Kurze Mitteilungen

Zusammenfassung Die Schlußfolgerungen, dieStegmann (J. Orn. 109, 1968, p. 441–445) für die verwandtschaftliche Stellung der Flughühner auf Grund seiner eigenen Untersuchungen und an Hand anderer Quellen zieht, werden kommentiert. Es wird gezeigt, daß einige dieser Schlußfolgerungen nur auf morphologischen Merkmalen beruhen und mit den zu anderen Resultaten gelangten ethologischen und physiologischen Untersuchungsergebnissen nicht zu vereinbaren sind. Außerdem erscheint es unwahrscheinlich, daß die Flughühner, deren Ursprung von bodenlebenden Nestflüchtern unbestritten ist, auf dem Umweg über die baum- und felsbewohnenden nesthockenden Tauben erst sekundär wieder zu bodenbewohnenden Nestflüchtern geworden sind. Es gibt kein einziges Flughuhn-Merkmal, das diese Hypothese stützen könnte.     

Methoden zum Solaninnachweis und zur Solaninbestimmung in Kartoffelzuchtmaterial

Zusammenfassung Es werden Methoden zum Nachweis und zur Bestimmung des Solanins in Kartoffelzuchtmaterial beschrieben. Mit den Nachweismethoden lassen sich Klone mit hohem, mittlerem und niedrigem Solaningehalt in Knollen und Blättern erkennen, die quantitativen Methoden gestatten eine genaue Bestimmung des Solaningehaltes in den Eliten und anderem Zuchtmaterial. Alle Verfahren, insbesondere die des Nachweises in Knollen und Blättern, eignen sich für Serienuntersuchungen und können für die Einengung des Zuchtmaterials vor der Geschmacksprüfung durch Ausscheidung solaninreicher, stark bitterer Klone herangezogen werden.Bei den qualitativen und quantitativen Methoden der Knollenuntersuchung wird von Knollensaft —durch Auspressen oder Zentrifugieren gewonnen —bzw. essigsauren Extrakten ausgegangen, bei den Blattuntersuchungen von Extrakten aus Blatt-trockensubstanz mit einem Gemisch von Essigsäure und Essigsäureäthylester. Den Nachweismethoden liegt die auf dem Papier ausgeführte Farbreaktion mit Antimontrichlorid, den Bestimmungsmethoden die Reaktion mit Paraformaldehyd-Phosphorsäure, die photometrisch ausgewertet wird, zugrunde.Brauchbarkeit und Grenzen der beschriebenen Ausleseverfahren werden an Hand von Ergebnissen diskutiert.Mit 5 AbbildungenHerrn Prof. Dr. Dr.H. Stubbe zum 60. Geburtstag.     

OX40 ligand and programmed cell death 1 ligand 2 expression on inflammatory dendritic cells regulates CD4 T cell cytokine production in the lung during viral disease

CD4 Th differentiation is influenced by costimulatory molecules expressed on conventional dendritic cells (DCs) in regional lymph nodes and results in specific patterns of cytokine production. However, the function of costimulatory molecules on inflammatory (CD11b(+)) DCs in the lung during recall responses is not fully understood, but it is important for development of novel interventions to limit immunopathological responses to infection. Using a mouse model in which vaccination with vaccinia virus vectors expressing the respiratory syncytial virus (RSV) fusion protein (rVVF) or attachment protein (rVVG) leads to type 1- or type 2-biased cytokine responses, respectively, upon RSV challenge, we found expression of CD40 and OX40 ligand (OX40L) on lung inflammatory DCs was higher in rVVF-primed mice than in rVVG-primed mice early after RSV challenge, whereas the reverse was observed later in the response. Conversely, programmed cell death 1 ligand 2 (PD-L2) was higher in rVVG-primed mice throughout. Inflammatory DCs isolated at the resolution of inflammation revealed that OX40L on type 1-biased DCs promoted IL-5, whereas OX40L on type 2-biased DCs enhanced IFN-γ production by Ag-reactive Th cells. In contrast, PD-L2 promoted IFN-γ production, irrespective of conditions, suppressing IL-5 only if expressed on type 1-biased DCs. Thus, OX40L and PD-L2 expressed on DCs differentially regulate cytokine production during recall responses in the lung. Manipulation of these costimulatory pathways may provide a novel approach to controlling pulmonary inflammatory responses.     

