High-Throughput Screening Enhances Kidney Organoid Differentiation from Human Pluripotent Stem Cells and Enables Automated Multidimensional Phenotyping

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Tobamovirus evolution: gene overlaps, recombination, and taxonomic implications

Tobamoviruses, mostly isolated from solanaceous plants, may represent ancient virus lineages that have codiverged with their hosts. Recently completed nucleotide sequences of six nonsolanaceous tobamoviruses allowed assessment of the codivergence hypothesis and support a third subgroup within tobamoviruses. The genomic sequences of 12 tobamoviruses and the partial sequences of 11 others have been analyzed. Comparisons of the predicted protein sequences revealed three clusters of tobamoviruses, corresponding to those infecting solanaceous species (subgroup 1), those infecting cucurbits and legumes (subgroup 2), and those infecting crucifers. The orchid-infecting odontoglossum ringspot tobamovirus was associated with subgroup 1 genomes by its coat and movement protein sequences, but with the crucifer-pathogenic tobamoviruses by the remainder of its genome, suggesting that it is the progeny of a recombinant. For four of five genomic regions, subgroup 1 and 3 genomes were equidistant from a subgroup 2 genome chosen for comparison, suggesting uniform rates of evolution. A phylogenetic tree of plant families based on the tobamoviruses they harbor was congruent with that based on rubisco sequences but had a different root, suggesting that codivergence was tempered by rare events of viruses of one family colonizing another family. The proposed subgroup 3 viruses probably have an origin of virion assembly in the movement protein gene, a large (25-codon) overlap of movement and coat protein open reading frames, and a comparably shorter genome. Codon-position- dependent base compositions and codon prevalences suggested that the coat protein frame of the overlap region was ancestral. Bootstrapped parsimony analysis of the nucleotides in the overlap region and of the sequences translated from the -1 frame (the subgroup 3 movement protein frame) of this region produced trees inconsistent with those deduced from other regions. The results are consistent with a model in which a no or short overlap organization was ancestral. Despite encoding of subgroup 2 and 3 movement protein C-termini by nonhomologous nucleotides, weak similarities between their amino acid sequences suggested convergent sequence evolution.      

Cytochemical demonstration of hydrogen peroxide in polymorphonuclear leukocyte phagosomes

Phagocytosis by polymorphonuclear leukocytes (PMN) is accompanied by specific morphological and metabolic events which may result in the killing of internalized micro-organism. Hydrogen peroxide is produced in increased amounts during phagocytosis (17) and in combination with myeloperoxidase and halide ions constitute a potent, microbicidal mechanism (8,9,11). There can be direct iodination of micro-organisms (10), or alternatively, other intermediate reaction products, i.e. chloramines and aldehydes (21), can exert a microbicidal effect. The H2O2-peroxidase-halide system is presumed to operate within the phagocytic vacuole (12,18). Myeloperoxidase, present in the primary granules of PMN, enters the phagocytic vacuole during degranulation (1,4,7), and halide ions are probably derived from the extracellular medium or are present in the PMN (see 11, 18). For the operation of this system in intact cells, the presence of H2O2 in the phagocytic vacuole is necessary, and indeed this has been suggested by the work of several investigators (12, 18, 21). In the present investigation, the diaminobenzidine reaction of Graham and Karnovsky (5), modified to utilize endogenous myeloperoxidase and hydrogen peroxide, has been applied to actively phagocytizing PMN to demonstrate cytochemically the presence of H2O2 in the phagocytic vacuole.     

