Biomedical subjects

Eggehard Holler

Publications and source records attributed to Eggehard Holler.

11 recordsLinked to original sources

PubMed2006

Inhibition of laminin-8 in vivo using a novel poly(malic acid)-based carrier reduces glioma angiogenesis.

We have previously shown that laminin-8, a vascular basement membrane component, was overexpressed in human glioblastomas multiforme and their adjacent tissues compared to normal brain. Increased laminin-8 correlated with shorter glioblastoma recurrence time and poor patient survival making it a potential marker for glioblastoma diagnostics and prediction of disease outcome. However, laminin-8 therapeutic potential was unknown because the technology of blocking the expression of multi-chain complex proteins was not yet developed. To inhibit the expression of laminin-8 constituents in glioblastoma in vitro and in vivo, we used Polycefin, a bioconjugate drug delivery system based on slime-mold Physarum polycephalum-derived poly(malic acid). It carries an attached transferrin receptor antibody to target tumor cells and to deliver two conjugated morpholino antisense oligonucleotides against laminin-8 alpha4 and beta1 chains. Polycefin efficiently inhibited the expression of both laminin-8 chains by cultured glioblastoma cells. Intracranial Polycefin treatment of human U87MG glioblastoma-bearing nude rats reduced incorporation of both tumor-derived laminin-8 chains into vascular basement membranes. Polycefin was thus able to simultaneously inhibit the expression of two different chains of a complex protein. The treatment also significantly reduced tumor microvessel density (p < 0.001) and area (p < 0.001) and increased animal survival (p < 0.0004). These data suggest that laminin-8 may be important for glioblastoma angiogenesis. Polycefin, a versatile nanoscale drug delivery system, was suitable for in vivo delivery of two antisense oligonucleotides to brain tumor cells causing a reduction of glioblastoma angiogenesis and an increase of animal survival. This system may hold promise for future clinical applications.

Angiogenesis Inhibitors↗

PubMed2006

Screening for beta-poly(L-malate) binding proteins by affinity chromatography.

Poly(beta-L-malic acid) is a cell type-specific polymer of myxomycetes (true slime molds) with the physiological role to organize mobility of certain proteins over the giant multinucleated plasmodia. We have developed an affinity chromatography employing 1,6-diamino-n-hexane-Sepharose-coupled poly(malic acid) to identify such proteins in cellular extracts of Physarum polycephalum. Molecular masses were measured by SDS-PAGE and non-denaturing PAGE after silver staining and/or Western blotting. Protein complexes/subunits were detected by 2-dimensional non-denaturing PAGE/SDS-PAGE. A simplified gel shift experiment displayed binding to fragmented calf thymus DNA. Nuclei were richest in poly(malate) binding proteins followed by cytoplasm and membranes. A protein of 370 kDa dissociated into 11 subunits of 11-29 kDa, indicative of a highly complex protein. This and other proteins displayed binding to nucleic acid in gel shift experiments. Poly(malate) is considered a structural and functional equivalent of long contiguous aspartate repeats in proteins of eukaryotes.

Animals↗

PubMed2006

Stage specific expression of poly(malic acid)-affiliated genes in the life cycle of Physarum polycephalum. Spherulin 3b and polymalatase.

Polymalic acid is receiving interest as a unique biopolymer of the plasmodia of mycetozoa and recently as a biogenic matrix for the synthesis of devices for drug delivery. The acellular slime mold Physarum polycephalum is characterized by two distinctive growth phases: uninucleated amoebae and multinucleated plasmodia. In adverse conditions, plasmodia reversibly transform into spherules. Only plasmodia synthesize poly(malic acid) (PMLA) and PMLA-hydrolase (polymalatase). We have performed suppression subtractive hybridization (SSH) of cDNA from amoebae and plasmodia to identify plasmodium-specific genes involved in PMLA metabolism. We found cDNA encoding a plasmodium-specific, spherulin 3a-like polypeptide, NKA48 (spherulin 3b), but no evidence for a PMLA-synthetase encoding transcript. Inhibitory RNA (RNAi)-induced knockdown of NKA48-cDNA generated a severe reduction in the level of PMLA suggesting that spherulin 3b functioned in regulating the level of PMLA. Unexpectedly, cDNA of polymalatase was not SSH-selected, suggesting its presence also in amoebae. Quantitative PCR then revealed low levels of mRNA in amoebae, high levels in plasmodia, and also low levels in spherules, in agreement with the expression under transcriptional regulation in these cells.

Amino Acid Sequence↗

PubMed2005

Laminin isoform expression in breast tumors.

Certain laminins of vascular basement membranes have been identified in human breast tumors and brain gliomas that share the same beta1 chain. These laminins are new carcinoma angiogenic markers and might represent potential targets for antiangiogenic therapy.

Angiogenesis Inhibitors↗

PubMed2005

Use of the giant multinucleate plasmodium of Physarum polycephalum to study RNA interference in the myxomycete.

