Phosphatidylcholine could be the source of 1,2-DAG which activates protein kinase C in EGF-stimulated colon carcinoma cells (HT29)

In our previous study (A. Balogh et al, Cell. Signalling 5 (6), 795-802, 1993.), we have shown that epidermal growth factor (EGF) increased protein kinase C (PKC) activities in colon carcinoma cell line (HT29), possibly through the increased 1,2-diacylglycerol (1,2-DAG) production via phosphatidylcholine (PC). Here we investigate the effect of the well-known PKC activator 12-O-tetradecanoyl-2 phorbol-13-acetate (TPA), on the levels of ³²P incorporation into EGF induced phosphatidylinositols (PI, PI4P, PI4, 5P₂) and different phospholipids (PC, PA, PS) as well as on induced tyrosine kinase activity. TPA significantly decreased the effects of EGF and it had the biggest inhibitory effect on EGF induced PC level. These data support our contention that PC plays an important role in the activation of PKC via 1,2-DAG production in the EGF stimulated pathway.     

Roadsides provide refuge for orchids: characteristic of the surrounding landscape

Seminatural habitats are declining throughout the world; thus, the role of small anthropogenic habitats in the preservation of plants is becoming increasingly appreciated. Here, we surveyed the orchid flora of roadside verges in five Central European countries (Austria, Hungary, Romania, Slovakia, and Slovenia) and tested how the surrounding landscape matrix affects the overall number of species and individuals, and also different functional groups of orchids. We found more than 2,000 individuals of 27 orchid species during our surveys. According to our results, the increasing coverage of agricultural and urban areas negatively affects both the number of orchid species and individuals on roadsides. Our study further suggests that differences in the surrounding habitats affect which species are found on roadsides, since the increasing coverage of grasslands or forested areas around orchid occurrences had a significant positive effect on the number of grassland or forest‐dwelling species and individuals, respectively. Most variance in orchid numerosity and diversity was explained by the cover of the suitable habitat types of the respective taxa in the surrounding landscape of the sampling points. This highlights the importance of roadsides acting as refugia for numerous species and valuable plant communities as well as in supporting biodiversity in general.     

Synthesis and pharmacological investigation of new N-hydroxyalkyl-2-aminophenothiazines exhibiting marked MDR inhibitory effect

Novel N-hydroxyalkyl-2-aminophenothiazines implying a tetrazole moiety at the alkyl chain have been synthesized by hydroboration–oxidation of dienes followed by Buchwald–Hartwig cross-coupling reaction. Also, some sulfoxide and sulfone derivatives have been prepared by selective oxidations. MDR inhibition studies on rat hepatocyte cell culture revealed that some derivatives exhibit marked biological efficacy exceeding that of the standard verapamil (e.g., 3h, 4h, 16). Selected derivatives were subjected to chemical resolution to provide both enantiomers which were shown of similar activity on P-gp interaction measurements. The new compounds exhibited no toxicity.     

Transmigration characteristics of breast cancer and melanoma cells through the brain endothelium: Role of Rac and PI3K

Brain metastases are common and devastating complications of both breast cancer and melanoma. Although mammary carcinoma brain metastases are more frequent than those originating from melanoma, this latter has the highest tropism to the brain. Using static and dynamic in vitro approaches, here we show that melanoma cells have increased adhesion to the brain endothelium in comparison to breast cancer cells. Moreover, melanoma cells can transmigrate more rapidly and in a higher number through brain endothelial monolayers than breast cancer cells. In addition, melanoma cells have increased ability to impair tight junctions of cerebral endothelial cells. We also show that inhibition of Rac or PI3K impedes adhesion of breast cancer cells and melanoma cells to the brain endothelium. In addition, inhibition of Rac or PI3K inhibits the late phase of transmigration of breast cancer cells and the early phase of transmigration of melanoma cells. On the other hand, the Rac inhibitor EHT1864 impairs the junctional integrity of the brain endothelium, while the PI3K inhibitor LY294002 has no damaging effect on interendothelial junctions. We suggest that targeting the PI3K/Akt pathway may represent a novel opportunity in preventing the formation of brain metastases of melanoma and breast cancer.     

Roadside verges and cemeteries: Comparative analysis of anthropogenic orchid habitats in the Eastern Mediterranean

Several important habitats have become threatened in the last few centuries in the Mediterranean Basin due to major changes adopted in land‐use practices. The consequent loss of natural and seminatural orchid habitats leads to the appreciation of small anthropogenic habitats, such as cemeteries and roadside verges. Colonization of cemeteries and roadside verges by orchids has long been known, but no study to date compared the suitability of these two anthropogenic habitats for orchids. Therefore, in this paper our aim was to survey cemeteries and roadside verges and to compare these two habitats regarding their role in conserving Mediterranean terrestrial orchids. We conducted field surveys in three Mediterranean islands, Cyprus, Crete, and Lesbos, where both cemeteries and roadside verges were sampled on a geographically representative scale. We found a total of almost 7,000 orchid individuals, belonging to 77 species in the two anthropogenic habitat types. Roadside verges hosted significantly more individuals than cemeteries in Crete and Lesbos, and significantly more species across all three islands. Our results suggest that although cemeteries have a great potential conservation value in other parts of the world, intensive maintenance practices that characterized cemeteries in these three islands renders them unable to sustain valuable plant communities. On the other hand, roadside verges play a prominent role in the conservation of Mediterranean orchids in Cyprus and Greece. The pioneer status of roadside verges facilitates their fast colonization, while roads serve as ecological corridors in fragmented landscapes.     

