Strong reduction of AGO2 expression in melanoma and cellular consequences.

BACKGROUND: Processing of microRNAs (miRNAs) is a highly controlled process. Deregulation of miRNA expression was observed in several types of cancer but changes in the miRNA-processing enzymes have not been analysed until today. In this study, we analysed Argonaute2 (AGO2, EIF2C2), as one main factor of the miRNA processing ensemble, in the context of cancer development, especially in melanoma. METHODS: We determined the AGO2 expression level in melanoma, as well as in other cancers, with biochemical approaches (qRT-PCR, western blot and immunofluorescence studies) and analysed the cell behaviour in migration assays. RESULTS: Specifically in melanoma, we revealed a strong reduction of AGO2 expression compared with primary melanocytes. The reduction of AGO2 expression was only found on protein level, whereas the mRNA level stayed unchanged hinting to post-transcriptional regulation. We could show that re-expression of AGO2 in melanoma leads to a strong improvement of regulatory effects due to increased functionality of small-interfering RNAs and short hairpin RNAs. CONCLUSION: We identified melanoma-specific downregulation of AGO2 and corresponding reduced RNAi efficiency. These findings will help to understand the molecular basis of malignant melanoma and can potentially lead to an improvement of therapeutic strategies.

A multiprotein complex mediates the ATP-dependent assembly of spliceosomal U snRNPs.

The spliceosomal snRNPs U1, U2, U4 and U5 contain a common RNP structure termed the Sm core that is formed by the binding of Sm proteins onto the U snRNA. Although isolated Sm proteins assemble spontaneously onto U snRNAs in vitro, there is increasing evidence that SMN and its interactor Gemin2 are involved in this process in vivo. Here, we describe a cell-free assay system for the assembly of U snRNPs that closely reproduces in vivo conditions. Using this system, we show that assembly of U1 snRNP depends on ATP. Immunodepletion of SMN-Gemin2 from the extract abolished assembly even though the extract contained high levels of Sm proteins. An affinity-purified macromolecular SMN complex consisting of 16 components including all Sm proteins restored assembly in the immunodepleted extract. These data provide the first direct evidence that a complex containing SMN and Gemin2 mediates the active assembly of spliceosomal U snRNPs.

Methylation of Sm proteins by a complex containing PRMT5 and the putative U snRNP assembly factor pICln.

Seven Sm proteins, termed B/B', D1, D2, D3, E, F, and G, assemble in an ordered manner onto U snRNAs to form the Sm core of the spliceosomal snRNPs U1, U2, U4/U6, and U5. The survival of motor neuron (SMN) protein binds to Sm proteins and mediates in the context of a macromolecular (SMN-) complex the assembly of the Sm core. Binding of SMN to Sm proteins is enhanced by modification of specific arginine residues in the Sm proteins D1 and D3 to symmetrical dimethylarginines (sDMAs), suggesting that assembly might be regulated at the posttranslational level. Here we provide evidence that the previously described pICln-complex, consisting of Sm proteins, the methyltransferase PRMT5, pICln, and two novel factors, catalyzes the sDMA modification of Sm proteins. In vitro studies further revealed that the pICln complex inhibits the spontaneous assembly of Sm proteins onto a U snRNA. This effect is mediated by pICln via its binding to the Sm fold of Sm proteins, thereby preventing specific interactions between Sm proteins required for the formation of the Sm core. Our data suggest that the pICln complex regulates an early step in the assembly of U snRNPs, possibly the transfer of Sm proteins to the SMN-complex.

Rapid spread of transposable p elements in experimental populations of Drosophila melanogaster.

The invasion of P elements in natural populations of Drosophila melanogaster was modeled by establishing laboratory populations with 1%, 5% and 10% P genomes and monitoring the populations for 20 generations. In one experiment, the ability of flies to either induce or suppress gonadal sterility in different generations was correlated with the amount of P element DNA. In a second experiment, the percentage of genomes that contained P elements, and the distribution of P elements among individual flies was monitored. The ability to induce gonadal dysgenesis increased rapidly each generation. However, the increase in P cytotype lagged behind by five to ten generations. The total amount of P element DNA and the frequency of flies containing P elements increased each generation. The number of P elements within individual genomes decreased initially, but then increased. Finally, the distribution of P elements within the genomes of individuals from later generations varied considerably, and this pattern differed from the parental P strain. These results suggest that the interaction between the assortment and recombination of chromosomal segments, and multiplicative transposition could result in the rapid spread of P elements in natural populations.

