Regeneration of a chimeric retina from single cells in vitro: cell-lineage-dependent formation of radial cell columns by segregated chick and quail cells

Summary We report here that similar to E6-chicken retinal cells, dissociated cells from 5.5-day-old (E5.5) quail retinae reaggregate in rotary culture, multiply about tenfold and reestablish histotypical areas. These cellular aggregates include all nuclear layers either with inversed or correct laminar polarity, depending on the local origin of the cells (called rosetted and laminar in-vitro-retinae (IVR), respectively; Layer and Willbold 1989). In combined cultures, chick and quail cells are evenly mixed only during the first two days of culture. Along with the assembly of single cells into rosettes and then into discrete laminae, sectors of chick and quail cells begin to segregate. They are delineated by borders running radially through all three nuclear layers. Thus, interspecies migration of cells at this advanced stage of differentiation is strongly inhibited. Concomitant with this segregation, coherent radial columns spanning all three layers but containing cells from either species only, can be traced histologically. We conclude that a weak segregation of chick and quail retinal cells takes place already at the single cell level, but that the permanent segregation of entire tissue parts must be due to clonal cellular proliferation within the IVR in conjunction with some developmental-structural mechanism retaining clonal progenies within a columnar order.Abbreviations ECM extracellular matrix - E5.5 days of embryonic age - GCL ganglion cell layer - GC's ganglion cells - i.c. in culture - INL inner nuclear layer - rosetted in-vitro-retina retinal cell organoid aggregated from single cells of the central retina - IPL inner plexiform layer - MRE marginal retinal epithelium - ONL outer nuclear layer - OPL outer plexiform layer - OS ora serrate - PR photoreceptor cell - laminated in-vitro-retina fully laminated retinal cellorganoid resembling an E15-retina aggregated from cells of the eye periphery including RPE - RPE retinal pigment epithelium     

Isolation of a crotoxin-like protein from the venom of a South American rattlesnake (Crotalus durissus collilineatus)

1. A crotoxin-like protein was isolated from the venom of a South American rattlesnake Crotalus durissus collilineatus. 2. Many of its properties are similar to those of crotoxin, including its non-covalent heterodimeric structure, electrophoretic mobility on SDS-PAGE, isoelectric focusing properties, toxicity in mice, immunological reactivity, multiple isoforms, phospholipase activity, peptide map, and instability on an anion-exchange column. 3. Results indicate that "collilineatus toxin" is strongly homologous with crotoxin, found in the venom of Crotalus durissus terrificus, and all other characterized rattlesnake neurotoxins.     

The postsynaptic 43K protein clusters muscle nicotinic acetylcholine receptors in Xenopus oocytes.

Nicotinic acetylcholine receptors (AChRs) are localized at high concentrations in the postsynaptic membrane of the neuromuscular junction. A peripheral membrane protein of Mr 43,000 (43K protein) is closely associated with AChRs and has been proposed to anchor receptors at postsynaptic sites. We have used the Xenopus oocyte expression system to test the idea that the 43K protein clusters AChRs. Mouse muscle AChRs expressed in oocytes after injection of RNA encoding receptor subunits are uniformly distributed in the surface membrane. Coinjection of AChR RNA and RNA encoding the mouse muscle 43K protein causes AChRs to form clusters of 0.5-1.5 microns diameter. AChR clustering is not a consequence of increased receptor expression in the surface membrane or nonspecific clustering of all membrane proteins. The 43K protein is colocalized with AChRs in clusters when the two proteins are expressed together and forms clusters of similar size even in the absence of AChRs. These results provide direct evidence that the 43K protein causes clustering of AChRs and suggest that regulation of 43K protein clustering may be a key step in neuromuscular synaptogenesis.     

Aminopyridazine derivatives and TXA2-PGI2 balance

Experiments carried out under the conditions adopted showed the strong affinity of aminopyridazine derivatives for the eicosanoids TXA2 and PGI2. But this affinity depended on the chemical structure of the molecule: a small change in the radical grafted onto the pyridazine ring could completely modify the pharmacological activity of the molecule. Consequently it should be possible to control the properties of pyridazine derivatives according to pharmacological needs. Thus: --pyridazin-3-one derivatives were mainly active on TXA2 biosynthesis: 2-aminoalkyl 5-arylidene 6-methyl (4H) pyridazin-3-ones inhibited the TXA2-synthesizing activity of cardiac tissue whereas 3-amino 4,6-diaryl pyridazin-3-ones were specific inhibitors of the TXA2 synthetase in vitro, but these effects were weak. --pyridazine derivatives were devoid of any effect on the TXA2-synthesizing activity of cardiac tissue: they acted on either TXA2 synthetase or PGI2 synthetase according to the radicals grafted onto the pyridazine ring. --none of the compounds under study was active on the PGI2-synthesizing activity of cardiac tissue.     