Radial basis function neural networks for modeling growth rates of the basidiomycetes Physisporinus vitreus and Neolentinus lepideus

A radial basis function (RBF) neural network was developed and compared against a quadratic response surface (RS) model for predicting the specific growth rates of the biotechnologically important basidiomycetous fungi, Physisporinus vitreus and Neolentinus lepideus, under three environmental conditions: temperature (10–30 °C), water activity (0.950–9.998), and pH (4–6). Both the RBF network and polynomial RS model were mathematically evaluated against experimental data using graphical plots and several statistical indices. The evaluation showed that both models gave reasonably good predictions, but the performance of the RBF neural network was superior to that of the classical statistical method for all three data sets used (training, testing, full). Sensitivity analysis revealed that of the three experimental factors the most influential on the growth rate of P. vitreus was water activity, followed by temperature and pH to a lesser extent. In contrast, temperature in particular and then water activity were the key determinants of the development of N. lepideus. RBF neural networks could be a powerful technique for modeling fungal growth behavior under certain parameters and an alternative to time-consuming, traditional microbiological techniques.     

Dosing of growth hormone in growth hormone deficiency

Growth hormone (GH) treatment of GH-deficient (GHD) children is to a certain extent standardized worldwide. Recombinant 22 kDa GH is injected once daily by the subcutaneous route, mostly in the evening. The amount of GH injected (calculated per kg body weight or body surface area, expressed in terms of IU or mg) in prepubertal children mimics the known production rate (approximately 0.02 mg [0. 06 IU]/kg body weight per day). However, there is a wide variation in dosage, the reasons for which are partly unknown and partly due to national traditions and regimes imposed by authorities regulating reimbursement. The situation during puberty is less standardized, with most clinicians still not increasing the dosage according to known production rates. The results of these approaches in terms of adult height outcome are not always satisfactory. In order to achieve optimal height development during childhood, puberty and adulthood, strategies must be developed to individualize GH dosing according to set therapeutical goals taking into account efficacy, safety and cost. The implementation of prediction algorithms will help us to reach these goals. In addition, other response variables will have to be monitored during treatment in order to correct for deficits resulting from GHD.     

Rare autosomal recessive cardiac valvular form of Ehlers-Danlos syndrome results from mutations in the COL1A2 gene that activate the nonsense-mediated RNA decay pathway

Splice site mutations in the COL1A2 gene of type I collagen can give rise to forms of Ehlers-Danlos syndrome (EDS) because of partial or complete skipping of exon 6, as well as to mild, moderate, or lethal forms of osteogenesis imperfecta as a consequence of skipping of other exons. We identified three unrelated individuals with a rare recessively inherited form of EDS (characterized by joint hypermobility, skin hyperextensibility, and cardiac valvular defects); in two of them, COL1A2 messenger RNA (mRNA) instability results from compound heterozygosity for splice site mutations in the COL1A2 gene, and, in the third, it results from homozygosity for a nonsense codon. The splice site mutations led to use of cryptic splice donor sites, creation of a downstream premature termination codon, and extremely unstable mRNA. In the wild-type allele, the two introns (IVS11 and IVS24) in which these mutations occurred were usually spliced slowly in relation to their respective immediate upstream introns. In the mutant alleles, the upstream intron was removed, so that exon skipping could not occur. In the context of the mutation in IVS24, computer-generated folding of a short stretch of mRNA surrounding the mutation site demonstrated realignment of the relationships between the donor and acceptor sites that could facilitate use of a cryptic donor site. These findings suggest that the order of intron removal is an important variable in prediction of mutation outcome at splice sites and that folding of the nascent mRNA could be one element that contributes to determination of order of splicing. The complete absence of pro alpha 2(I) chains has the surprising effect of producing cardiac valvular disease without bone involvement.     

