Phosphatidylinositol 3-kinase interacts with the adaptor protein Dab1 in response to Reelin signaling and is required for normal cortical lamination

Reelin is a large secreted signaling protein that binds to two members of the low density lipoprotein receptor family, the apolipoprotein E receptor 2 and the very low density lipoprotein receptor, and regulates neuronal positioning during brain development. Reelin signaling requires activation of Src family kinases as well as tyrosine phosphorylation of the intracellular adaptor protein Disabled-1 (Dab1). This results in activation of phosphatidylinositol 3-kinase (PI3K), the serine/threonine kinase Akt, and the inhibition of glycogen synthase kinase 3beta, a protein that is implicated in the regulation of axonal transport. Here we demonstrate that PI3K activation by Reelin requires Src family kinase activity and depends on the Reelin-triggered interaction of Dab1 with the PI3K regulatory subunit p85alpha. Because the Dab1 phosphotyrosine binding domain can interact simultaneously with membrane lipids and with the intracellular domains of apolipoprotein E receptor 2 and very low density lipoprotein receptor, Dab1 is preferentially recruited to the neuronal plasma membrane, where it is phosphorylated. Efficient Dab1 phosphorylation and activation of the Reelin signaling cascade is impaired by cholesterol depletion of the plasma membrane. Using a neuronal migration assay, we also show that PI3K signaling is required for the formation of a normal cortical plate, a step that is dependent upon Reelin signaling.     

Erythropoietin therapy for acute stroke is both safe and beneficial

BACKGROUND: Erythropoietin (EPO) and its receptor play a major role in embryonic brain, are weakly expressed in normal postnatal/adult brain and up-regulated upon metabolic stress. EPO protects neurons from hypoxic/ ischemic injury. The objective of this trial is to study the safety and efficacy of recombinant human EPO (rhEPO) for treatment of ischemic stroke in man. MATERIALS AND METHODS: The trial consisted of a safety part and an efficacy part. In the safety study, 13 patients received rhEPO intravenously (3.3 X 10(4) IU/50 ml/30 min) once daily for the first 3 days after stroke. In the double-blind randomized proof-of-concept trial, 40 patients received either rhEPO or saline. Inclusion criteria were age <80 years, ischemic stroke within the middle cerebral artery territory confirmed by diffusion-weighted MRI, symptom onset <8 hr before drug administration, and deficits on stroke scales. The study endpoints were functional outcome at day 30 (Barthel Index, modified Rankin scale), NIH and Scandinavian stroke scales, evolution of infarct size (sequential MRI evaluation using diffusion-weighted [DWI] and fluid-attenuated inversion recovery sequences [FLAIR]) and the damage marker S100ss. RESULTS: No safety concerns were identified. Cerebrospinal fluid EPO increased to 60-100 times that of nontreated patients, proving that intravenously administered rhEPO reaches the brain. In the efficacy trial, patients received rhEPO within 5 hr of onset of symptoms (median, range 2:40-7:55). Admission neurologic scores and serum S100beta concentrations were strong predictors ofoutcome. Analysis of covariance controlled for these two variables indicated that rhEPO treatment was associated with an improvement in follow-up and outcome scales. A strong trend for reduction in infarct size in rhEPO patients as compared to controls was observed by MRI. CONCLUSION: Intravenous high-dose rhEPO is well tolerated in acute ischemic stroke and associated with an improvement in clinical outcome at 1 month. A larger scale clinical trial is warranted.     

Effect of bradykinin analogues on the B1 receptor of rat ileum

The longitudinal muscle of isolated rat ileum is a sensitive bioassay suitable for testing compounds with antagonistic effects on the B(1) receptor. Bradykinin analogues with replacement of proline by alkyl-substituted phenylalanine at position 7 are effective on this receptor as entire molecules and have a stronger antagonistic effect than on the B(2) receptor. A corresponding desArg(9)-compound has a specific effect on the B(1) receptor and a very high antagonistic potency. [LNMPhe(2)]bradykinin as a compound without any replacement at position 7 or 8 shows antagonistic activity as well.     

