Isolation and characterization of new microsatellites at the nm23-H1 and nm23-H2 gene loci and application for loss of heterozygosity (LOH) analysis

 The nm23-H1 gene has been suggested to be a metastasis suppressor gene. Studies about the events of loss of heterozygosity (LOH) at the nm23 locus and its correlation to metastasis are controversially discussed. To optimize detection of LOH at the nm23 locus, we screened two P1 clones for additional microsatellites. Tumor and normal DNA from 37 colorectal, 16 gastric, and 8 germ cancer patients were examined for LOH. We found two new CA repeats, one 5′ to nm23-H1 and another 3′ to nm23-H2. Using these nm23 locus-specific CA repeats and five other chromosome 17 loci (D17S1522, D17S1566, D17S855, D17S515, and TP53), allele loss was observed in 4/32 (12.5%) patients with colon cancer, 2/14 (14.3%) with gastric cancer, and 1/7 (14%) with germ cancer. No isolated LOH of the nm23 region was observed. Received: 5 May 1997 / Accepted: 2 June 1997     

Synthesis, characterization and potential application of monoacyl-cyclodextrins

Although the preparation of cyclodextrin (CD) monoesters with a variety of carboxylic acids has been already described in the literature, the direct regioselective CD acylation has proved to be critical, often requiring to be replaced with a more elaborate synthetic process. In this paper we describe the one-step preparation of several monoacylated CDs from acyclic or aromatic carboxylic acid derivatives. The ability of β-CD to enclose cupric ions in a sandwich-type manner was exploited to lead to high regioselectivity in the acylation of β-CD with benzoyl chloride, cinnamoyl chloride and phenyl acetyl chloride in water. Long chain aliphatic monoesters of α-, β- and γ-CD were best prepared in DMF. The results of our study showed that solvent and general conditions determined an overwhelming regioselectivity of acylation. ¹H, ¹³C and 2D NMR experiments could easily discriminate the position of the ester. Monoacylated CDs were evaluated as a carrier of silibinin, the inclusion complexes were prepared and characterized by thermal analysis.     

Brief communication: stability and catalytic activity of novel circular DNAzymes

DNAzymes represent a new generation of catalytic nucleic acids for specific RNA targeting in order to inhibit protein translation from the specifically cleaved mRNA. The 10-23 DNAzyme was found to hydrolyze RNA in a sequence-specific manner both in vitro and in vivo. Although single-stranded DNAzymes may represent the most effective nucleic acid drug to date, they are nevertheless sensitive to nuclease degradation and require modifications for in vivo application. However, previously used stabilization of DNAzymes by site-specific phosphorothioate (PT) modifications reduces the catalytic activity, and the PTO displays toxic side effects when applied in vivo. Thus, improving the stability of DNAzymes without reducing their catalytic activity is essential if the potential of these compounds should be realized in vivo. RESULTS: The Circozyme was tested targeting the mRNA of the most common genetic rearrangement in pediatric acute lymphoblastic leukemia TEL/AML1 (ETV6/RUNX1). The Circozyme exhibits a stability comparable to PTO-modified DNAzymes without reduction of catalytic activity and specificity and may represent a promising tool for DNAzyme in vivo applications. CONCLUSION: The inclusion of the catalytic site and the specific mRNA binding sequence of the DNAzyme into a circular loop-stem-loop structure (Circozyme) of approximately 70 bases presented here represents a new effective possibility of DNAzyme stabilization.     

Diversity,biogeography and host specificity of kelp endophytes with a focus on the genera Laminarionema and Laminariocolax (Ectocarpales,Phaeophyceae)

Endophytic filamentous brown algae are known to invade stipes and fronds of kelps with potentially negative effects for the hosts. They have simple filamentous thalli and are difficult to identify based on morphology. We investigated the molecular diversity of 56 endophytes isolated from seven different kelp species from Europe, Chile, Korea and New Zealand by sequencing two unlinked molecular markers (5’COI and ITS1). A majority of 49 of the isolated endophytes (88%) belonged to the genera Laminarionema and Laminariocolax. The endophyte Laminarionema elsbetiae was isolated from Saccharina latissima and S. japonica tissues in Europe and Korea, respectively, and showed highly similar sequences in both regions. Three different species of the genus Laminariocolax were identified, the most common of which was L. aecidioides, an endophyte with a worldwide distribution and a broad host range. The other two species, L. tomentosoides and a species described here as Laminariocolax atlanticus sp. nov., were associated with different kelp species in the northern hemisphere and the North Atlantic, respectively. Our results suggest that specific host-endophyte patterns could exist locally, as found in kelps in Brittany where all endophytes isolated from S. latissima were L. elsbetiae, all endophytes isolated from Laminaria digitata were Laminariocolax tomentosoides, and those isolated from Laminaria hyperborea were Laminariocolax atlanticus and L. aecidioides. However, this pattern was not consistent with the results from other places, such as Western Scotland and Helgoland where the same kelp species are present.     

Mycobacteria exploit nitric oxide‐induced transformation of macrophages into permissive giant cells

Immunity to mycobacteria involves the formation of granulomas, characterized by a unique macrophage (MΦ) species, so‐called multinucleated giant cells (MGC). It remains unresolved whether MGC are beneficial to the host, that is, by prevention of bacterial spread, or whether they promote mycobacterial persistence. Here, we show that the prototypical antimycobacterial molecule nitric oxide (NO), which is produced by MGC in excessive amounts, is a double‐edged sword. Next to its antibacterial capacity, NO propagates the transformation of MΦ into MGC, which are relatively permissive for mycobacterial persistence. The mechanism underlying MGC formation involves NO‐induced DNA damage and impairment of p53 function. Moreover, MGC have an unsurpassed potential to engulf mycobacteria‐infected apoptotic cells, which adds a further burden to their antimycobacterial capacity. Accordingly, mycobacteria take paradoxical advantage of antimicrobial cellular efforts by driving effector MΦ into a permissive MGC state.     

Molecular phylogenetics employing modern and ancient DNA

Comparative studies of DNA in recent populations and characterisation of ancient hereditary material have contributed very interesting facts to our understanding of evolution of modern mankind. Analysis of DNA homology in related species, assessment of mutations and polymorphisms in various populations and new DNA sequence data from prehistoric finds allowed - via sophisticated DNA extraction techniques, PCR, sequencing and digitalised processing of genetic information - insights into possible roots of Homo sapiens and related species, migration patterns and ancient cultural habits, thus enrhing the palaeoanthropological discipline. However, a presentation of this development would not be complete without pointing towards the methodological limitations and manifold presentations burdened with artifacts, data misinterpretation and unjustified conclusions. Presently, this modern field of research is in its consolidation phase and new parameters for quality control and authentication are being implemented to avoid spectacular but unfounded reports. It is expected that most of the problems connected to old biomolecules may be closely related to fossilisation parameters. The future challenge will be the full understanding of the complex and multi-faceted processes underlying diagenesis, including the elucidation of nucleic acid postmortem damage".