[Incidence of posthepatectomy liver failure and biliary leakage : A cohort study]. / Inzidenz von Leberversagen und Galleleckagen nach Leberteilresektion : Eine Kohortenstudie.

BACKGROUND: The International Study Group of Liver Surgery (ISGLS) defined posthepatectomy liver failure as pathological values for the international normalized ratio (INR) and bilirubin 5 days after liver resection. The occurrence of biliary leakage was defined as a drainage bilirubin to serum bilirubin ratio > 3 at day 3 or later after resection or interventional surgical revision due to biliary peritonitis. A confirmatory explorative analysis was carried out. PATIENTS AND METHODS: The study involved an evaluation of primary liver resection from the years 2009 and 2010. Primary endpoints were the incidence of posthepatectomy liver failure and biliary leakage in accordance with the ISGLS definition. Secondary endpoints were complications and 90-day mortality. Results are displayed as median values (minimum and maximum). RESULTS: A total of 214 liver resections were included from the years 2009 and 2010. Patients were an average of 61.5 years old (min. 18, max. 83 years). The incidence of liver failure was 7.4 % (16 out of 214) and fatal in 7 patients. In 31 % (65 out of 214) a biliary leakage occurred, 14 (23 %) patients developed a type B, 1 patient(5 %) a type C leakage and 50 leakages were clinically inapparent. The incidence of clinically relevant biliary leakages was 7 % (15 out of 214). The sensitivity of the definition was 100 % and the specificity 75 %. The incidence of Dindo-Calvien complications > 3b was 10.2 %, of sepsis 5.6 % and the 90-day mortality was 6.5 %. Multivariate analysis did not reveal independent predictive factors for biliary leakage or liver failure. CONCLUSION: The definition for posthepatectomy liver failure was found to be valid in this cohort. The incidence of postoperative biliary leakage is over-estimated with the current definition and delivers a large number of false positive results without clinical relevance.

Simple plaque hybridization method for the detection of differentially represented repetitive DNA.

A differential DNA hybridization method of detecting moderately repetitive strain-specific or species-specific DNA is described. Two Drosophila melanogaster strains, one with and one without transposable elements, were utilized as a model system to demonstrate the effectiveness of this procedure. A genomic library was constructed from flies of the pi 2 strain, which contains both P and hobo transposable elements. Duplicate plaque lifts of this library were probed with DNA from the same strain and with DNA from the Canton-S strain, which contains neither of these two families of transposable elements. Plaques that hybridized stronger to the genomic DNA that contained elements were noted, and then the filters were stripped and reprobed with P and hobo element DNA. Many of the differentially hybridizing plaques were shown to contain DNA homologous to one of the two known elements. This method should allow the isolation and cloning of any repetitive DNA present in one species or isolate, but absent or present in reduced copy number in another species or isolate. By analogy to the recent invasion of D. melanogaster by P elements, such differentially represented DNA is likely to represent recently invading transposable elements that are actively mobile.

Prediction of HIV peptide epitopes by a novel algorithm.

Identification of promiscuous or multideterminant T cell epitopes is essential for HIV vaccine development, however, current methods for T cell epitope identification are both cost intensive and labor intensive. We have developed a computer-driven algorithm, named EpiMer, which searches protein amino acid sequences for putative MHC class I- and/or class II-restricted T cell epitopes. This algorithm identifies peptides that contain multiple MHC-binding motifs from protein sequences. To evaluate the predictive power of EpiMer, the amino acid sequences of the HIV-1 proteins nef, gp160, gag p55, and tat were searched for regions of MHC-binding motif clustering. We assessed the algorithm's predictive power by comparing the EpiMer-predicted peptide epitopes to T cell epitopes that have been published in the literature. The EpiMer method of T cell epitope identification was compared to the standard method of synthesizing short, overlapping peptides and testing them for immunogenicity (overlapping peptide method), and to an alternate algorithm that has been used to identify putative T cell epitopes from primary structure (AMPHI). For the four HIV-1 proteins analyzed, the in vitro testing of EpiMer peptides for immunogenicity would have required the synthesis of fewer total peptides than either AMPHI or the overlapping peptide method. The EpiMer algorithm proved to be more efficient and more sensitive per amino acid than both the overlapping peptide method and AMPHI. The EpiMer predictions for these four HIV proteins are described. Since EpiMer-predicted peptides have the potential to bind to multiple MHC alleles, they are strong candidates for inclusion in a synthetic HIV vaccine.