Active site closure facilitates juxtaposition of reactant atoms for initiation of catalysis by human dUTPase

Human dUTPase, essential for DNA integrity, is an important survival factor for cancer cells. We determined the crystal structure of the enzyme:alpha,beta-imino-dUTP:Mg complex and performed equilibrium binding experiments in solution. Ordering of the C-terminus upon the active site induces close juxtaposition of the incoming nucleophile attacker water oxygen and the alpha-phosphorus of the substrate, decreasing their distance below the van der Waals limit. Complex interactions of the C-terminus with both substrate and product were observed via a specifically designed tryptophan sensor, suitable for further detailed kinetic and ligand binding studies. Results explain the key functional role of the C-terminus.     

Identification and validation of colorectal neoplasia-specific methylation markers for accurate classification of disease

Aberrant DNA methylation occurs early in oncogenesis, is stable, and can be assayed in tissues and body fluids. Therefore, genes with aberrant methylation can provide clues for understanding tumor pathways and are attractive candidates for detection of early neoplastic events. Identification of sequences that optimally discriminate cancer from other diseased and healthy tissues is needed to advance both approaches. Using well-characterized specimens, genome-wide methylation techniques were used to identify candidate markers specific for colorectal neoplasia. To further validate 30 of these candidates from genome-wide analysis and 13 literature-derived genes, including genes involved in cancer and others with unknown functions, a high-throughput methylation-specific oligonucleotide microarray was used. The arrays were probed with bisulfite-converted DNA from 89 colorectal adenocarcinomas, 55 colorectal polyps, 31 inflammatory bowel disease, 115 extracolonic cancers, and 67 healthy tissues. The 20 most discriminating markers were highly methylated in colorectal neoplasia (area under the receiver operating characteristic curve > 0.8; P < 0.0001). Normal epithelium and extracolonic cancers revealed significantly lower methylation. Real-time PCR assays developed for 11 markers were tested on an independent set of 149 samples from colorectal adenocarcinomas, other diseases, and healthy tissues. Microarray results could be reproduced for 10 of 11 marker assays, including eight of the most discriminating markers (area under the receiver operating characteristic curve > 0.72; P < 0.009). The markers with high specificity for colorectal cancer have potential as blood-based screening markers whereas markers that are specific for multiple cancers could potentially be used as prognostic indicators, as biomarkers for therapeutic response monitoring or other diagnostic applications, compelling further investigation into their use in clinical testing and overall roles in tumorigenesis.     

dUTPase and nucleocapsid polypeptides of the Mason-Pfizer monkey virus form a fusion protein in the virion with homotrimeric organization and low catalytic efficiency

Betaretroviruses encode dUTPase, an essential factor in DNA metabolism and repair, in the pro open reading frame located between gag and pol. Ribosomal frame-shifts during expression of retroviral proteins provide a unique possibility for covalent joining of nucleocapsid (NC) and dUTPase within Gag-Pro polyproteins. By developing an antibody against the prototype betaretrovirus Mason-Pfizer monkey virus dUTPase, we demonstrate that i) the NC-dUTPase fusion protein exists both within the virions and infected cells providing the only form of dUTPase, and ii) the retroviral protease does not cleave NC-dUTPase either in the virion or in vitro. We show that recombinant betaretroviral NC-dUTPase and dUTPase are both inefficient catalysts compared with all other dUTPases. Dynamic light scattering and gel filtration confirm that the homotrimeric organization, common among dUTPases, is retained in the NC-dUTPase fusion protein. The betaretroviral dUTPase has been crystallized and single crystals contain homotrimers. Oligonucleotide and Zn2+ binding is well retained in the fusion protein, which is the first example of acquisition of a functional nucleic acid binding module by the DNA repair factor dUTPase. Binding of the hexanucleotide ACTGCC or the octanucleotide (TG)4 to NC-dUTPase modulates enzymatic function, indicating that the low catalytic activity may be compensated by adequate localization.     

Organometallic flavonoid derivatives as spectroscopic probes

Derivatives of naringenin have been synthesized with organometalcarbonyl reporting groups for IR spectroscopy attached at C-2, C-3′, or C-6, and the products have been tested for the induction of nod gene expression using a Rhizobium leguminosarum strain which contains the Escherichia coli lacZ (β-galactosidase) gene fused to nodABC. Derivatives with an OMe substituent within the reporting group moiety showed residual gene induction activity.