Biomedical subjects

T Röhl

Publications and source records attributed to T Röhl.

6 recordsLinked to original sources

PubMed2006

RNA interference in the mouse vascular endothelium by systemic administration of siRNA-lipoplexes for cancer therapy.

RNA interference (RNAi) entails the potential for novel therapeutic strategies through the silencing of disease-causing genes in vivo. However, recent studies have raised an issue regarding applicable routes of administration for small interfering RNA (siRNA) molecules as therapeutics. In this study, we demonstrate that liposomally formulated siRNA molecules, the so-called siRNA-lipoplexes, but not naked siRNAs, are delivered to the tumor endothelial cells in vivo by microscopy. In addition, functional intracellular delivery of formulated siRNA targeting the tumor suppressor PTEN is shown in endothelial cells of the liver and tumor. Finally, the therapeutic potential of systemically administered siRNA(CD31)-lipoplexes is established by inhibition of tumor growth in two different xenograft mouse models. Our findings corroborate the applicability of this liposomal siRNA delivery technology for inducing RNAi to modulate gene expression levels in angiogenesis-dependent processes. In addition, our results advocate CD31 as a promising therapeutic target for antiangiogenic intervention. Therefore, our study provides a basis for the development of antiangiogenic cancer therapies based on RNAi.

3T3 Cells↗

PubMed2001

In vitro reconstitution of insertion and processing of cytochrome f in a homologous chloroplast translation system.

Using a homologous chloroplast translation system, we have reconstituted insertion and processing of the chloroplast-encoded thylakoid protein cytochrome f (pCytf). Cross-linking demonstrated that pCytf nascent chains when attached to the 70 S ribosome tightly interact with cpSecA, but this is strictly dependent on thylakoid membranes and a functional signal peptide. This indicates that cpSecA is only operative in pCytf biogenesis when it is bound to the membrane, most likely as part of the Sec translocon. No evidence for interaction between the 54-kDa subunit of the chloroplast signal recognition particle (cpSRP) and the pCytf nascent chain could be detected, suggesting that pCytf, in contrast to the polytopic D1 protein, does not require cpSRP for targeting. Insertion of pCytf occurred only co-translationally, resulting in processing and accumulation of both the processed signal peptide and the mature protein in the thylakoid. This co-translational membrane insertion and processing required a functional signal peptide and was inhibited by azide, demonstrating that cpSecA is essential for translocation of the soluble luminal domain. pCytf also associated post-translationally with thylakoids, but the soluble N-terminal domain could not be translocated into the lumen. This is the first study in which synthesis, targeting, and insertion of a chloroplast-encoded thylakoid membrane protein is reconstituted from exogenous transcripts and using the chloroplast translational machinery.

Base Sequence↗

PubMed2001

Complementation of bacterial SecE by a chloroplastic homologue.

The SecE protein is an essential component of the SecAYE-translocase, which mediates protein translocation across the cytoplasmic membrane in bacteria. In the thylakoid membranes of chloroplasts, a protein homologous to SecE, chloroplastic (cp) SecE, has been identified. However, the functional role of cpSecE has not been established experimentally. In this report we show that cpSecE in cells depleted for bacterial SecE (i) supports growth, (ii) stabilizes, just like bacterial SecE, the Sec-translocase core component SecY, and (iii) supports Sec-dependent protein translocation. This indicates that cpSecE can functionally replace bacterial SecE in vivo, and strongly suggests that the thylakoid membrane contains a SecAYE-like translocase with functional and structural similarities to the bacterial complex. This study further underscores the evolutionary link between chloroplasts and bacteria.

Adenosine Triphosphatases↗

PubMed2000

Identification of the pore-forming region of the outer chloroplast envelope protein OEP16.

The chloroplast outer envelope protein OEP16 forms a cation-selective high conductance channel with permeability to amines and amino acids. The region of OEP16 directly involved in channel formation has been identified by electrophysiological analysis of a selection of reconstituted OEP16 mutants. Because analysis of these mutants depended on the use of recombinant protein, we evaluated the electrophysiological properties of OEP16 isolated directly from pea chloroplasts and of the recombinant protein produced in Escherichia coli. The results show that the basic properties like conductance, selectivity, and open probability of the channel formed by native pea OEP16 are comparable with the channel activity formed by the recombinant source of the protein. Following electrophysiological analysis of OEP16 mutants we found that point mutations and insertion of additional amino acid residues in the region of the putative helix 1 (Glu(73) to Val(91)) did not change the properties of the OEP16 channel. The only exception was a Cys(71)-->Ser mutation, which led to a loss of the CuCl(2) sensitivity of the channel. Analysis of N- and C-terminal deletion mutants of OEP16 and mutants containing defined shuffled domains indicated that the minimal continuous region of OEP16, which is able to form a channel in liposomes, lies in the first half of the protein between amino acid residues 21 and 93.

Amines↗

PubMed1999

The outer envelope protein OEP24 from pea chloroplasts can functionally replace the mitochondrial VDAC in yeast.

The chloroplastic outer envelope protein OEP24 from pea forms a high-conductance low specificity solute channel as shown by in vitro studies. In order to establish its function also in an in vivo-like system, the gene encoding OEP24 was transformed into a yeast strain which lacks the general mitochondria solute channel porin, also known as voltage-dependent anion channel (VDAC). Transformation of the yeast VDAC(-) strain with the OEP24 gene resulted in the recovery of a phenotype indistinguishable from the wild-type. The OEP24 polypeptide is targeted to the mitochondrial outer membrane in this heterologous system. We conclude that OEP24 forms a solute channel in pea chloroplasts in planta.

Chloroplasts↗

PubMed1997

Postimport methylation of the small subunit of ribulose-1,5-bisphosphate carboxylase in chloroplasts.

Electron impact mass spectronomy analysis of the amino-terminal amino acid of the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase (Rubisco) showed that the amino-terminal methionine residue is post-translationally modified to N-methyl-methionine. Modification of the amino-terminal methionine residue was found in mature SSU proteins from the dicotyledonous plants pea and spinach as well as the monocotyledonous plants barley and corn. SSU methyltransferase is a soluble protein in the chloroplast stroma and accepts heterologously expressed non-methylated SSU as a substrate using S-adenosylmethionine as methyl-group donor. We show that this modification occurs after post-translational uptake of the precursor form of SSU into chloroplasts and processing to its mature size. This reaction represents a new step in the import and assembly pathway of Rubisco holoenzyme.

Chloroplasts↗