Synthesis,characterization and antitumor activity of platinum(II) complexes with diethyl and monoethyl 2-quinolylmethylphosphonates

A series of new platinum(II) complexes with diethyl (2-dqmp) and monoethyl (2-Hmqmp) 2-quinolylmethylphosphonates have been prepared and studied. Both organophosphorus ligands by reaction with [PtX(4)](2-) (X=Cl, Br) form either the molecular or ionic complexes depending on the acidity of the reaction solution. Dihalide adducts, trans-[PtL(2)X(2)] (L=2-dqmp, 2-Hmqmp), with N-bonded ligand through the quinoline nitrogen were obtained in the neutral medium, while under acidic conditions at pH<3 were isolated the ion-pair salt complexes, [LH](2)[PtX(4)], containing the protonated quinoline ligand as cation and tetrahaloplatinate complex as anion. In addition, 2-Hmqmp at pH approximately 3.5 forms quinolinium hexahalodiplatinum salt complexes, [2-H(2)mqmp](2)[Pt(2)X(6)], while the chelate complex, [Pt(2-mqmp)(2)].2H(2)O, with N,O-bonded ligand through the quinoline nitrogen and the deprotonated phosphonic acid oxygen was obtained at pH>6. The new complexes were characterized on the basis of elemental and thermogravimetric analyses, conductometric measurements, and by infrared and (1)H NMR spectral studies. As a preliminary assessment of their biological activity, complexes were evaluated for their in vitro cytostatic activity in an epidermoid human carcinoma (KB) and murine leukemia (L1210) cell lines. The results obtained were compared with those obtained for the corresponding Pd(II) complexes.     

Automated identification of putative methyltransferases from genomic open reading frames

We have analyzed existing methodologies and created novel methodologies for the automatic assignment of S-adenosylmethionine (AdoMet)-dependent methyltransferase functionality to genomic open reading frames based on predicted protein sequences. A large class of the AdoMet-dependent methyltransferases shares a common binding motif for the AdoMet cofactor in the form of a seven-strand twisted beta-sheet; this structural similarity is mirrored in a degenerate sequence similarity that we refer to as methyltransferase signature motifs. These motifs are the basis of our assignments. We find that simple pattern matching based on the motif sequence is of limited utility and that a new method of "sensitized matrices for scoring methyltransferases" (SM2) produced with modified versions of the MEME and MAST tools gives greatly improved results for the Saccharomyces cerevisiae yeast genome. From our analysis, we conclude that this class of methyltransferases makes up approximately 0.6-1.6% of the genes in the yeast, human, mouse, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, and Escherichia coli genomes. We provide lists of unidentified genes that we consider to have a high probability of being methyltransferases for future biochemical analyses.     

Application of semi-industrial monolithic columns for downstream processing of clotting factor IX

It has been shown in a previous study that monolithic columns can be used for downstream processing of different concentrates of clotting factor IX [K. Branovi? et al., J. Chromatogr. A 903 (2000) 21]. This paper demonstrates that such supports are useful tools also at an early stage of the purification process of factor IX from human plasma. Starting with the eluate after solid-phase extraction with DEAE-Sephadex, the use of monolithic columns has allowed much better purification than that achieved with conventional anion-exchange supports. The period of time required for separation is also much reduced. In up-scaling experiments, separations are carried out with 8, 80 and 500 ml columns. A volume of 1830 ml of DEAE-Sephadex eluate, containing a total of 27.6 g of protein and 48500 IU of factor IX is applied to the 500 ml monolithic column. This corresponds to a separation on a pilot scale. The results of this separation after up-scaling are comparable to those obtained with the 8 ml column on a laboratory scale.     

The use of HPLC-MS in T-cell epitope identification

The hunt for T-cell epitopes is going on because hopes are set on such peptide sequences for diagnosis and vaccine development in the fight against infectious and tumor diseases. In addition to a variety of other techniques used in T-cell epitope identification, mass spectrometers coupled to microcapillary liquid chromatography have now become an important and sensitive tool in separation, detection, and sequence analysis of highly complex natural major histocompatibility complex (MHC) ligand mixtures. In this article, we review the basics of mass spectrometric techniques and their on-line coupling to microcapillary liquid chromatography (microcap-LC). Furthermore, we introduce current strategies for the identification of new T-cell epitopes using microcapillary liquid chromatography-mass spectrometry (microcap-LC-MS).     

