Sperma-übertragung (und andere merkmale) in ihrer bedeutung für das phylogenetische system der sternorrhyncha (insecta,hemiptera) Phylogenetische studien an hemiptera I. psylliformes (psyllina und aleyrodina) als monophyletische gruppe

The method for the search for synapomorphies (in the sense of Hennig) is demonstrated by the study of the system of sperm-transfer. The ground plan of sperm-transfer in Homoptera (and other insect groups) is peristaltic movement of circular muscles surrounding the ejaculatory duct. In contrast, Psyllina and Aleyrodina exhibit a completely different structure and function of the ejaculatory duct, being extremely specialized, and principally identical in both the groups. This is a synapomorphic complex which proves Psyllina and Aleyrodina as most closely related. Other synapomorphies confirm this statement.     

Growth hormone and insulin-like growth factor I concentrations in bulls of various growth hormone genotypes

A leucine/valine substitution at amino acid position 127 was identified by the polymerase chain reaction and restriction fragment length polymorphism in the bovine growth hormone gene. Genotyping was performed in 84 AI bulls of three different breeds, in which plasma concentrations of growth hormone (GH) and insulin-like growth factor I (IGF-1) were also measured. Gene frequencies of variants L (leucine) und V (valine) were 0.80/0.20 (Black and White), 0.90/0.10 (Brown), 0.71/0.29 (Simmental). Hormone concentrations were measured during different physiological conditions (normal feeding, fasting, realimentation) in the majority of animals. Generally, genotype LL was associated with higher concentrations of GH than LV. This difference was significant in Black and White bulls (P < 0.05). In contrast, IGF-1 concentrations were higher in LV than in LL animals. This was most pronounced in mature, realimented Simmental bulls. We conclude that the various GH alleles influence the circulating concentrations of GH and IGF-1.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Effect of sulphite on adenine nucleotides of the green alga Trebouxia

The amounts of AMP, ADP and ATP were determined after sulfite incubation in photo-organotrophically cultivated cells of the green alga Trebouxia. D     

A Human In Vitro Whole Blood Assay to Predict the Systemic Cytokine Response to Therapeutic Oligonucleotides Including siRNA

Therapeutic oligonucleotides including siRNA and immunostimulatory ligands of Toll-like receptors (TLR) or RIG-I like helicases (RLH) are a promising novel class of drugs. They are in clinical development for a broad spectrum of applications, e.g. as adjuvants in vaccines and for the immunotherapy of cancer. Species-specific immune activation leading to cytokine release is characteristic for therapeutic oligonucleotides either as an unwanted side effect or intended pharmacology. Reliable in vitro tests designed for therapeutic oligonucleotides are therefore urgently needed in order to predict clinical efficacy and to prevent unexpected harmful effects in clinical development. To serve this purpose, we here established a human whole blood assay (WBA) that is fast and easy to perform. Its response to synthetic TLR ligands (R848: TLR7/8, LPS: TLR4) was on a comparable threshold to the more time consuming peripheral blood mononuclear cell (PBMC) based assay. By contrast, the type I IFN profile provoked by intravenous CpG-DNA (TLR9 ligand) in humans in vivo was more precisely replicated in the WBA than in stimulated PBMC. Since Heparin and EDTA, but not Hirudin, displaced oligonucleotides from their delivery agent, only Hirudin qualified as the anticoagulant to be used in the WBA. The Hirudin WBA exhibited a similar capacity as the PBMC assay to distinguish between TLR7-activating and modified non-stimulatory siRNA sequences. RNA-based immunoactivating TLR7/8- and RIG-I-ligands induced substantial amounts of IFN-α in the Hirudin-WBA dependent on delivery agent used. In conclusion, we present a human Hirudin WBA to determine therapeutic oligonucleotide-induced cytokine release during preclinical development that can readily be performed and offers a close reflection of human cytokine response in vivo.     

