Interactions of Platinum and Ruthenium Coordination Complexes with Pancreatic Phospholipase A2 and Phospholipids Investigated by MALDI TOF Mass Spectrometry

Phospholipase A₂ is involved in propagation of inflammatory processes and carcinogenesis through its role in phospholipid metabolism, and release of arachidonic acid and lysophospholipids. Recent findings on correlation between elevated PLA₂ activity and metastatic cancer render this enzyme an attractive target for cancer therapy. On the other hand, due to a broad range of oxidation states under physiological conditions and a high affinity for protein binding, platinum and ruthenium coordination complexes are promising candidates for PLA₂ inhibitors. In this article, we discuss the interactions of Pt and Ru coordination complexes with PLA₂ and phospholipids, as well as the application of MALDI‐TOF mass spectrometry for screening PLA₂ inhibitors. Owing to the ability of this technique to simultaneously detect and monitor changes in substrate and product concentrations, the inhibitor mechanisms of both Pt and Ru complexes with various ligands were determined.     

Combinatorial pathway analysis for improved L-tyrosine production in Escherichia coli: identification of enzymatic bottlenecks by systematic gene overexpression

Combinatorial overexpression of aromatic amino acid biosynthesis (AAAB) genes in the L-tyrosine producing Escherichia coli strains T1 and T2 was employed to search for AAAB reactions limiting L-tyrosine production. All AAAB genes except aroG and tyrA, which were substituted by their feedback resistant derivatives in the host strains, were cloned and overexpressed. A total of 72 different strains overexpressing various AAAB gene combinations were generated and from those strains with improved phenotype, enzymatic bottlenecks of the AAAB pathway could be inferred. The two major gene overexpression targets for increased L-tyrosine production in E. coli were ydiB and aroK, coding for a shikimate dehydrogenase and a shikimate kinase, respectively, and the combination of ydiB and aroK for overexpression resulted in the best L-tyrosine producing strains in this study, yielding 45% for strain T1 and 26% for strain T2, respectively, higher L-tyrosine titers. Interestingly, overexpression studies with combinations of more than one gene revealed that new gene targets could be identified when overexpessed together with other genes but not alone as single gene overexpression. For example, tyrB encoding the last enzyme of the AAAB pathway, an aromatic amino acid transaminase, improved L-tyrosine production significantly when co-overexpressed together with ydiB or aroK, but not when overexpressed alone. It is also noteworthy that E. coli T1, which generally yielded less L-tyrosine, was amenable to greater improvements than strain T2, i.e. E. coli T1 exhibited generally more space for phenotype improvement.     

A Surface-Induced Asymmetric Program Promotes Tissue Colonization by Pseudomonas aeruginosa

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Heterokaryon Identification Through Simultaneous Fluorescence of Tetramethylrhodamine Isothiocyanate and Fluorescein Isothiocyanate Labelled Protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein iso-thiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emisson spectrum, but not both, was possible for any single filter set.     

Ultrastructural and immunofluorescence studies of basal-lamina alterations during mouse-lung morphogenesis

Epithelial differentiation during lung development appears to be influenced by mesenchyme-derived instructions coupled with hormonal regulations. The basal lamina which is associated with progenitor and differentiating epithelia during mouse embryogenesis (Theiler-stages 16-28) was examined by transmission electron microscopy and indirect-immunofluorescence microscopy. During the embryonic phase of lung development, progenitor epithelia for the pulmonary acinus projected microvilli or cytoplasmic "feet" through the basal lamina, which resulted in discontinuities and a close approximation of the adjacent mesenchymal-cell processes. These changes were also associated with the transitory polarization of mesenchymal cells perpendicular to the plane of the basal lamina, which resulted in a sheet of cuboidal mesenchymal cells adjacent to the developing acinar-tubule epithelium. During the embryonic phase of lung development, these specific interstitial or mesenchymal cells stained for heparan-sulfate proteoglycans; no other cell types were immunostained. By Theiler-stage 25, the acinar-tubule epithelia had differentiated into type-II pneumonocytes which contained lamellar bodies and significant amounts of glycogen. Fibronectin, laminin, and heparan-sulfate proteoglycan were localized in the basement membranes during the embryonic, canalicular, and terminal sac phases of lung morphogenesis. A diffuse localization of fibronectin of the interstitial cell surfaces was observed. These observations indicate that major changes in the structure and composition of basal lamina occur during the embryonic and fetal phases of pulmonary-acinus epithelial-cell differentiation and the production of pulmonary surfactant. The major changes in the basal lamina may be partly mediated by mesenchyme-derived instructions for type-II epithelial-cell differentiation.     

