1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-formulated, immune-stimulatory vascular endothelial growth factor a small interfering RNA (siRNA) increases antitumoral efficacy in murine orthotopic hepatocellular carcinoma with liver fibrosis

Most experimental therapy studies are performed in mice that bear subcutaneous or orthotopic hepatoma but are otherwise healthy and nonfibrotic. The majority of hepatocellular carcinoma (HCC), however, develops in patients suffering from preexisting liver fibrosis. We investigated the efficacy of a standard experimental therapeutic approach to interrupt the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) cascade via VEGF-A silencing, with or without 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP; cationic lipid) formulation, in HCC mice with preexisting liver fibrosis. The data show that intraperitoneal treatment with naked VEGF-A small interfering RNA (siRNA; 200 microg/kg) was inefficient to treat HCC implanted into fibrotic livers. VEGF-A siRNA containing an immunostimulatory motif in combination with DOTAP formulation significantly reduced hepatic VEGF-A expression and additionally activated the innate and adapted immune system as shown by an increased intrahepatic interferon type 1 response (68-fold increased beta-interferon expression). DOTAP-formulated VEGF-A siRNA markedly improved VEGF-A siRNA uptake and enhanced the antitumor response. This study shows for the first time the therapeutic feasibility of using synergistic effects (gene silencing and activation of the immune system) united in one siRNA sequence to reduce HCC growth and metastasis in mice with preexisting liver fibrosis. We expect that these results will help to direct and improve future experimental gene-silencing approaches and establish more efficient antitumoral therapies against HCC.     

Isolation of region-specific probes from pig Chromosome 6 by coincidence cloning

Coincidence cloning is a technique that permits the isolation of sequences common to two independent sources of complex DNA, and this method has been used to isolate a set of probes from a region of porcine Chromosome (Chr) 6 containing the loci for glucosephosphate isomerase (GPI) and the skeletal muscle calcium release channel (CRC). Porcine DNA was specifically PCR-amplified from a pigxhamster hybrid cell line containing the centromere region (p1.2–q1.2) of pig Chr 6 and other pig chromosome fragments by use of a porcine SINE specific primer with an EcoRI site in the 5-end. Flow-sorted Chr 6 preparations were amplified with the same SINE primer, but with a SalI site in the 5-end. The products were digested with EcoRI and SalI respectively, combined, denatured, and reannealed. The heteroduplex molecules, containing both an EcoRI and a SalI cohensive end, were selected by cloning in SalI/EcoRI-digested pUC13. Approximately 40% of the primary clones contained a single SalI/EcoRI-insert, indicating that they are coincidence clones. The average insert size was 1.4 kb. Fluorescence in situ hybridization of a pool of 34 coincidence clones to pig chromosomes showed a preferential labeling of the centromere region and of the q2.5–q2.7 region of pig Chr 6. Nineteen coincidence clones were hybridized to SINE-PCR products from flow-sorted pig Chr 6 and to pigxrodent hybrid cell lines. Eighteen clones gave positive signals correlated with the GPI/CRC content of the source DNAs.     

Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.

The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by -1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta-glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a -1 frameshift occurs at a frequency of approximately 1%. Site-directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem-loop structure. Part of this stem-loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid-binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template-binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.     

Plastid division: across time and space

Plastid division is executed by the coordinated action of at least two molecular machineries--an internal machinery situated on the stromal side of the inner envelope membrane that was contributed by the cyanobacterial endosymbiont from which plastids evolved, and an external machinery situated on the cytosolic side of the outer envelope membrane that was contributed by the host. Here we review progress in defining the components of the plastid division complex and understanding the mechanisms of envelope constriction and division-site placement in plants. We also highlight recent work identifying the first molecular linkage between the internal and external division machineries, shedding light on how their mid-plastid positioning is coordinated across the envelope membranes. Little is known about the mechanisms that regulate plastid division in plant cells, but recent studies have begun to hint at potential mechanisms.     

Dorsiventralität der Statocyten in plagiogeotropen Seitenwurzeln von Lepidium sativum L.

