Peter Lorenz

MicroRNA let-7b targets important cell cycle molecules in malignant melanoma cells and interferes with anchorage-independent growth

A microRNA expression screen was performed analyzing 157 different microRNAs in laser-microdissected tissues from benign melanocytic nevi (n = 10) and primary malignant melanomas (n = 10), using quantitative real-time PCR. Differential expression was found for 72 microRNAs. Members of the let-7 family of microRNAs were significantly downregulated in primary melanomas as compared with benign nevi, suggestive for a possible role of these molecules as tumor suppressors in malignant melanoma. Interestingly, similar findings had been described for lung and colon cancer. Overexpression of let-7b in melanoma cells in vitro downregulated the expression of cyclins D1, D3, and A, and cyclin-dependent kinase (Cdk) 4, all of which had been described to play a role in melanoma development. The effect of let-7b on protein expression was due to targeting of 3′-untranslated regions (3′UTRs) of individual mRNAs, as exemplified by reporter gene analyses for cyclin D1. In line with its downmodulating effects on cell cycle regulators, let-7b inhibited cell cycle progression and anchorage-independent growth of melanoma cells. Taken together, these findings not only point to new regulatory mechanisms of early melanoma development, but also may open avenues for future targeted therapies of this tumor.

Enhanced thoracic gene delivery by magnetic nanobead-mediated vector

Systemic gene delivery is limited by the adverse hydrodynamic conditions on the collection of gene carrier particles to the specific area. In the present study, a magnetic field was employed to guide magnetic nanobead (MNB)/polymer/DNA complexes after systemic administration to the left side of the mouse thorax in order to induce localized gene expression.

The functional −443T/C osteopontin promoter polymorphism influences osteopontin gene expression in melanoma cells via binding of c‐Myb transcription factor

In the present report, the possible role of a recently described functional polymorphism of the osteopontin (OPN) promoter at position −443 (−443T/C) for OPN expression in melanoma cells was addressed. As shown by real‐time PCR analysis, melanoma metastases that were homozygous for the −443C allele expressed significantly higher levels of OPN mRNA compared with those that were either heterozygous (−443T/C) or homozygous for the −443T allele. In line with this, immunoblotting showed significantly enhanced baseline and bFGF‐induced OPN protein expression in melanoma cell lines which were homozygous for the −443C allele, compared with cell lines with other allelic variants. Similar results were obtained in in vitro luciferase assays. Chromatin immunoprecipitation (ChIP) demonstrated binding of c‐Myb to the −443 OPN promoter region, and binding could significantly be enhanced after bFGF stimulation. Moreover, as shown by electrophoretic mobility shift assays (EMSA), recombinant DNA‐binding domain of c‐Myb bound in a sequence‐specific manner to this region. Finally, the role of c‐Myb for OPN gene regulation via binding to the −443 promoter region could be further substantiated by ectopic overexpression of c‐Myb in melanoma cells, using different reporter gene constructs. Taken together, it is demonstrated that the −443 promoter region exerts influence on OPN gene expression in melanoma cells, and differential binding of c‐Myb transcription factor appears to play a major role in this process. These findings might be a feasible explanation for different OPN expression levels in metastatic tumors and may also have prognostic and therapeutic relevance.

Computational analysis of high-density peptide microarray data with application from systemic sclerosis to multiple sclerosis.

Auto-antibodies are implicated in the pathophysiology of various autoimmune diseases. High-density peptide microarrays incubated with human serum can detect antibody reactivities against thousands of peptides. This enables the identification of new auto-antigens and the determination of the parts of protein antigens (epitopes) that are recognized by antibody paratopes. We discuss the utility of peptide microarrays to investigate epitope-antibody-recognitions (EAR) from systemic sclerosis to multiple sclerosis. The technology can help to establish reliable diagnostic and prognostic biomarkers employing a combination of antigenic peptides. We describe the specifics of peptide microarray data and present bioinformatic methods for their analysis. Quality control, data pre-processing and the filtering of specific peptides are demonstrated on an example data set. Peptide microarrays representing 24 selected proteins by 3235 overlapping 15mer peptides were used to measure antibodies in serum of 10 patients with limited cutaneous systemic sclerosis (SSC) and 10 healthy blood donors. The data showed a sparse and skewed distribution, and we observed strong individual differences since many peptide sequences were bound by antibodies of only one serum sample. In the sera of the SSc patients, but not of the healthy controls, we found antibodies to three peptides MGPRRRSRKPEAPRR, TPTPGPSRRGPSLGA and GPSRRGPSLGASSHQ that share a similar sequence motif (GP-R/S-RR). These peptides map to two known linear epitopes at the N-terminus of centromere protein A (CENPA), demonstrating the utility of peptide microarrays. Presented experimental and bioinformatic approach can be applied in the same manner for multiple sclerosis research.