CD studies of peptides that mimic the four helicoidal sequences of the uteroglobin monomer

Summary Short peptides spanning the helicoidal sequences of the uteroglobin monomer (crystal forms P2₁ and C222₁) were synthesized and studied by circular dichroism spectroscopy. None of them showed any secondary structure in the absence of HFIP. However, most peptides achieved a helical conformation when this structuring agent was used, with the exception of the analogue corresponding to the helicoidal fragment 19–24 (helix II, crystal P2₁). These results indicate that other factors, such as interchain interactions, have to contribute to helix stabilization in the molecule. On the other hand, while peptides corresponding to N- and C-terminal fragments that contain the first and fourth helices of the monomer, respectively (1–14 and 48–70) achieved a -like structure when 10–15% of HFIP was used, this behaviour was not observed when TFE was used. Moreover, substitution of cysteine by -aminobutyric acid at position 3 increased both the helicity of fragment 1–14 and its ability to adopt a -like structure, but the opposite effect was observed for fragment 48–70 when -aminobutyric acid was introduced at position 69. These results indicate that this part of the protein might be sensitive to the chemical environment it is exposed to and that the two cysteine residues at positions 3 and 69 of the monomer could play a different role in the folding process.     

Single embryo transfer in IVF to prevent multiple pregnancies.

As the success rates of IVF clinics improve, one of the adverse consequences is the increased incidence of twins, due largely to the number of embryos transferred. Even if the number of embryos transferred is restricted to two, the twinning rate can exceed 40% of the pregnancies. An obvious way to reduce this high twin rate would be to transfer only one embryo. This would require that cryopreservation of the supernumerary embryos be efficacious enough so that the chance of achieving an ongoing pregnancy is not diminished by transferring a single embryo in the stimulated cycle. Previous studies utilising embryos on day 2 and 3 of development have shown that the pregnancy rates can be acceptable (about 40%) and that the cumulative rate can be up to 60%. Most of these studies, however, do not include a comparison with the cumulative pregnancy rate with two embryos transferred in the stimulated cycle. Therefore, the efficacy has not been proven. We present clinical data from the past few years to illustrate the increase in success rates and the concomitant increase in twinning rates. The increased success in the cryopreservation program has enabled us to trial a single embryo transfer program and compare the results to the transfer of two embryos. The results strongly suggest that the transfer of a single embryo is the better clinical option.     

Gene expression patterns in ovarian carcinomas

We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers.     

Mechanisms of resistance in the rice cultivar Manikpukha to the rice stem nematode Ditylenchus angustus

The incompatible interaction between the rice cultivar Manikpukha and the rice stem nematode Ditylenchus angustus has been reported recently. This research focuses on the underlying mechanisms of resistance in Manikpukha. Invasion, post‐infection development and reproduction of D. angustus were compared in compatible and incompatible interactions to identify the stage in which resistance occurs. The results indicate that resistance in Manikpukha is associated with reduced development and reproduction, implying that resistance acts post‐invasion. We studied the possible involvement of three classical defence hormones, salicylic acid (SA), jasmonic acid (JA) and ethylene (ET), in response to infection in a compatible interaction using biosynthesis/signalling‐deficient transgenic rice lines. All three hormones appear to have an influence on the basal defence of Nipponbare against the stem nematode. Although hormone application increases basal defences, expression studies and hormone analyses after nematode infection in Manikpukha did not show a clear involvement of the hormone defense pathways for SA, ET and JA. However, it seems that OsPAL1 plays a pivotal role in resistance, indicating that the phenylpropanoid pathway and its products might be key players in the incompatible interaction. Lignin measurement showed that, although basal levels are similar, Manikpukha had a significantly higher lignin content on nematode infection, whereas it was decreased in the susceptible cultivar. The results presented here show that SA, ET and JA are involved in basal defences, but the resistance of Manikpukha against D. angustus probably relies on products of the phenylpropanoid pathway.