Malaria and World War II: German malaria experiments 1939-45

The epidemiological and pharmacological fight against malaria and German malaria research during the Nazi dictatorship were completely under the spell of war. The Oberkommando des Heeres (German supreme command of the army) suffered the bitter experience of unexpected high losses caused by malaria especially at the Greek front (Metaxes line) but also in southern Russia and in the Ukraine. Hastily raised anti-malaria units tried to teach soldiers how to use the synthetic malaria drugs (Plasmochine, Atebrine) properly. Overdoses of these drugs were numerous during the first half of the war whereas in the second half it soon became clear that it would not be possible to support the army due to insufficient quantities of plasmochine and atebrine. During both running fights and troop withdrawals at all southern and southeastern fronts there was hardly any malaria prophylaxis or treatment. After war and captivity many soldiers returned home to endure heavy malaria attacks. In German industrial (Bayer, IG-Farben) and military malaria laboratories of the Heeres-Sanit?ts-Akademie (Army Medical Academy) the situation was characterised by a hasty search for proper dosages of anti-malaria drugs, adequate mechanical and chemical prophylaxis (Petroleum, DDT, and other insecticides) as well as an anti-malaria vaccine. Most importantly, large scale research for proper atebrine and plasmochine dosages was conducted in German concentration camps and mental homes. In Dachau Professor Claus Schilling tested synthetic malaria drugs and injected helpless prisoners with high and sometimes lethal doses. Since the 1920s he had been furiously looking for an anti-malaria vaccine in Italian mental homes and from 1939 he continued his experiments in Dachau. Similar experiments were also performed in Buchenwald and in a psychiatric clinic in Thuringia, where Professor Gerhard Rose tested malaria drugs with mentally ill Russian prisoners of war. Schilling was put to death for his criminal research in 1946, Rose was condemned to lifelong imprisonment in 1947, though, not for his malaria research but for his dreadful experiments with epidemic typhus sera which he also had performed in concentration camps and with prisoners of war in Russia.     

Transduction and transfection of difficult-to-transfect cells: Systematic attempts for the transfection of protozoa Leishmania

Cell-penetrating peptides (CPPs) are used to internalize different cargoes, including DNA, into live mammalian and plant cells. Despite many cells being easily transfected with this approach, other cells are rather “difficult” or “hard to transfect,” including protist cells of the genus Leishmania. Based on our previous results in successfully internalizing proteins into Leishmania tarentolae cells, we used single CPPs and three different DNA-binding proteins to form protein-like complexes with plasmids covered with CPPs. We attempted magnetofection, electroporation, and transfection using a number of commercially available detergents. While complex formation with negatively charged DNA required substantially higher amounts of CPPs than those necessary for mostly neutral proteins, the cytotoxicity of the required amounts of CPPs and auxiliaries was thoroughly studied. We found that Leishmania cells were indeed susceptible to high concentrations of some CPPs and auxiliaries, although in a different manner compared with that for mammalian cells. The lack of successful transfections implies the necessity to accept certain general limitations regarding DNA internalization into difficult-to-transfect cells. Only electroporation allowed reproducible internalization of large and rigid plasmid DNA molecules through electrically disturbed extended membrane areas, known as permeable membrane macrodomains.     

Ipangulines and minalobines, chemotaxonomic markers of the infrageneric Ipomoea taxon subgenus Quamoclit, section Mina

A comprehensive GC-MS analysis of 8 Ipomoea species belonging to the subgenus Quamoclit, section Mina revealed that the members of this taxon form combinations of two necine bases with rare necic acids resulting in unique pyrrolizidine alkaloids. The occurrence and diversity of these metabolites show remarkable variations: Some species, especially Ipomoea hederifolia and Ipomoea lobata, are able to synthesize a large number of alkaloids whereas others, especially Ipomoea coccinea and Ipomoea quamoclit, are poor synthesizers with only a few compounds. However, these metabolites are apparently chemotaxonomic markers of this infrageneric taxon in general. They represent either esters of (-)-platynecine (altogether 48 ipangulines and 4 further esters including results of a previous study) or esters of (-)-trachelanthamidine, an additional novel structural type called minalobines (altogether 21 alkaloids). Both types are characterized by section-specific rare necic acids, e.g., ipangulinic/isoipangulinic acid, phenylacetic acid. The alkaloids of Ipomoea cholulensis, I. coccinea, I. hederifolia, Ipomoea neei, and Ipomoea quamoclit were mono and diesters of platynecine. Minalobines turned out to be metabolites of I. lobata (Cerv.) Thell. (syn.: Mina lobata Cerv.) lacking ipangulines. The major alkaloid of this species, minalobine R, has been isolated and identified as 9-O-(threo-2-hydroxy-2-methyl-3-phenylacetoxy-butyryl)-(-)-trachelanthamidine on the basis of spectral data. Apparently only two of the species included in this study, Ipomoea cristulata and Ipomoea sloteri, are able to synthesize both, ipangulines as well as minalobines. Minalobine O could be isolated as a major alkaloid of I. cristulata, its structure has been established as 9-O-(erythro-2-hydroxy-2-methyl-3-tigloyloxy-butyryl)-(-)-trachelanthamidine on the basis of spectral data.     