Mechanisms involved in the induction of apoptosis by T-2 and HT-2 toxins in HL-60 human promyelocytic leukemia cells

T-2 and HT-2 toxins belong to a group of mycotoxins that are widely encountered as natural contaminants known to elicit toxic responses in hematopoietic cells. In the present study, HL-60 cells were used to characterize the apoptotic effects of T-2 and a major metabolite, HT-2, and to examine the mechanisms involved. Apoptotic cells were identified microscopically by chromatin condensation and nuclear fragmentation, by flow cytometric analysis, and by DNA gel electrophoresis. T-2 and HT-2 induced concentration-dependent apoptosis after 24 h in HL-60 cells, starting at concentrations of 3.1 and 6.25 ng/ml respectively. An increased number of apoptotic cells could be observed 4–6 h after exposure to 12.5 ng/ml of toxin. Little cytotoxicity (plasma membrane damage) was observed even after exposure to concentrations of toxins (25–50 ng/ml) inducing apoptosis in 60–100% of the cells. The apoptotic process was almost completely blocked in the presence of the general caspase inhibitor zVAD.fmk. In contrast, no or only minor effects were observed with the more specific caspase inhibitors DEVD.CHO, IETD.fmk, and DEVD.fmk. As judged by Western blotting, the levels of several procaspases (-3, -7, -8, -9, but not -12) were reduced 3–6 h after exposure to toxin. Substantial increases in the presumed active form(s) of caspase-8 and -9 were observed. Furthermore, poly(ADP-ribose) polymerase (PARP) was already markedly cleaved 3 h after toxin treatment, indicative of active caspase-3 and -7. No or only minor changes in Bcl-2, Bcl-XL and Bax levels were observed. BAPTA-AM and ZnCl₂ blocked the degradation of procaspases, the fragmentation of PARP, and the induction of apoptosis. In summary, both T-2 and HT-2 induced apoptosis, with T-2 being somewhat more potent than HT-2. The divalent calcium concentration, [Ca²⁺], appears to be involved in the activation of several caspases, resulting in DNA fragmentation, chromosomal condensation, and nuclear fragmentation.     

Analysis of ligand-stimulated CC chemokine receptor 5 (CCR5) phosphorylation in intact cells using phosphosite-specific antibodies

Human CC chemokine receptor 5 (CCR5), a member of the superfamily of G protein-coupled receptors, regulates the activation and directed migration of leukocytes and serves as the main coreceptor for the entry of R5 tropic strains of human immunodeficiency viruses. We have previously shown that RANTES/CCL5 binding to CCR5 induces GPCR kinase (GRK)- and protein kinase C (PKC)-mediated phosphorylation of four distinct C-terminal serine residues. To study these phosphorylation events in vivo, we have generated monoclonal antibodies, which specifically react only with either phosphorylated or nonphosphorylated CCR5. These phosphosite-specific antibodies reveal that following ligand stimulation of the receptor serine 337 is exclusively phosphorylated by a PKC-mediated mechanism, while GRKs phosphorylate serine 349. GRK-mediated receptor phosphorylation proceeds in a regular time-dependent manner (t(12) approximately 2 min) with an apparent EC(50) of 5 nm. In contrast, PKC phosphorylates serine 337 at 50-fold lower concentrations and in a very rapid, albeit transient manner. Protein phosphatases that are active at neutral pH and are inhibited by okadaic acid rapidly dephosphorylate phosphoserine 337, but less efficiently phosphoserine 349, in intact cells and in an in vitro assay. Immunofluorescence microscopy demonstrates that phosphorylated receptors accumulate in a perinuclear compartment, which resembles recycling endosomes. This study is the first to analyze in detail the spatial and temporal dynamics of GRK- versus PKC-mediated phosphorylation of a G protein-coupled receptor and its subsequent dephosphorylation on the level of individual phosphorylation sites.