Effect of ionizing radiation on artificial (planar) lipid membranes. II. The ion carriers valinomycin and nonactin as probes for radiation induced structural changes of the membrane

Planar lipid membranes in the presence of the ion carriers valinomycin or nonactin were irradiated with 14 MeV electrons from a linear accelerator. A large increase of the membrane conductance by up to more than two orders of magnitude was found. The effect is virtually abolished either at high pH, or in the absence of oxygen, or in the presence of the radical scavenger ethanol. A further prerequisite for the effect is the presence of unsaturated fatty acid residues. A kinetic analysis of the carrier transport model based on current-voltage curves and on voltage-jump relaxation experiments was performed as a function of radiation dose. Only the translocation rate constant, kMS, of the charged carrier-ion complex was found to be influenced by irradiation. The effect is interpreted as an increase of the polarity (dielectric constant) of the membrane interior induced by the presence of polar products of lipid peroxidation. A combined action of OH- and HO2-radicals seems to be responsible for the phenomena. At large radiation doses (greater than or equal to 10(3) Gy) a reduction of the membrane conductance was observed. This is interpreted as an increased microviscosity, possibly caused by cross-linking of fatty acid residues. Ion carriers represent sensitive probes of radiation induced membrane damage.     

Evolution of the Larval Cuticle Proteins Coded by the Secondary Sex Chromosome Pair: X2 and Neo-Y of Drosophila miranda: I. Comparison at the DNA Sequence Level

The larval cuticle protein genes (Lcps) represent a multigene family located at the right arm of the metacentric autosome 2 (2R) in Drosophila melanogaster. Due to a chromosome fusion the Lcp locus of Drosophila miranda is situated on a pair of secondary sex chromosomes, the X2 and neo-Y chromosome. Comparing the DNA sequences from D. miranda and D. melanogaster organization and the gene arrangement of Lcp1–Lcp4 are similar, although the intergene distances vary considerably. The greatest difference between Lcp1 and Lcp2 is due to the occurrence of a pseudogene in D. melanogaster which is not present in D. miranda. Thus the cluster of the four Lcp genes existed already before the separation of the melanogaster and obscura group. Intraspecific homogenizations of different cluster units must have occurred repeatedly between the Lcp1/Lcp2 and Lcp3/Lcp4 sequence types. The most obvious example is exon 2 of the Lcp3 gene in D. miranda, which has been substituted by the corresponding section of the Lcp4 gene rather recently. The homogenization must have occurred before the translocation which generated the neo-Y chromosome. Lcp3 of D. melanogaster has therefore no orthologous partner in D. miranda. Rearrangements in the promoter regions of the D. miranda Lcp genes have generated new, potentially functional CAAT-box motifs. Since three of the Lcp alleles on the neo-Y are not expressed and Lcp3 is expressed only at a reduced level, it is suggestive to speculate that the rearrangements might be involved as cis-regulatory elements in the up-regulation of the X2-chromosomal Lcp alleles, in Drosophila an essential process for dosage compensation. The Lcp genes on the neo-Y chromosome have accumulated more base substitutions than the corresponding alleles on the X2. Received: 27 December 1995 / Accepted: 30 April 1996     

Integrin-linked kinase controls microtubule dynamics required for plasma membrane targeting of caveolae

Caveolae are specialized compartments of the plasma membrane that are involved in signaling, endocytosis, and cholesterol transport. Their formation requires the transport of caveolin-1 to the plasma membrane, but the molecular mechanisms regulating the transport are largely unknown. Here, we?identify a critical role for adhesion-mediated signaling through β1 integrins and integrin-linked kinase (ILK) in caveolae formation. Mice lacking β1 integrins or ILK in keratinocytes have dramatically reduced numbers of plasma membrane caveolae in?vivo, which is due to impaired transport of caveolin-1-containing vesicles along microtubules (MT) to the plasma membrane. Mechanistically, ILK promotes the recruitment of the F-actin binding protein IQGAP1 to the cell cortex, which, in turn, cooperates with its?effector mDia1 to locally stabilize MTs and to allow?stable insertion of caveolae into the plasma membrane. Our results assign an important role to the integrin/ILK complex for caveolar trafficking to the cell surface.     