The plasmodium of Physarum polycephalum harbors billions of synchronized nuclei in a single cell of complex structure. Due to its synchrony and extreme size, it is used as a model to study events on a single cell level, such as cell cycle and differentiation. We show here for the first time that this model, despite its enormous size and structural complexity, is accessible to RNA interference by simple injection of dsRNA or siRNA. The targeted gene is that of polymalatase, an intracellular adapter of poly(beta-l-malate) involved in the maintenance of the synchrony and functioning as an extracellular hydrolase of this polymer. Real-time reverse transcriptase polymerase chain reaction analysis revealed that the specific mRNA was knocked down to about 10% of the original level. The suppression of a single injection lasted for approximately 14 cell cycles (144 h) and could be prolonged for any time by repeated dsRNA injections. Western blots indicated that the knockdown of RNA was paralleled by a strong reduction in polymalatase synthesis. However, a change in the phenotype of the plasmodium could not be clearly observed. In principle, the plasmodium offers an easy system for studying gene knockdown by RNA interference.

Animals↗

PubMed2005

Low-molecular-weight poly(alpha-methyl beta,L-malate) of microbial origin: synthesis and crystallization.

Low-molecular-weight poly(alpha-methyl beta,L-malate) made of approximately 25-30 units was prepared from microbial poly(beta,L-malic acid) by treatment with diazomethane. The thermal characterization of the polymalate methyl ester was carried out and its crystalline structure was preliminary examined. Its ability to crystallize both from solution and from the melt was comparatively evaluated.

Animals↗

PubMed2004

Injection of poly(beta-L-malate) into the plasmodium of Physarum polycephalum shortens the cell cycle and increases the growth rate.

Poly(beta-L-malate) (PMLA) has been reported as an unconventional, physiologically important biopolymer in plasmodia of myxomycetes, and has been proposed to function in the storage and transport of nuclear proteins by mimicking the phospho(deoxy)ribose backbone of nucleic acids. It is distributed in the cytoplasm and especially in the nuclei of these giant, multinucleate cells. We report here for the first time an increase in growth rate and a shortening of the cell cycle after the injection of purified PMLA. By comparing two strains of Physarum polycephalum that differed in their production levels of PMLA, it was found that growth activation and cell cycle shortening correlated with the relative increases of PMLA levels in the cytoplasm or the nuclei. Growth rates of a low PMLA producer strain (LU897 x LU898) were increased by 40-50% while those of a high producer strain (M(3)CVIII) were increased by only 0-17% in comparison with controls. In both strains, shortening of the cell cycle occurred to a similar extent (7.2-9.5%), and this was associated with similar increases in nuclear PMLA levels. The effects showed saturation dependences with regard to the amount of injected PMLA. A steep rise of intracellular PMLA shortly after injection was followed by the appearance of histone H1 in the cytoplasm. The increase in growth rate, the shortening of the cell cycle duration and the appearance of H1 in the cytoplasm suggest that PMLA competes with nucleic acids in binding to proteins that control translation and/or transcription. Thus, PMLA could play an important role in the coordination of molecular pathways that are responsible for the synchronous functioning of the multinucleate plasmodium.

Animals↗

PubMed2003

Localization of fluorescence-labeled poly(malic acid) to the nuclei of the plasmodium of Physarum polycephalum.

The nuclei in the plasmodium of Physarum polycephalum, as of other myxomycetes, contain high amounts of polymalate, which has been proposed to function as a scaffold for the carriage and storage of several DNA-binding proteins [Angerer, B. and Holler, E. (1995) Biochemistry 34, 14741-14751]. By delivering fluorescence-labeled polymalate into a growing plasmodium by injection, we observed microscopic staining of nuclei in agreement with the proposed function. The fluorescence intensity was highest during the reconstruction phase of the nuclei. To examine whether the delivery was under the control of polymalatase or related proteins [Karl, M. & Holler, E. (1998) Eur. J. Biochem.251, 405-412], the cellular distribution of these proteins was also examined by staining with antibodies against polymalatase. Double-stained plasmodia revealed a fluorescent halo around each fluorescent nucleus during the reconsititution. Fluorescent nuclei were not observed when the hydroxyl terminus of polymalate, known to be essential for the binding of polymalatase, was blocked by labeling with fluorescein-5-isothiocyanate. By immune precipitation, it was shown that polymalate and polymalatase or related proteins were in the precipitate. It is concluded that polymalate is delivered to the surface of nuclei in the complex with polymalatase or related proteins. The complex dissociates, and polymalate translocates into the nucleus, while polymalatase or related proteins remain at the surface.

Active Transport, Cell Nucleus↗

PubMed2002

The DNA-polymerase inhibiting activity of poly(beta-l-malic acid) in nuclear extract during the cell cycle of Physarum polycephalum.