Summary The calyptra of plagiotropic lateral roots of Lepidium sativum L. is composed of three rows of cells. Movable amyloplates, possibly functioning as statoliths, are located only a few central cells of the ontogenetic youngest cell row. Beside the lateral root axis the two innermost statocytes contain a stable complex of rough endoplasmic reticulum, which is preferentially located in the central distal cell corner. In the statocytes lying above the lateral root axis the amyloplasts are sedimented on the ER-complex during growth in direction of the geotropic liminal angle. In the statocyte below the axis the ER-complex is free of amyloplasts. Thus a dorsiventrality exists in the statocytes located above and below the root axis in regard to the arrangement of their organelles.

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Herrn Professor Dr. Maximilian Steiner zum 70. Geburtstag.     

Transmission Electron Microscopical Observations on Stomatogenesis during Metamorphosis of Eufolliculina uhligi (Ciliophora: Heterotrichida)1

Stomatogenesis during metamorphosis of the marine loricate ciliate, Eufolliculina uhligi, was observed by transmission electron microscopy. Kinetosome proliferation in the stomatogenic territory leads to the formation of an anarchic field. This separates into the left adoral and the right paroral primordia. Both primordia consist of pairs of kinetosomes. One kinetosome of a pair is associated with one transverse and two postciliary microtubules; the other has one transverse microtubule. The postciliary microtubules of the adoral kinetosomes become divergent; those of the paroral kinetosomes become convergent. The adoral kinetosomes arrange in promembranelles. Then a third row of kinetosomes is produced anteriorly to each promembranelle. This third row is short at the peristome but longer in the buccal area. The paroral kinetosomes form a stichodyad. The buccal part of the paroral primordium is resorbed during formation of the buccal cavity. Stomatogenesis ends with the development of a functioning cytostome. During this process, the postciliary microtubules of the buccal adoral membranelles elongate and become associated with cytopharyngeal vesicles. Fusion of these vesicles with the cytostome has been observed some time after the completion of the oral structures.     

A multilocus phylogeny of Malagasy scincid lizards elucidates the relationships of the fossorial genera Androngo and Cryptoscincus

A phylogeny for 29 species of scincine lizards from Madagascar, based on 3693 bp of six mitochondrial and five nuclear genes, revealed multiple parallel evolution of adaptations for a burrowing life, and unexpected relationships of the monotypic genera Androngo and Cryptoscincus. Androngo trivittatus was sister to Pygomeles braconnieri, and Cryptoscincus minimus was deeply nested within the genus Paracontias, all of these being fossorial taxa of elongated bodies and partly or fully reduced limbs. To account for these results, we place Cryptoscincus as a junior synonym of Paracontias, and discuss possible taxonomic consequences that may affect the status of Androngo, once additional data become available.     

Metagenome survey of biofilms in drinking-water networks

Most naturally occurring biofilms contain a vast majority of microorganisms which have not yet been cultured, and therefore we have little information on the genetic information content of these communities. Therefore, we initiated work to characterize the complex metagenome of model drinking water biofilms grown on rubber-coated valves by employing three different strategies. First, a sequence analysis of 650 16S rRNA clones indicated a high diversity within the biofilm communities, with the majority of the microbes being closely related to the Proteobacteria: Only a small fraction of the 16S rRNA sequences were highly similar to rRNA sequences from Actinobacteria, low-G+C gram-positives and the Cytophaga-Flavobacterium-Bacteroides group. Our second strategy included a snapshot genome sequencing approach. Homology searches in public databases with 5,000 random sequence clones from a small insert library resulted in the identification of 2,200 putative protein-coding sequences, of which 1,026 could be classified into functional groups. Similarity analyses indicated that significant fractions of the genes and proteins identified were highly similar to known proteins observed in the genera Rhizobium, Pseudomonas, and Escherichia: Finally, we report 144 kb of DNA sequence information from four selected cosmid clones, of which two formed a 75-kb overlapping contig. The majority of the proteins identified by whole-cosmid sequencing probably originated from microbes closely related to the alpha-, beta-, and gamma-Proteobacteria: The sequence information was used to set up a database containing the phylogenetic and genomic information on this model microbial community. Concerning the potential health risk of the microbial community studied, no DNA or protein sequences directly linked to pathogenic traits were identified.