Control of an outbreak of mouse coccidiosis in a closed colony.

In 1979 severe coccidiosis occurred within a closed colony of C57bl/6J Bom mice. The coccidiosis caused diarrhoea, weight loss and deaths. Pathogenicity and response to chemotherapy were studied in experimentally infected Bor: CFW1 (SPF) mice. Histological and other studies indicated that infection was caused by more than 1 Eimeria species which have not yet been determined. Treatment with different anticoccidial drugs was unsuccessful, but use of 2 triazinone-derivatives (Bay g 7183, Bay i 9142) added to the food (15 ppm) succeeded in completely eradicating the organisms from the breeding houses. Experimental findings on the sensitivity of the coccidia to other anticoccidial drugs are described.

MicroRNA miR-125b controls melanoma progression by direct regulation of c-Jun protein expression.

A fundamental event in the development and progression of malignant melanoma is the deregulation of cancer-relevant transcription factors. We recently showed that c-Jun is a main regulator of tumor progression in melanoma and thus the most important member of the AP-1 transcription factor family for this disease. Interestingly, we revealed that c-Jun expression was regulated on the post-transcriptional level and therefore speculated that miRNAs could be involved in c-Jun regulation. We determined seed sequences for miR-125b and miR-527 in the coding region of c-Jun mRNA that hints at the direct involvement of miRNA-dependent regulation on the protein level. We found that the expression of miR-125b was significantly reduced in malignant melanoma cell lines and tissue samples compared with melanocytes, whereas miR-527 remained unchanged. In further functional experiments, treatment of melanoma cells with pre-miR-125b resulted in strong suppression of cellular proliferation and migration, supporting the role of miR-125b in melanoma. In addition, transfection of pre-miR-125b led to strong downregulation of c-Jun protein but not mRNA expression in melanoma cells. Luciferase assays using reporter plasmids containing the miR-125b seed sequence in the luciferase coding region confirmed the direct interaction with miR-125b. Furthermore, immunoprecipitation of Ago-2 revealed that c-Jun mRNA accumulated in the RNA-induced silencing complex after pre-miR-125b transfection in melanoma cells. In summary, we identified an important role for miR-125b in malignant melanoma. Moreover, we demonstrated post-transcriptional regulation of c-Jun by this miRNA and showed that c-Jun is a main mediator of the effects of miR-125b on melanoma cells.

Regulation of microRNA biogenesis and function.

MicroRNAs (miRNAs) are considered as key regulators of literally all cellular pathways. Therefore, miRNA biosynthesis and their individual cellular functions must be tightly regulated as well. MiRNAs are transcribed as primary transcripts, which are processed to mature miRNAs in two consecutive maturation steps. Finally, the mature miRNA is incorporated into a miRNA-protein complex, where it directly interacts with a member of the Argonaute (Ago) protein family. The miRNA guides such protein complexes to partial complementary target sites, which are typically located in the 3' untranslated region (UTR) of mRNAs leading to inhibition of gene expression. MiRNA activity and abundance is regulated on various levels ranging from transcription and processing to target site binding and miRNA stability. Recent advances in our understanding of how miRNA activity is regulated in mammalian cells are summarised and discussed in this review article.

Concurrent antiviral and immunosuppressive activities of leflunomide in vivo.

We previously reported that the immunosuppressive malononitrileamides leflunomide and FK778 exert antiviral activity against cytomegalovirus (CMV). In the current investigation, we tested the hypothesis that leflunomide exerts concurrent antiviral activity and immune suppression in CMV-infected cardiac allograft recipients. Lewis rats were transplanted with Brown Norway hearts and then inoculated with rat CMV. Plaque assay demonstrated that leflunomide (30 mg/kg/day) reduced viral loads by 4-6 logs, and that the reduction in viral load was unaffected by administration of uridine. Leflunomide was as effective as cyclosporine A (CsA) or tacrolimus in preservation of allograft integrity through day 28. These studies directly demonstrate the bifunctionality of leflunomide as concurrently immunosuppressive and antiviral, enhancing the promise of this agent as a clinical option for treatment of transplant recipients.

