength (LSS) model. With the obtained regression constant values (log k0, RM0) in mind, we selected molecular parameters influencing retention mechanism in the considered chromatographic systems. Lipophilicity plays an important role in the TLC mode with the acetonitrile‒methanol mobile phase, while geometrical characteristics of the test compounds (ring complexity, number of multiple bonds) play a predominant role in the TLC mode with the acetonitrile‒water mobile phase. It was also established that the hydrophilic mechanisms are different for the non-aqueous TLC vs. aqueous TLC systems and for the TLC vs. HPLC systems. As a result, different elution orders and separation performances can be expected for the imidazoline and serotonin receptor ligands, depending on the chromatographic system employed.

Mutual relationships have been studied between seven plant end seed characteristics (plant height, number of leaves per plant, 1000-seed mass, hectoliter mass, kernel content, seed thickness, seed width) and their correlation with oil content in seed. Significant of highly significant correlations have been found between oil content on one side and 1000-seed mass, hectoliter mass, kernel content, and seed thickness on the other. Anon-significant negative correlation was registered for the number of leaves per plant, non-significant positive correlations for plant height and seed width. The path analysis has revealed a highly significant positive direct effect of hectoliter mass and kernel content and a significant negative direct effect of seed width on oil content in seed. The coefficient of determination has shown that 79.5% of the total variability for oil content were controlled by the factors studied.

8-Chloroadenosine 3',5'-monophosphate (8-Cl-cAMP) is a potential new anticancer agent, but its mechanism of action is not clearly defined. In this work we have studied the effect of various heat inactivated and heat untreated human sera in the absence or in the presence of a nonspecific phosphodiesterase (PDE) inhibitor, IBMX, or of nucleoside transport inhibitor and cGMP-specific PDE inhibitor dipiridamole (DP), or of inosine-monophosphatedehydrogenase (IMPDH) inhibitor, tiazofurin, (T), on the antiproliferative 8-Cl-cAMP action towards two human malignant cell lines, K562 and HeLa cells, in vitro. Cell survival was determined 72 hrs after the agents action, using MTT assay. The results obtained, indicated the similar inhibitory effect of 8-Cl-cAMP on HeLa cell survival in the presence of four different heat untreated human sera (IC50 = 4-4.8 microM). Serum heat inactivation caused decrease in 8-Cl-cAMP antiproliferative action depending on the blood donor (IC50 = 23 microM, 15 microM, 19 microM, and 9 microM) and suggesting that some thermolabile ingredient(s) present in sera is involved, at least partially, in the induction or permittance of antiproliferative 8-Cl-cAMP action. K562 Cells were not as much resistant to 8-Cl-cAMP as HeLa cells, or mouse melanoma B16 cells; in the presence of heat untreated FBS, IC50 = 16 microM, while for B16 cells IC50 was 8 microM. Different human sera show different effect on 8-Cl-cAMP action on K562 cells: IC50 = 7.5 microM and 16.5 microM. In the presence of heat inactivated human sera 8-Cl-cAMP IC50 concentrations were higher, with relevant mutual differences. The effect of different sera on 8-Cl-cAMP action was only partly abrogated in the presence of a nonspecific PDE inhibitor, IBMX, suggesting that the serum PDE action is one of the various factors contributing to the induction of 8-Cl-cAMP antiproliferative action. Nucleoside transport inhibitor and cGMP-specific PDE inhibitor dipiridamole inhibited the antiproliferative 8-Cl-cAMP action to HeLa and K562 cells. Tiazofurin and 8-Cl-cAMP acted as antagonists on HeLa, but not on K562 cells.

A simple and rapid in vitro method for qualitative and quantitative estimation of the G alpha-subunits interaction with the third intracellular loop of human D2s dopamine receptor has been developed. For this purpose, D2s-CL3 was cloned in pGEX-2T vector and expressed in E. coli BL21 DE3 as a fusion protein with glutathione-S-transferase (D2s-CL3-GST). The resulting soluble construct was purified by affinity chromatography on glutathione-Sepharose. G alpha-subunits were expressed and purified as His-tagged proteins. For the assay of G alpha/D2s-CL3-GST interactions, varying concentrations of pure His-tagged G alpha-proteins were immobilized on His-Bind Resin and titrated with D2s-CL3-GST fusion protein. G alpha/D2s-CL3-GST interactions were quantified by GST activity determination assay. It was shown that the fusion protein interacts specifically with different G alpha proteins, especially with G alpha(i) proteins. Based on saturation binding analyses, Kd values were determined revealing the highest affinity of His-G alpha(i,2) binding to the fusion protein. The affinities for G alpha(i)/D2s-CL3-GST protein interactions estimated in this way were in nanomolar range of concentrations.