Synthesis and SAR of highly potent and selective dopamine D3-receptor antagonists: variations on the 1H-pyrimidin-2-one theme

In our efforts to further pursue one of the most selective dopamine D(3)-receptor antagonists reported to date, we now describe the synthesis and SAR of novel and highly selective dopamine D(3) antagonists based on a 1H-pyridin-2-one or on a urea scaffold. The most potent compounds exhibited K(i) values toward the D(3) receptor in the nano- to subnanomolar range and high selectivity versus the related D(2) dopamine receptor. Thus, 1H-pyridin-2-one 7b displays oral bioavailability (F=37%) as well as brain penetration (brain plasma ratio 3.7) in rat. Within the urea series, an excellent D(3) versus D(2) selectivity (>100-fold) could be achieved by removal of one NH group (compound 6), although bioavailability (rat) was suboptimal (F<10%). These data significantly enhance our understanding of the D(3) pharmacophore and are expected to lead to novel approaches for the treatment of schizophrenia.     

Simulierung der Röntgenstrahlenwirkung durch freie Radikale

Zusammenfassung Die in einer Hochfrequenzentladung in Wasserdampf entstehenden OH-Radikale bewirken eine Oxydation von Eisen (II) und eine Reduktion von Ger (IV), während H-Atome aus der Wasserstoffentladung Hexacyanoferrat (III), Gold (III) und Silber (I) reduzieren. Diese Ergebnisse werden mit der Röntgenbestrahlung verglichen und die Reaktionsgleichungen diskutiert.Es gelingt so, die Wirkungen der OH-Radikale und H-Atome zu trennen, die bei Bestrahlung immer gemeinsam auftreten.Es ergibt sich, daß OH-Radikale stärker für die sekundären, chemischen Effekte der Bestrahlung verantwortlich sind als H-Atome.Herrn Prof. Dr.-Ing. H.Engelhard möchte ich für sein stets förderndes Interesse am Fortgang dieser Arbeit meinen herzlichen Dank aussprechen.Der Deutschen Forschungsgemeinschaft und dem Herrn Bundesminister für wissenschaftliche Forschung danke ich für die zur Verfügung gestellten Mittel.     

The Multi-PDZ domain protein MUPP1 as a lipid raft-associated scaffolding protein controlling the acrosome reaction in mammalian spermatozoa

The success of acrosomal exocytosis, a complex process with a variety of interrelated steps, relies on the coordinated interaction of participating signaling molecules. Since scaffolding proteins are known to spatially organize sequential signaling pathways, we examined whether the Multi-PDZ domain protein MUPP1, recently identified in mammalian spermatozoa, is functionally active in controlling acrosomal secretion in mammalian sperm cells. To address this question, permeabilized mouse sperm were loaded with inhibitory antibodies against MUPP1 as well as with a photosensitive Ca(2+) chelator which allows a controlled release of acrosomal Ca(2+). The results revealed that MUPP1 controls initial tethering and docking of the acrosomal vesicle, whereas syntaxin 2, a t-SNARE protein also expressed in the acrosomal cap of mammalian spermatozoa, appears to take part in the final process of acrosomal fusion. Interestingly, using immunogold electron microscopy, it was found that MUPP1 is detectable in the region of the periacrosomal membrane. Furthermore, in isolated detergent-insoluble glycolipid-enriched membrane domains from epididymal spermatozoa, MUPP1 was found to show a striking association with the Triton X-100 insoluble membrane fraction, which did not change significantly upon sperm capacitation or partial chemical extraction of cholesterol. This evidence points to a role of MUPP1 as a membrane raft-associated molecular organizer, and suggests that mammalian spermatozoa may use a scaffolding protein and distinct membrane subdomains to spatially organize components involved in the process of acrosomal exocytosis.     

An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice

We describe a transgenic mouse line, Pax8-rtTA, which, under control of the mouse Pax8 promoter, directs high levels of expression of the reverse tetracycline-dependent transactivator (rtTA) to all proximal and distal tubules and the entire collecting duct system of both embryonic and adult kidneys. Using crosses of Pax8-rtTA mice with tetracycline-responsive c-MYC mice, we established a new, inducible model of polycystic kidney disease that can mimic adult onset and that shows progression to renal malignant disease. When targeting the expression of transforming growth factor beta-1 to the kidney, we avoided early lethality by discontinuous treatment and successfully established an inducible model of renal fibrosis. Finally, a conditional knockout of the gene encoding tuberous sclerosis complex-1 was achieved, which resulted in the early outgrowth of giant polycystic kidneys reminiscent of autosomal recessive polycystic kidney disease. These experiments establish Pax8-rtTA mice as a powerful tool for modeling renal diseases in transgenic mice.