RIG-I Detects Triphosphorylated RNA of Listeria monocytogenes during Infection in Non-Immune Cells

The innate immune system senses pathogens by pattern recognition receptors in different cell compartments. In the endosome, bacteria are generally recognized by TLRs; facultative intracellular bacteria such as Listeria, however, can escape the endosome. Once in the cytosol, they become accessible to cytosolic pattern recognition receptors, which recognize components of the bacterial cell wall, metabolites or bacterial nucleic acids and initiate an immune response in the host cell. Current knowledge has been focused on the type I IFN response to Listeria DNA or Listeria-derived second messenger c-di-AMP via the signaling adaptor STING. Our study focused on the recognition of Listeria RNA in the cytosol. With the aid of a novel labeling technique, we have been able to visualize immediate cytosolic delivery of Listeria RNA upon infection. Infection with Listeria as well as transfection of bacterial RNA induced a type-I-IFN response in human monocytes, epithelial cells or hepatocytes. However, in contrast to monocytes, the type-I-IFN response of epithelial cells and hepatocytes was not triggered by bacterial DNA, indicating a STING-independent Listeria recognition pathway. RIG-I and MAVS knock-down resulted in abolishment of the IFN response in epithelial cells, but the IFN response in monocytic cells remained unaffected. By contrast, knockdown of STING in monocytic cells reduced cytosolic Listeria-mediated type-I-IFN induction. Our results show that detection of Listeria RNA by RIG-I represents a non-redundant cytosolic immunorecognition pathway in non-immune cells lacking a functional STING dependent signaling pathway.     

FLT3 Mutations in Early T-Cell Precursor ALL Characterize a Stem Cell Like Leukemia and Imply the Clinical Use of Tyrosine Kinase Inhibitors

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.     

Prediction of quaternary structure by analysis of hot spot residues in protein-protein interfaces: the case of anthranilate phosphoribosyltransferases

It is an important goal of computational biology to correctly predict the association state of a protein based on its amino acid sequence and the structures of known homologues. We have pursued this goal on the example of anthranilate phosphoribosyltransferase (AnPRT), an enzyme that is involved in the biosynthesis of the amino acid tryptophan. Firstly, known crystal structures of naturally occurring homodimeric AnPRTs were analyzed using the Protein Interfaces, Surfaces, and Assemblies (PISA) service of the European Bioinformatics Institute (EBI). This led to the identification of two hydrophobic “hot spot” amino acids in the protein-protein interface that were predicted to be essential for self-association. Next, in a comprehensive multiple sequence alignment (MSA), naturally occurring AnPRT variants with hydrophilic or charged amino acids in place of hydrophobic residues in the two hot spot positions were identified. Representative variants were characterized in terms of thermal stability, enzymatic activity, and quaternary structure. We found that AnPRT variants with charged residues in both hot spot positions exist exclusively as monomers in solution. Variants with hydrophilic amino acids in one hot spot position occur in both forms, monomer and dimer. The results of the present study provide a detailed characterization of the determinants of the AnPRT monomer-dimer equilibrium and show that analysis of hot spots in combination with MSAs can be a valuable tool in prediction of protein quaternary structures.     

The N terminus of ClpB from Thermus thermophilus is not essential for the chaperone activity

ClpB from Thermus thermophilus belongs to the Clp/Hsp100 protein family and reactivates protein aggregates in cooperation with the DnaK chaperone system. The mechanism of protein reactivation and interaction with the DnaK system remains unclear. ClpB possesses two nucleotide binding domains, which are essential for function and show a complex allosteric behavior. The role of the N-terminal domain that precedes the first nucleotide binding domain is largely unknown. We purified and characterized an N-terminal shortened ClpB variant (ClpBDeltaN; amino acids 140-854), which remained active in refolding assays with three different substrate proteins. In addition the N-terminal truncation did not significantly change the nucleotide binding affinities, the nucleotide-dependent oligomerization, and the allosteric behavior of the protein. In contrast casein binding and stimulation of the ATPase activity by kappa-casein were affected. These results suggest that the N-terminal domain is not essential for the chaperone function, does not influence the binding of nucleotides, and is not involved in the formation of intermolecular contacts. It contributes to the casein binding site of ClpB, but other substrate proteins do not necessarily interact with the N terminus. This indicates a substantial difference in the binding mode of kappa-casein that is often used as model substrate for ClpB and other possibly more suitable substrate proteins.     

Detection of a G/T base substitution in two bleak (Alburnus alburnus) populations by RAPD

A G/T substitution has been found in a 462 bp fragment produced by random PCR amplification (RAPD) of genomic DNA in bleak ( Alburnus alburnus ). Two bleak populations from the rivers Main and Danube were screened for the two variants by allele-specific PCR. The allele frequencies were 0.62 and 0.31 for variant T, and 0.38 and 0.69 for variant G in the river populations of Main and Danube, respectively. Allele frequencies differed significantly (P < 0.01).