Several 2-methylthiomethylbenzimidazoles (3a-e) and the corresponding sulphinyl derivatives 4a-e were synthesized and evaluated measuring their in vitro binding affinity at the D1 and D2 dopamine (source: synaptosomal membranes of the bovine nucleus caudatus) and 5-HT1A serotonin (source: synaptosomal membranes of the bovine hippocampus) receptors. [3H]SCH 23390, [3H]spiperone, and [3H]-8-OH-DPAT were employed as specific radioligands for the D1, D2 and 5-HT1A receptors, respectively. None of the compounds except for 3b acting as a moderate [3H]SCH 23390, competitor, expressed binding affinity at the D1 receptor. Compounds 4a and 4e were inactive displacers of both [3H]spiperone and [3H]-8-OH-DPAT. Ligands 4b, 3d and 4d acted as weak to moderate [3H]spiperone competitors and 3a was a weak [3H]-8-OH-DPAT displacer. The remaining ligands expressed binding affinity at the corresponding receptors in a nanomolar concentration range. Among them, compound 3b with Ki of 14.2 nM and 8.4 nM in [3H]spiperone and [3H]-8-OH-DPAT binding assay, respectively, was the most potent mixed dopaminergic/serotonergic ligand. Although sterically similar, the two classes of ligands differ with regard to electronic properties of substituents in position 2 of the benzimidazole ring. Oxidation of 2-(methylthiomethyl)benzimidazoles afforded ligands devoid of binding affinity at the 5-HT1A receptor and significantly reduced binding affinity at the D2 receptor. This points to the importance of electronic properties of substituents in position 2 of benzimidazole ring for the D2/5-HT1A affinity ratio of this type of ligands.

With an aim of creating new, mixed D-2/5-HT1A ligands, sixteen different compounds were synthesized. For this purpose 1-arylpiperazines attached through N-4 nitrogen to the dopaminergic pharmacophores of 2-(5-benzimidazole)-ethyl-, 2-(5-benztriazole)-ethyl-, 2-[5-(benzimidazole-2-thione]-ethyl- and 2-[6-(1,4-dihydroquinoxaline-2,3-dione)]-ethyl-type were selected according to their structural diversity (phenyl, substituted phenyl, heteroaryl and naphthyl). All new compounds were checked for in vitro binding affinity at the dopamine (D-1 and D-2) and serotonin (5-HT1A) receptors. Synaptosomal membranes prepared from fresh bovine caudate nuclei and hippocampus were used as a source of dopamine and serotonin receptors, respectively, [3H]SCH 23390 (D-1 selective), [3H]spiperone (D-2 selective) and 8-OH-[3H]DPAT (5-HT1A selective) were employed as the radioligands. All compounds were inactive competitors of [3H]SCH 23390, 1c and 2b being inactive in the two other binding assays, as well. Derivatives of 1-(2-pyridyl)piperazine 1b and 2a and of 1-phenylpiperazine 1a expressed moderate to low affinity for both D-2 and 5-HT1A receptors, while 1-(2-pyridyl)-piperazines 3b and 4b and 4-(1-phenylethyl)-1-(1-naphthyl)-piperazine 10 were moderate [3H]spiperone, but potent 8-OH-[3H]DPAT competitors. Among them, derivatives of 1-(1-naphthyl)-piperazine 1e, 2d and 3e and of 1-(2,3-dimethylphenyl)-piperazine 1d, 2c and 3d were the strongest competitors at both D-2 and 5-HT1A receptors.