Proteome analysis of diseased joints from mice suffering from collagen-induced arthritis.

Abstract Strains of mice that are susceptible to autoimmunity have provided experimental models to analyze the molecular basis for the complex multifactorial inheritance of human autoimmune disease. In this study proteins associated with collagen-induced arthritis (CIA) in mice were experimentally identified using a global proteomics approach. Two-dimensional gels of proteins from inflamed and non-inflamed joints showed a distinguished protein profile visualizing about 530 Coomassie-stained protein spots in the pH 4–7 range. A total of 76 spots were identified by peptide mass fingerprinting with good confidence. They included proteins of cytoskeletal origin, chaperones, enzymes and also some signal transduction molecules. Comparison to gels from non-inflamed paws pointed to proteins that were differentially expressed between the control and diseased state. Ferritin light chain and antioxidant protein 2 were slightly more abundant, lymphoid enhancer binding factor 1 slightly, but significantly, less abundant in inflamed paws. Fourteen of the identified proteins were the products of genes that had increased transcript levels in the diseased state. However, on the protein level no significant differences were found in comparison to the controls. This study provides us with the framework for more detailed approaches to understanding the complex disease arthritis that go beyond global proteomics.

Transcriptional repression mediated by the KRAB domain of the human C2H2 zinc finger protein Kox1/ZNF10 does not require histone deacetylation.

Abstract The KRAB domain of human Kox1, a member of the KRAB C2H2 zinc finger family, confers strong transcriptional repressor activities even to remote promoter positions. Here, HDAC inhibitors were used to demonstrate that histone deacetylation is not required for mediating transcriptional repression of KRAB zinc finger proteins. Two reporter systems with either stably integrated or transiently transfected templates, both under control of strong viral promoters, were analyzed. Under all circumstances, HDAC inhibition did not alter the repression potential of the KRAB domain. In case of the stably integrated luciferase reporter gene system, neither expression levels of the KRAB fusion protein nor complex formation with its putative corepressor TIF1β were significantly changed. Furthermore, the TIF1β/KRAB complex was devoid of mSin3A and HDAC1. In the transient transfection system, the transcriptional repression induced by TIF1β and HP1α was not diminished by HDAC inhibitors, whereas the repressory activity of TIF1α was significantly affected. Thus, KRAB, TIF1β and HP1α are likely to be functionally linked. In conclusion, HDAC activity is not essential for the strong transcriptional repressor activity mediated by the KRAB domain of Kox1 in particular and, presumably, by KRAB domains in general. This feature might be helpful in identifying and characterizing target genes under the control of KRAB zinc finger proteins.

Investigations into the Phenolic Constituents of Dog's Mercury (Mercurialis perennis L.) by LC-MS/MS and GC-MS analyses

INTRODUCTION Dogs mercury (Mercurialis perennis L.) is a traditional European medicinal plant considered as a rich source of bioactive natural products. Yet phytochemical data of the plant are scant. OBJECTIVE This study aimed to identify the hydrophilic phenolic constituents from M. perennis by aqueous and hydroalcoholic extraction. METHODOLOGY Extracts of herbal parts were investigated in-depth by HPLC(DAD)-MS/MS and GC/MS analyses. In addition, a novel compound was isolated and fully characterised by 1- and 2D-NMR experiments. RESULTS Several conjugates of caffeic, p-coumaric and ferulic acids together with glucaric or 2-hydroxyglutaric acids (depsides) were detected in the aqueous extracts from aerial plant parts by use of LC-MS/MS techniques as well UV-spectral data. By implementation of preparative chromatography on polyamide pretreated with formic acid followed by vacuum liquid chromatography on reversed-phase C(18) -silica, one of the predominant depsides was isolated as a pure compound. The NMR spectra ((1) H and (13) C NMR) together with 2D-hetereonuclear multiple bond correlation NMR experiments (gHMBC and gHSQC) and chiral GC investigation, allowed identification of this compound as (-)-(E)-caffeoyl-2-(R)-oxoglutarate. This structure was additionally supported by GC/MS data after silylation and methylation reactions. The hydroalcoholic extract from aerial parts was separated by solvent partition between ethyl acetate and n-butanol. The latter fraction (n-butanol) yielded a mixture of mono- and oligo-glycosides of kaempferol and quercetin, all of them being assigned by LC-MS/MS. CONCLUSIONS The present investigation constitutes the first comprehensive report on the hydrophilic constituents of the rarely studied plant Mercurialis and thus completes the phytochemical knowledge on M. perennis.