The naturally synchronous plasmodia of myxomycetes synthesize poly(beta-l-malic acid), which carries out cell-specific functions. In Physarum polycephalum, poly(beta-l-malate) [the salt form of poly(beta-l-malic acid)] is highly concentrated in the nuclei, repressing DNA synthetic activity of DNA polymerases by the formation of reversible complexes. To test whether this inhibitory activity is cell-cycle-dependent, purified DNA polymerase alpha of P. polycephalum was added to the nuclear extract and the activity was measured by the incorporation of [3H]thymidine 5'-monophosphate into acid precipitable nick-activated salmon testis DNA. Maximum DNA synthesis by the reporter was measured in S-phase, equivalent to a minimum of inhibitory activity. To test for the activity of endogenous DNA polymerases, DNA synthesis was followed by the highly sensitive photoaffinity labeling technique. Labeling was observed in S-phase in agreement with the minimum of the inhibitory activity. The activity was constant throughout the cell cycle when the inhibition was neutralized by the addition of spermidine hydrochloride. Also, the concentration of poly(beta-l-malate) did not vary with the phase of the cell cycle [Schmidt, A., Windisch, C. & Holler, E. (1996) Nuclear accumulation and homeostasis of the unusual polymer poly(beta-l-malate) in plasmodia of Physarum polycephalum. Eur. J. Cell Biol. 70, 373-380]. To explain the variation in the cell cycle, a periodic competition for poly(beta-l-malate) between DNA polymerases and most likely certain histones was assumed. These effectors are synthesized in S-phase. By competition they displace DNA polymerase from the complex of poly(beta-l-malate). The free polymerases, which are no longer inhibited, engage in DNA synthesis. It is speculated that poly(beta-l-malate) is active in maintaining mitotic synchrony of plasmodia by playing the mediator between the periodic synthesis of certain proteins and the catalytic competence of DNA polymerases.

Animals↗

PubMed1998

Molecular constituents of the replication apparatus in the plasmodium of Physarum polycephalum: identification by photoaffinity labelling.

The plasmodium of Physarum polycephalum has long been considered a model system for syncytically growing cells, but important details of the DNA replication apparatus, such as the DNA polymerase epsilon and other replication factors, have not been detected. In this study, a new variation of photoaffinity labelling and immunoblotting was used to detect DNA polymerases and other factors in nuclear extracts of P. polycaphalum. Proteins were specifically cross-linked with photoreactive arylazido-dCMP residues incorporated during extension of template-primer DNA. The DNA synthesized in situ was 32P-labelled. After nucleolytic removal of protruding DNA, the proteins were separated by SDS-gel electrophoresis, electroblotted on membranes and subjected to autoradiography. The alpha, delta, epsilon and beta-like DNA polymerases were labelled, as were histones and replication-factor-like proteins. Cytoplasmic extracts were devoid of these species. Abundant proliferating-cell nuclear antigen and replication protein A large subunit were labelled and found to be of unusual mass. A number of subunits of purified DNA polymerase holoenzymes were labelled. In contrast, only the DNA-polymerizing subunits could be labelled in nuclear extracts. Higher-order complexes in the nuclear extract may make subunits inaccessible to photo-cross-linking. Complex formation is promoted by beta-poly(L-malate), a plasmodium-specific putative storage and carrier molecule that supports DNA replication in the synchronized nuclei. Percoll, a polyvinylpyrrolidone-coated colloidal silica, partially disrupted these complexes. A 200 kDa fragment of DNA polymerase epsilon and a 135 kDa beta-like DNA polymerase did not participate in the complexes, suggesting functions unlike those of the other polymerases. DNA polymerase molecules were intact during proliferation of plasmodia, but were nicked before their clearance from the nuclei at growth arrest.

Animals↗

PubMedDate not supplied

Polycefin, a new prototype of a multifunctional nanoconjugate based on poly(beta-L-malic acid) for drug delivery.

A new prototype of nanoconjugate, Polycefin, was synthesized for targeted delivery of antisense oligonucleotides and monoclonal antibodies to brain tumors. The macromolecular carrier contains: 1. biodegradable, nonimmunogenic, nontoxic beta-poly(L-malic acid) of microbial origin; 2. Morpholino antisense oligonucleotides targeting laminin alpha4 and beta1 chains of laminin-8, which is specifically overexpressed in glial brain tumors; 3. monoclonal anti-transferrin receptor antibody for specific tissue targeting; 4. oligonucleotide releasing disulfide units; 5. L-valine containing, pH-sensitive membrane disrupting unit(s), 6. protective poly(ethylene glycol); 7. a fluorescent dye (optional). Highly purified modules were conjugated directly with N-hydroxysuccinimidyl ester-activated beta-poly(L-malic acid) at pendant carboxyl groups or at thiol containing spacers via thioether and disulfide bonds. Products were chemically validated by physical, chemical, and functional tests. In vitro experiments using two human glioma cell lines U87MG and T98G demonstrated that Polycefin was delivered into the tumor cells by a receptor-mediated endocytosis mechanism and was able to inhibit the synthesis of laminin-8 alpha4 and beta1 chains at the same time. Inhibition of laminin-8 expression was in agreement with the designed endosomal membrane disruption and drug releasing activity. In vivo imaging showed the accumulation of intravenously injected Polycefin in brain tumor tissue via the antibody-targeted transferrin receptor-mediated endosomal pathway in addition to a less efficient mechanism known for high molecular mass biopolymers as enhanced permeability and retention effect. Polycefin was nontoxic to normal and tumor astrocytes in a wide range of concentrations, accumulated in brain tumor, and could be used for specific targeting of several biomarkers simultaneously.

Animals↗