Substructural specificity and polyvalent carbohydrate recognition by the Entamoeba histolytica and rat hepatic N- acetylgalactosamine/galactose lectins

Both the Entamoeba histolytica lectin, a virulence factor for the causative agent of amebiasis, and the mammalian hepatic lectin bind to N-acetylgalactosamine (GalNAc) and galactose (Gal) nonreducing termini on oligosaccharides, with preference for GalNAc. Polyvalent GalNAc- derivatized neoglycoproteins have >1000-fold enhanced binding affinity for both lectins (Adler,P., Wood,S.J., Lee,Y.C., Lee,R.T., Petri,W.A.,Jr. and Schnaar,R.L.,1995, J. Biol. Chem ., 270, 5164-5171). Substructural specificity studies revealed that the 3-OH and 4-OH groups of GalNAc were required for binding to both lectins, whereas only the E.histolytica lectin required the 6-OH group. Whereas GalNAc binds with 4-fold lower affinity to the E.histolytica lectin than to the mammalian hepatic lectin, galactosamine and N-benzoyl galactosamine bind with higher affinity to the E. histolytica lectin. Therefore, a synthetic scheme for converting polyamine carriers to poly-N-acyl galactosamine derivatives (linked through the galactosamine primary amino group) was developed to test whether such ligands would bind the E.histolytica lectin with high specificity and high affinity. Contrary to expectations, polyvalent derivatives including GalN6lys5, GalN4desmosine, GalN4StarburstTMdendrimer, and GalN8StarburstTMdendrimer demonstrated highly enhanced binding to the mammalian hepatic lectin but little or no enhancement of binding to the E.histolytica lectin. We propose that the mammalian hepatic lectin binds with greatest affinity to GalNAc "miniclusters," which mimic branched termini of N-linked oligosaccharides, whereas the E.histolytica lectin binds most effectively to "maxiclusters," which may mimic more widely spaced GalNAc residues on intestinal mucins.      

Effect of selenium on resistance of hemoglobin to photooxidative processes

On an example of a guinea pig it is shown that exogenous selenium (0.5 mg Na2SeO3 per 1 kg of the animal weight) during 2-hour exposition in the animal organism increases the resistance to the photo-induced oxidation of haemoglobin in erythrocyte lysates without additional stimulation of glutathione peroxidase mechanism of haemoglobin protection by exogenous selenium. It is shown that the saturation of haemoglobin fractions by selenium hampers the oxidative modification of haemoglobin. Using pregnancy of women as a natural model of selenium-deficiency condition, it has been shown that physiological debilitation of saturation erythrocytes with selenium, including haemoglobin fractions of lysates erythrocytes caused debilitation of resistance of haemoglobin to photooxidative destruction. Under these conditions not only activity of enzyme glutathione peroxidise in erythrocyte lysates, but also the peroxidase activity of haemoglobin (in the presence of glutathione) were decreased. It is more characteristic of erythrocyte lysates with a less content of selenium, i.e. for the erythrocytes of women on late terms of pregnancy that testifies to the presence of certain relation between haemoglobin saturation with selenium and its peroxidase activity (in the presence of glutathione).     

Serotonin-induced changes in the responses of the electroexcitable membrane of snail neurons

The effects of serotonin (5-HT) added to the washing solution on the plastic properties of the electroexcitable membrane of nonidentified neurons of the parietal ganglion and identified neurons RPa2 are studied on the isolated nervous system of the snail. The neurons of the first group, which became rapidly habituated to the intracellular stimulation, are shown to manifest the ability to restore action potential generation in the presence of 5-HT. In contrast, the neurons possessing endogenous rhythmic activity (RPa2) in the presence of 5-HT generate burst activity against the background of the development of slow waves of the membrane potential. A comparative analysis of the effect of 5-HT and compounds with a known effect on Ca²⁺ and the calcium-dependent potassium channels (quinine, CoCl₂ and CdCl₂) points to the existence of various mechanisms providing for the serotoninergic regulation of the plasticity of the electroexcitable neuron membrane. This difference stems from the processes of activation and blockade of the Ca-dependent mechanisms.A. I. Karaev Institute of Physiology Azerbaizhan Academy of Sciences, Baku. Translated from Neirofiziologiya, Vol. 24, No. 3, pp. 286–290, May–June, 1992.