Immunocytochemical study of the glucocorticoid receptor in rat liver nuclei after hyperthermic stress

The presence of glucocorticoid receptors (GR) in rat liver nuclei over a 24 h time period following hyperthermic stress at 41 degrees C was immunocytologically studied using unfixed nuclear smears. Liver nuclei in unstressed animals were found to be immunonegative for GR. However, intense GR immunopositivity followed by a subsequent gradual decrease in receptor levels was observed in the nuclei of test animals during the first 2 h after stress. This stress-related increase in the receptor nuclear level was greater than the increase seen after dexamethasone administration. These results suggest that hyperthermic stress could potentiate the hormonal stimulation of receptor nuclear translocation.     

Expression of one sponge Iroquois homeobox gene in primmorphs from Suberites domuncula during canal formation

Sponges (Porifera) represent the evolutionary oldest multicellular animals. They are provided with the basic molecules involved in cell-cell and cell-matrix interactions. We report here the isolation and characterization of a complementary DNA from the sponge Suberites domuncula coding for the sponge homeobox gene, SUBDOIRX-a. The deduced polypeptide with a predicted Mr of 44,375 possesses the highly conserved Iroquois-homeodomain. We applied in situ hybridization to localize Iroquois in the sponge. The expression of this gene is highest in cells adjacent to the canals of the sponge in the medulla region. To study the expression of Iroquois during development, the in vitro primmorph system from S. domuncula was used. During the formation of these three-dimensional aggregates composed of proliferating cells, the expression of Iroquois depends on ferric iron and water current. An increased expression in response to water current is paralleled with the formation of canal-like pores in the primmorphs. It is suggested that Iroquois expression is involved in the formation of the aquiferous system, the canals in sponges and the canal-like structures in primmorphs.     

Synthesis and antitumor activity of N-sulfonyl derivatives of nucleobases and sulfonamido nucleoside derivatives

The introduction of sulfonamido group on the C-2 position of pyrimidine nucleosides was achieved by ring opening of 2,2'- and 2,3'-anhydronucleosides. N-sulfonyl derivatives of nucleobases and sulfonamido derivatives of nucleosides were assayed for in vitro antitumor activity.     

Pentylenetetrazol-induced seizures and kindling: changes in free fatty acids,superoxide dismutase,and glutathione peroxidase activity

The whole brain free fatty acid (FFA) level, as well as the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were determined in the frontal cortex, cerebellum, hippocampus, and pons-medulla region of the single pentylenetetrazol (PZT)-treated and PZT-kindled Hannover-Wistar rats. PZT administration in the convulsive dose caused significant increase of the brain FFA content. Decreased SOD activity was detected in the frontal cortex of PZT-kindled rats, whereas decreased GPX activity was found in the frontal cortex and cerebellum of all treated rats, as well as in the hippocampus and pons-medulla of PZT-kindled rats. Kindling caused distinctive change of antioxidative defense in the frontal cortex, hippocampus, and pons-medulla region.     

Localization of carboxypeptidase A-like enzyme in rat kidney

In this study we demonstrate that carboxypeptidase A (CPA)-like enzyme is expressed in rat kidney. The major metabolites of angiotensin (Ang) I by the rat renal mesangial cell extract at 37 degrees C, pH 7.4, were Ang 1-9 and Ang II. Quinaprilat did not influence the formation of Ang 1-9, but it inhibited formation of Ang II. The formation of Ang 1-9 was inhibited by potato carboxypeptidase inhibitor, 1,10-phenanthroline or EDTA. Lowering the pH from 7.4 to 4.0 also inhibited the formation of this nonapeptide. These findings suggest that a metallocarboxypeptidase is responsible for Ang 1-9 production. Using monoclonal antibodies to CPA, Western blot showed the presence of CPA-like enzyme in the extracts prepared from the mesangial cells or kidney cortex of the rat. Immunohistochemistry showed that CPA-like enzyme is localized in the mesangial glomerular cells and adventitia of kidney blood vessels, whereas it was absent in the renal tubules. Our data suggest that a CPA-like enzyme could be added to a repertoire of enzymes present in the rat mesangial cells and adventitia of renal blood vessels.