Structural biology of RNA silencing and its functional implications.

We outline structure-function contributions from our laboratories on protein-RNA recognition events that monitor siRNA length, 5 -phosphate and 2-nucleotide 3 overhangs, as well as the architecture of Argonaute, its externally bound siRNA complex, and Argonaute-based models involving guide-strand-mediated mRNA binding, cleavage, and release.

[A method to detect microfilariae of Dipetalonema witei in the peripheral blood of mastomys natalensis (author's transl)]. / Eine Methode zum Nachweis von Mikrofilarien von Dipetalonema witei im peripheren Blut von Mastomys natalensis.

A new cheap and time saving procedure is described for the detection of microfilariae of Dipetalonema witei in the peripheral blood of Mastomys natalensis. A special apparatus delivering an ether-air mixture is used to anesthetize the experimental animals for a short period in a very careful way and without side effects. The ether wapour induces a rapid shift of microfilariae within one minute from the lung to the peripheral blood.

Biodegradation of N-methylmorpholine-N-oxide.

N-Methylmorpholine-N-oxide (NMMO) is capable of dissolving cellulose without any further addition of chemicals. The solution can be used to produce cellulosic staple fibres by pressing it through spinning jets into an aqueous spinning bath. Because of results from conventional biodegradation tests using non-adapted activated sludge, the solvent is generally considered being persistent. The object of the described work was to show, whether and how activated sludge can be adapted to N-methylmorpholine-N-oxide and whether it is possible to purify NMMO-containing wastewaters in conventional wastewater treatment plants. The experiments showed that the sludge can be adapted within about 15-20 days. Adapted sludge can degrade the substance itself and its most important metabolites to concentrations below their detection levels and retain this ability even during limited periods without solvent being present in the wastewater. The main requirement for a successful adaptation is a high sludge age. The degradation takes place in several steps. First, NMMO is reduced to N-methylmorpholine. The next step is a demethylation of N-methylmorpholine to morpholine. This step is crucial for the adaptation process. Once morpholine has been formed, the adaptation proceeds very quickly until none of the substances in question can be detected any longer. So the next step must be the cleavage of the morpholine ring structure.

Rapid spread of a P element/Adh gene construct through experimental populations of Drosophila melanogaster.

Transposable elements may be potential tools for the dispersal of engineered DNA through target insect populations. The utility of this hypothesis is predicted on the ability of transposable elements carrying a large DNA insert to rapidly disperse through a population. In addition, the inserted DNA must be replicated with a high degree of fidelity during this dispersal. We have monitored the ability of a transposable element with an inserted gene to spread through experimental populations and tested whether the passenger gene retains its ability to encode an active protein. Several Drosophila melanogaster laboratory populations were initiated with female flies that were null for alcohol dehydrogenase activity and contained no P elements. Most of the females were mated to males of the same strain; however, 1 or 10% of the females were mated to males from a strain that had previously been transformed with a helper P element and a P element/Adh gene construct. The dispersal of P elements to new genomes was monitored at each generation by randomly selecting females and performing DNA hybridization assays on dissected ovarian tissue. In addition, each female was tested for alcohol dehydrogenase activity using a simple histochemical assay. We find that, despite an approximate threefold increase in size, the P element constructs containing a functioning gene are still capable of rapid dispersal through the experimental populations. We also show that many of the inserted Adh genes still encode an active product.

[Results of the follow-up of varus osteotomy in Perthes disease with reference to the hip value]. / Behandlungsergebnisse der Varisierungsosteotomie bei Morbus Perthes unter Berücksichtigung des Hüftwertes.

A total of 89 patients with 105 hip joints affected by Perthes' disease at Catterall's stages II to IV, which had been treated by varus osteotomy, were re-examined clinically and radiologically between two and ten years after surgery. The clinical and subjective results of treatment were generally good. The best clinical and radiologic results were found in patients who contracted the disease before the age of seven and in whom necrosis was not yet at an advanced stage. The results in children who did not undergo treatment until after the age of eight and those with the disease at Catterall's stage IV were significantly poorer as far as arthrotic deformation of the hip joint was concerned. Radiologic evaluation of the hips examined, with regard to severity of arthrosis, epiphyseal index, and hip value show that the hip value is the best means of distinguishing prearthrotically deformed hip joints from normal findings. For example, at the time of follow-up severe prearthrotic changes were found in 40 of 105 hip joints when the hip values were analyzed. Therefore, in cases of Perthes' disease also, the hip value is of special importance in prognostic counseling of patients as regards the extent to which the hip joint can be subjected to stress either at work or in sports.