Brevican isoforms associate with neural membranes

Brevican is a neural-specific proteoglycan of the brain extracellular matrix, which is particularly abundant in the terminally differentiated CNS. It is expressed by neuronal and glial cells, and as a component of the perineuronal nets it decorates the surface of large neuronal somata and primary dendrites. One brevican isoform harbors a glycosylphosphatidylinositol anchor attachment site and, as shown by ethanolamine incorporation studies, is indeed glypiated in stably transfected HEK293 cells as well as in oligodendrocyte precursor Oli-neu cells. The major isoform is secreted into the extracellular space, although a significant amount appears to be tightly attached to the cell membrane, as it floats up in sucrose gradients. Flotation is sensitive to detergent treatment. Brevican is most prominent in the microsomal, light membrane and synaptosomal fractions of rat brain membrane preparations. The association with the particulate fraction is in part sensitive to chondroitinase ABC and phosphatidylinositol-specific phospholipase C treatment. Furthermore, brevican staining on the surface of hippocampal neurons in culture is diminished after hyaluronidase or chondroitinase ABC treatment. Taken together, this could provide a mechanism by which perineuronal nets are anchored on neuronal surfaces.     

Endo‐ and epilithic faunal succession in a Pliocene–Pleistocene cave on Rhodes,Greece: record of a transgression

A fossil cave and associated sediments and fossil fauna located on the Greek island of Rhodes in the eastern Aegean Sea is reported here, and the depositional history discussed. The sediments were deposited during the late Pliocene, in the interstitial space between basement boulders of up to 1500 tons. The depositional history of the cave comprises eight stages. From initial flooding, the basin experienced a continuous transgression with sea‐level rise in excess of 500 m, followed by a rapid, forced regression of similar magnitude. The recognition of a succession of fossil communities illustrates this transgression, with a seemingly abrupt shift from endolithic to epilithic biota dominance late in the transgressive cycle. The communities recording the increasing water depth from 0 to >150 m are: The Gatrochaenolithes torpedo (bivalve boring) and Entobia gonioides (sponge boring) ichnocoenosis, with peak distribution between 0 and 1 m water depth; the E. gonioides – E. magna ichnocoenosis, with 1–5 m depth peak distribution; the exclusive E. magna ichnocoenosis, with 5–40 m depth peak distribution; and the E. gigantea ichnocoenosis, with a peak distribution approaching 150–200 m. Below this depth, an epilithic community without boring organisms takes over, characterized by the calcareous sponge Merlia cf. normani, and the inarticulate brachiopod Novocrania turbinata. Simultaneously with the succession of the endo‐ and epilithic cave wall fossil communities, skeletal calcarenite accumulated on the cave floor; the erosional remnants of this sediment are insufficient to further expand the overall transgression–regression model.     

4-amidinobenzylamine-based inhibitors of urokinase

A series of 4-amidinobenzylamine-based peptidomimetic inhibitors of urokinase was synthesized. The most potent one, benzylsulfonyl-D-Ser-Ala-4-amidinobenzylamide 16, inhibits uPA with a K(i) of 7.7 nM but is less selective than 10 with a Gly as P2 residue. Hydroxyamidine and carbonate prodrugs were prepared, which are rapidly converted into the active inhibitors in rats after subcutaneous application.