Twelve different N-n-propyl-N-(2-arylethyl)-5-(1H-benzimidazol-2-thione)-ethy lamines and 12 related heterocyclic congeners were synthesized with the aim of creating new high affinity dopaminergic ligands. Upon thorough chemical analysis they were evaluated for their affinity towards the D-1 and the D-2 dopamine receptors (DAR) by in vitro binding assay using synaptosomal membranes of the bovine caudate nuclei and [3H]SCH 23390 (R (+)-7-chloro-8-hydroxy-3-methyl-1-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride) (D-1 selective) and [3H]spiperone (D-2 selective) as the radioligands. None of the synthesized compounds expressed affinity for the binding to the D-1 DAR Compounds 11e, 11j, 11l, 12b, 12d, 13a, 13d, 14a, and 14d were also inactive competitors of [3H] spiperone binding to the D-2 DAR. However, 1H-benzimidazol-2-thione derivatives 11k, 11h, and 11f and 1, 4-di-hydroquinoxalin-2,3-dione derivative 12c (in this rank order of potencies) acted as strong competitors of [3H] spiperone binding to the D-2 receptors under conditions that prevented radioligand binding to serotonin 5HT(2) receptors. Relationship between the structure and affinities of the new ligands for the binding to the D-2 DAR as well as the demands of the receptor itself for the interaction with the new compounds are discussed.

Four semirigid dopaminergic biosiosteres (N,N-di-n-propyl-5,6,7,8-tetrahydro-1 H-benz[f]benzimidazol-6-yl-amine [sequence: see text] and three related congeners) were synthesized and chemically characterized. The affinity and selectivity of these compounds for D-1 and D-2 classes of the dopamine receptor were determined in competition binding experiments using synaptosomal membranes of the bovine caudate nuclei and [3H]SCH 23390 (D-1 selective) and [3H]spiperone (D-2 selective) as radioligands. None of the four compounds tested expressed significant affinity for D-1 receptors and all of them competed moderately with [3H]spiperone binding to D-2 receptors under conditions that prevented radioligand binding to serotonin 5HT2 receptors. Some aspects of structure affinity relationship for this class of dopaminergic ligands are discussed.

Four different heterocyclic dopamine bioisosteres were synthesized. The affinity and selectivity of these compounds for the D-1 and D-2 classes of the dopamine receptor were determined in competition binding experiments using synaptosomal membranes from bovine caudate nuclei and [3H]SCH 23390 (D-1 selective) and [3H]spiperone (D-2 selective) as radioligands. None of these compounds expressed significant affinity for the D-1 receptors, while compounds 3, 4, 2 and 1 in this order of potency strongly competed with [3H]spiperone binding to D-2 receptors under conditions that prevented radioligand binding to serotonin 5HT2 receptors (50 nM ketanserin). Compounds 3 and 4 behave as agonists as judged by the data obtained in competition binding experiments in the presence of Gpp(NH)p, the former (3) expressing a very high affinity for D-2 receptors.

Two series of non-catechol dopamine (DA) bioisosteres with the hydroxyl groups on the benzene ring replaced by N-H groups were synthesized using phenethylamine as a parent molecule. Compounds from the first series (1-9) contained a primary amine group, while those from the second one (10-19) had a N,N-di-propylamino group introduced into the side chain of the phenethylamine. The affinity and selectivity of these compounds for the D-1 and D-2 subtypes of the DA receptor were determined in competition binding assays using synaptosomal membranes from bovine caudate nuclei and [3H]SCH 23390 (D-1 selective) and [3H]spiperone (D-2 selective) as radioligands. None of the 19 compounds examined expressed significant affinity for D-1 receptors, while compounds 15, 17 and 19, in this order of potency, strongly competed with [3H]spiperone binding to D-2 receptors under conditions that prevented radioligand binding to serotonin 5HT2 receptors. Varying affinities of the compounds examined for the DA receptors can be ascribed to different acidity of the N-H groups introduced at meta- and para-positions of the phenethylamine molecule.