Width of the specular peak perpendicular to the principal plane for rough surfaces.

The bidirectional reflectance distribution function (BRDF) model developed by Torrance and Sparrow [J. Opt. Soc. Am. 57, 1105-1114 (1967)] is used to describe the specular reflection of rough surfaces. We compare this model with the BRDF measurements of four manmade surfaces with different roughnesses. The model can be used to describe the basic features of the measured BRDFs. We found that the width of the specular peak perpendicular to the principal plane decreases strongly with an increasing illumination zenith angle in the data as well as in the model. A model analysis shows that the width is approximately proportional to the cosine of the illumination angle theta(i), and the deviations are determined by the roughness of the surface. This relationship is accompanied by an increase in reflectance in the specular direction in the principal plane that is 1/cos theta(i) stronger than the increase for a perfectly smooth surface.

Neostigmine combined with bupivacaine, clonidine, and sufentanil for spinal labor analgesia.

UNLABELLED: We previously found that spinal clonidine prolongs labor analgesia when combined with spinal bupivacaine and sufentanil. We sought to determine whether the addition of spinal neostigmine to these drugs would further enhance labor analgesia. By use of a combined spinal/epidural technique, 36 patients were randomized to receive a hyperbaric spinal injection of bupivacaine 2.5 mg plus clonidine 50 microg and sufentanil 10 microg with or without neostigmine 10 microg. Pain, maternal hemodynamics, fetal heart rate, nausea, pruritus, sedation, motor block, sensory levels to pinprick, and maternal oxygen saturation were assessed at regularly specified intervals after spinal injection until additional analgesia was requested. The duration of spinal analgesia was similar between groups (215 +/- 60 min in the Control group versus 205 +/- 62 min in the Neostigmine group). Likewise, pain scores, the duration of labor, Apgar scores, and side effects were similar between groups except that patients administered neostigmine experienced significantly more nausea and vomiting (53% vs 7%, P = 0.01). We conclude that spinal neostigmine 10 microg produces severe nausea and does not potentiate the duration of spinal analgesia in laboring women from spinal bupivacaine, clonidine, and sufentanil. IMPLICATIONS: Spinal neostigmine 10 microg as an adjunct to spinal bupivacaine, clonidine, and sufentanil produces severe nausea and fails to potentiate analgesia in laboring women.

A comparison of epidural analgesia with 0.125% ropivacaine with fentanyl versus 0.125% bupivacaine with fentanyl during labor.

UNLABELLED: We previously found that the extent of an epidural motor block produced by 0.125% ropivacaine was clinically indistinguishable from 0.125% bupivacaine in laboring patients. By adding fentanyl to the 0. 125% ropivacaine and bupivacaine solutions in an attempt to reduce hourly local anesthetic requirements, we hypothesized that differences in motor block produced by the two drugs may become apparent. Fifty laboring women were randomized to receive either 0. 125% ropivacaine with fentanyl 2 microg/mL or an equivalent concentration of bupivacaine/fentanyl using patient-controlled epidural analgesia (PCEA) with settings of: 6-mL/hr basal rate, 5-mL bolus, 10-min lockout, 30-mL/h dose limit. Analgesia, local anesthetic use, motor block, patient satisfaction, and side effects were assessed until the time of delivery. No differences in verbal pain scores, local anesthetic use, patient satisfaction, or side effects between groups were observed; however, patients administered ropivacaine/fentanyl developed significantly less motor block than patients administered bupivacaine/fentanyl. Ropivacaine 0.125% with fentanyl 2 microg/mL produces similar labor analgesia with significantly less motor block than an equivalent concentration of bupivacaine/fentanyl. Whether this statistical reduction in motor block improves clinical outcome or is applicable to anesthesia practices which do not use the PCEA technique remains to be determined. IMPLICATIONS: By using a patient-controlled epidural analgesia technique, ropivacaine 0.125% with fentanyl 2 microg/mL produces similar analgesia with significantly less motor block than a similar concentration of bupivacaine with fentanyl during labor. Whether this statistical reduction in motor block improves clinical outcome or is applicable to anesthesia practices which do not use the patient-controlled epidural analgesia technique remains to be determined.