Comparative effects of some 3-amino 4,6-diarylpyridazines on the biosynthesis in vitro of TXA2 and PGI2- and on the TXA2- and PGI2-synthesizing activities of cardiac tissue

Some 3-amino 4,6-diarylpyridazine derivatives were tested for their effects on TXA2 and PGI2 biosyntheses in vitro and on the TXA2- and PGI2-synthesizing activities of cardiac tissue. Horse platelet and aorta microsomes were used as sources of thromboxane and prostacyclin synthetases respectively. The TXA2- and PGI2-synthesizing activities of cardiac tissue were studied on isolated perfused rabbit hearts (the heart microsomes being used both as TXA2 synthetase and PGI2 synthetase sources). TXB2 and 6-keto PGF1 alpha were determined by RIA. Among the compounds under study, 3-morpholino 4,6-diphenylpyridazine (III) was shown to inhibit specifically the TXA2 synthetase. Substitution of the morpholino group by a dimethylamino one (I) reinforced the inhibiting effects on TXA2 synthetase but it also revealed a slight anti-prostacyclin synthetase action of the molecule. Replacement of 3-morpholino moieties by either a 3-hydrazino (IV), or a 2-dimethylaminoethylamino (V), or a 2-morpholinoethylamino group (VI) abolished completely the effects of the molecule on TXA2 and PGI2 synthetases. Likewise the addition of chlorine on the para-position on the phenyl ring of I neutralized all its inhibitory effects both on TXA2 and PGI2 synthetases in vitro. None of the 3-amino 4,6-diarylpyridazine derivatives was active on either the TXA2- or PGI2-synthesizing activities of cardiac tissue.     

Synthesis and biological evaluation of pNPY fragments

Peptide fragments of pNPY corresponding to the C-terminal segments (13-36) and (25-36), the N-terminal segments (1-12) and (1-24), the segments (6-14) and (7-20), which contain a putative beta-turn, and the internal segments (13-24) and (20-30) were synthesized using solid phase methodology. These fragments were assayed for NPY receptor binding activity in the rat hypothalamus membrane preparation, enhancement of food intake in the rat following ivt administration and inhibition of electrically stimulated muscle contraction in the rat vas deferens. Only the C-terminal fragment (13-36) retained some of the activities of pNPY, appearing to act as a weak agonist, having an additive effect with pNPY on the inhibition of muscle contraction and prolonging the duration of action of pNPY in the feeding assay. It also had considerable alpha-helical character, as did pNPY. None of the other peptide fragments had any agonist or antagonist activity. These results suggest that the expression of full biological NPY activity requires both the C- and the N-terminal segments as well as a putative amphiphilic alpha-helical segment (14-31).     

The contribution of olfactory receptor neurons to the perception of pheromone component ratios in male redbanded leafroller moths

Summary (Z)-11-tetradecenyl acetate (Z-11, 14:AC) must be in a 1009 ratio with (E)-11-tetradecenyl acetate (E-11,14:AC) to produce maximal wing fanning and attraction in male redbanded leafrollers. Earlier electrophysiological studies had indicated that mixtures of these pheromone components elicited responses from olfactory receptor neurons that appeared to differ from those expected on the basis of the responses to the individual components. Here we evaluate whether the behavioral sensitivity to particular ratios of Z- and E-11,14:AC has a correlate in the response properties of olfactory receptor neurons.The stimuli included the ratios of Z- and E-11, 14:AC used in earlier behavioral work plus several different mixtures of the seven components found in the pheromone blend, and equivalent amounts of the individual components. These stimuli were presented over a range of intensities to individual trichoid sensilla on the male antenna. In common with earlier results, the receptor neuron with the larger amplitude action potential responded most strongly to Z-11,14:AC, whereas the companion receptor neuron in the sensillum responded most strongly to E-11,14:AC. In contrast with earlier results, each receptor neuron responded exclusively to its own most effective stimulus, without regard to the presence of any other compound. They failed to respond uniquely to any of the other five compounds in the female pheromone blend, or to any of the tested combinations of these compounds. These minor components also failed to modulate the responses elicited in receptor neurons by appropriate ratios of Z- and E-11,14:AC. Thus, the responses of the two types of olfactory receptor neurons found in trichoid sensilla failed to show an optimum at the pheromone ratio known to elicit peak behavioral activity.Abbreviation RBLR redbanded leafroller moth     

Cross-Homologies and Structural Differences Between Human Cholinesterases Revealed by Antibodies Against cDNA-Produced Human Butyrylcholinesterase Peptides

To study the polymorphism of human cholinesterases (ChEs) at the levels of primary sequence and three-dimensional structure, a fragment of human butyrylcholinesterase (BuChE) cDNA was subcloned into the pEX bacterial expression vector and its polypeptide product analyzed. Immunoblot analysis revealed that the clone-produced BuChE peptides interact specifically with antibodies against human and Torpedo acetylcholinesterase (AChE). Rabbit polyclonal antibodies prepared against the purified clone-produced BuChE polypeptides interacted in immunoblots with denatured serum BuChE as well as with purified and denatured erythrocyte AChE. In contrast, native BuChE tetramers from human serum, but not AChE dimers from erythrocytes, interacted with these antibodies in solution to produce antibody-enzyme complexes that could be precipitated by second antibodies and that sedimented faster than the native enzyme in sucrose gradient centrifugation. Furthermore, both AChE and BuChE dimers from muscle extracts, but not BuChE tetramers from muscle, interacted with these antibodies. To reveal further whether the anti-cloned BuChE antibodies would interact in situ with ChEs in the neuromuscular junction, bundles of muscle fibers were microscopically dissected from the region in fetal human diaphragm that is innervated by the phrenic nerve. Muscle fibers incubated with the antibodies and with 125I-Protein A were subjected to emulsion autoradiography, followed by cytochemical ChE staining. The anti-cloned BuChE antibodies, as well as anti-Torpedo AChE antibodies, created patches of silver grains in the muscle endplate region stained for ChE, under conditions where control sera did not. These findings demonstrate that the various forms of human AChE and BuChE in blood and in neuromuscular junctions share sequence homologies, but also display structural differences between distinct molecular forms within particular tissues, as well as between similarly sedimenting molecular forms from different tissues.