The ancient mammalian KRAB zinc finger gene cluster on human chromosome 8q24.3 illustrates principles of C2H2 zinc finger evolution associated with unique expression profiles in human tissues

BackgroundExpansion of multi-C2H2 domain zinc finger (ZNF) genes, including the Krüppel-associated box (KRAB) subfamily, paralleled the evolution of tetrapodes, particularly in mammalian lineages. Advances in their cataloging and characterization suggest that the functions of the KRAB-ZNF gene family contributed to mammalian speciation.ResultsHere, we characterized the human 8q24.3 ZNF cluster on the genomic, the phylogenetic, the structural and the transcriptome level. Six (ZNF7, ZNF34, ZNF250, ZNF251, ZNF252, ZNF517) of the seven locus members contain exons encoding KRAB domains, one (ZNF16) does not. They form a paralog group in which the encoded KRAB and ZNF protein domains generally share more similarities with each other than with other members of the human ZNF superfamily. The closest relatives with respect to their DNA-binding domain were ZNF7 and ZNF251. The analysis of orthologs in therian mammalian species revealed strong conservation and purifying selection of the KRAB-A and zinc finger domains. These findings underscore structural/functional constraints during evolution. Gene losses in the murine lineage (ZNF16, ZNF34, ZNF252, ZNF517) and potential protein truncations in primates (ZNF252) illustrate ongoing speciation processes. Tissue expression profiling by quantitative real-time PCR showed similar but distinct patterns for all tested ZNF genes with the most prominent expression in fetal brain. Based on accompanying expression signatures in twenty-six other human tissues ZNF34 and ZNF250 revealed the closest expression profiles. Together, the 8q24.3 ZNF genes can be assigned to a cerebellum, a testis or a prostate/thyroid subgroup. These results are consistent with potential functions of the ZNF genes in morphogenesis and differentiation. Promoter regions of the seven 8q24.3 ZNF genes display common characteristics like missing TATA-box, CpG island-association and transcription factor binding site (TFBS) modules. Common TFBS modules partly explain the observed expression pattern similarities.ConclusionsThe ZNF genes at human 8q24.3 form a relatively old mammalian paralog group conserved in eutherian mammals for at least 130 million years. The members persisted after initial duplications by undergoing subfunctionalizations in their expression patterns and target site recognition. KRAB-ZNF mediated repression of transcription might have shaped organogenesis in mammalian ontogeny.

Analysis of aqueous humour proteins of eyes with and without pseudoexfoliation syndrome

Pseudoexfoliation syndrome (PEX) has been suggested to represent a blood-aqueous barrier impairment leading to a higher protein content in aqueous humour of eyes with PEX. However, the nature of a prospective PEX protein has not yet been described. We set out to re-evaluate protein content and examine protein composition for prospective PEX protein candidates in aqueous humour of eyes with PEX syndrome. Aqueous humour of 52 patients with PEX and 38 without PEX signs was sampled during cataract or glaucoma surgery. Total aqueous protein concentration in the samples was analysed in 43 PEX specimens and 32 non-PEX specimens according to Bradford. Aqueous protein composition of all samples was determined by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS PAGE) and silver staining. Screening for amyloids was performed in nine PEX samples and six non-PEX samples by Congo Red staining and polarised light microscopy. Aqueous protein concentration was not significantly increased in PEX eyes in comparison with non-PEX eyes. Furthermore, we could not detect any characteristic difference in protein band sizes of the two groups after SDS PAGE. However, we were able to show the presence of amyloid exclusively in aqueous humour of PEX patients. Conclusion: our results do not confirm a generally higher protein concentration in pseudoexfoliation syndrome eyes. This does not necessarily contradict a blood-aqueous barrier impairment but illustrates the variance in protein concentration between and within the two groups. No characteristic protein band allocatable to pseudoexfoliation syndrome proteins could be detected in any of the samples. However, our findings support the theory that the pseudoexfoliation syndrome is associated with an amyloid of a serum protein.

Long-lived plasma cells and memory B cells produce pathogenic anti-GAD65 autoantibodies in Stiff Person Syndrome.

Stiff person syndrome (SPS) is a rare, neurological disorder characterized by sudden cramps and spasms. High titers of enzyme-inhibiting IgG autoantibodies against the 65 kD isoform of glutamic acid decarboxylase (GAD65) are a hallmark of SPS, implicating an autoimmune component in the pathology of the syndrome. Studying the B cell compartment and the anti-GAD65 B cell response in two monozygotic twins suffering from SPS, who were treated with the B cell-depleting monoclonal anti-CD20 antibody rituximab, we found that the humoral autoimmune response in SPS is composed of a rituximab-sensitive part that is rapidly cleared after treatment, and a rituximab-resistant component, which persists and acts as a reservoir for autoantibodies inhibiting GAD65 enzyme activity. Our data show that these potentially pathogenic anti-GAD65 autoantibodies are secreted by long-lived plasma cells, which may either be persistent or develop from rituximab-resistant memory B lymphocytes. Both subsets represent only a fraction of anti-GAD65 autoantibody secreting cells. Therefore, the identification and targeting of this compartment is a key factor for successful treatment planning of SPS and of similar autoimmune diseases.