The effects of several compounds acting through adenylate cyclase system and/or influencing prostaglandin biosynthesis on spermine-FBS cytotoxicity to human myelogenous leukemia K562 cells were studied. Salbutamol, a beta 2-adrenoceptor agonist inhibited to a certain extent spermine-FBS cytotoxic action to K562 cells, and propranolol, a beta 2-adrenoceptor antagonist, did not affect this inhibition. Aminophylline, an inhibitor of cyclic nucleotide phosphodiesterase, acted suppressing spermine-FBS cytotoxicity to K562 cells. Pretreatment of the cells with dexamethasone did not significantly alter salbutamol-related inhibition of spermine-FBS cytotoxicity. Indomethacin, an inhibitor of cyclooxygenases directly involved in prostaglandin biosynthesis, did not interfere with protective terbutaline effects against spermine-FBS cytotoxicity to K562 cells during the 24-hour period.

Interaction of a series of 99m Tc-radiopharmaceuticals with human blood proteins was investigated from two aspects: total protein binding, and specificity of binding to certain classes of proteins. After in vitro labelling of human sera with 99m Tc-radiopharmaceuticals, total protein binding was determined for thirteen 99m Tc-compounds (complexes of: HMPAO, GH, PAHIDA, MAG 3 , DTPA, PYP, MDP, DPD, EHIDA, IODIDA, Phytate and both complexes of DMSA) by a precipitation method (perchloric and trichloroacetic acid), dialysis and ultrafiltration. Selective binding to individual protein classes was determined by agarose gel electrophoresis after in vitro and in vivo labelling with the above-mentioned radiopharmaceuticals. The following carrier proteins were detected: α 1 -antitrypsin for 99m Tc-HMPAO, 99m Tc-DPD, 99m Tc(III)-DMSA renal agent, 99m Tc-GH, 99m Tc-DTPA, 99m Tc-PAHIDA and 99m Tc-EHIDA; albumin for 99m Tc(V)-DMSA tumorotropic agent and 99m Tc-MDP; transferrin for 99m Tc-phytate and 99m Tc-IODIDA; α 2 -globulin for 99m Tc-MAG 3 . Some agents were bound to two protein classes such as 99m Tc-PYP to α 1 -antitrypsin and transferrin; 99m Tc(III)-DMSA renal complex and 99m Tc-PAHIDA to α 1 -antitrypsin and α 2 -globulin. Some relationships between protein binding and biolocalization were noted for two 99m Tc-DMSA complexes and for three skeletal agents.

The effects of spermine and beta-adrenoceptor agonists (epinephrine, terbutaline and orciprenaline) in the presence and in the absence of fetal bovine serum (FBS) on human myelogenous leukemia K562 cells viability (V) and survival (N/Nc) were examined. Spermine-FBS significantly decreased both V and N/Nc of K562 cells. Aminoguanidine (AG), an amine oxidase inhibitor, and reduced form of glutathione abolished this effect demonstrating that the spermine-FBS action was amine oxidase-mediated. Epinephrine expressed a strong cytotoxicity to K562 cells which was abolished by pargyline, a specific monoamine oxidase (MAO) inhibitor, as well as by reduced form of glutathione. Terbutaline and orciprenaline exerted no cytotoxic activity to K562 cells cultured in FBS-supplemented medium, independently on the presence of spermine. However, terbutaline at concentrations of over 1 mmol strongly inhibited the cytotoxic effect on spermine-FBS. The relationship between cytotoxicity and chemical structure of beta-adrenoceptor agonists was discussed especially with respect to their stability toward oxidation.

The influence of glucocorticoid status of the rats (absence of glucocorticoid hormones achieved by adrenalectomy and substitution by dexamethasone 5 mg/kg b.w.) on the response of striatal dopamine-D2 receptors to environmentally induced hyperthermia and treatment with a local inflammatory agent turpentine (1 ml/kg b.w.) was studied. Both stress situations affected the density of D2 receptors in opposite directions. While hyperthermia led to an increase, turpentine treatment reduced Bmax value. The changes in the D2 receptor binding affinity were expressed as a certain decrement, which was statistically significant in sham-adrenalectomized hyperthermic animals and in both turpentine treated groups (sham-operated and adrenalectomized) administered dexamethasone. The absence of glucocorticoids in the circulation caused an elevation of Kd values in the control and both stressed groups and dexamethasone attenuated these changes. Dexamethasone also attenuated stress-related alterations of Bmax, as well as Kd increase upon adrenalectomy in control and hyperthermic animals. These results corroborate the evidence on the role of D2 receptors in thermoregulation and stress and demonstrate the significance of glucocorticoid hormones in the control of these processes.