Growth curves,morphology and ultrastructure of ten Paracoccidioides brasiliensis isolates

The yeast phase of ten P. brasiliensis isolates were studied to characterize their growth pattern, morphology and ultrastructure. Growth curves were determined after counts of total and viable fungi units (FU) during 20 days. Three growth patterns were observed: slow, reaching approximately 10–30× 10⁶ FU/tube (Pb 18, Pb 265 and PB 2); intermediate, reaching 60–150×10⁶ FU/tube (IVIC Pb 9, IVIC Pb 267, Pb SN, Pb Vitor and Pb Campo Grande) and fast, reaching 180–370×10⁶ FU/tube (Pb 2052 and Pb 192). The highest percentage of viable cells occurred on the 6th day of culture for Pb 192, Pb Campo Grande, Pb 2052 and IVIC Pb 9; on the 8th day for Pb Vitor, Pb SN, Pb 18 and IVIC Pb 267; on the 10th day for Pb 265 and on the 12th day of culture for Pb 2. Mean generation times varied from approximately 21.2 (Pb 2052) to 102.6 hours (Pb 265). The isolates showed similar morphology, except IVIC Pb 267 which did not present a typical yeast-phase at 35°C and the two fast-growing isolates (Pb 2052 and Pb 192) that presented smaller cell sizes and less tendency to clump. The ultrastructure of the isolates was similar: the cell walls presented a width of 0.1 to 0.2 °; the mitochondria presented few cristae and had equivalent patterns of distribution and morphology; the endoplasmic reticulum was scanty, presenting narrow cisternae; the vacuoles, empty or filled with electrondense material, were numerous and two to five nuclei with pores were constantly observed.     

Trinuclear iridium and rhodium complexes: the solution to a puzzle involving the multiple coordination possibilities of 1,8-naphthyridine-2-one ligands

The multiple coordination possibilities of 1,8-naphthyridine-2-one (HOnapy) and 5,7-dimethyl-1,8-napthyridine-2-one (HOMe₂napy) ligands allow the synthesis of a variety of tri- di- and mononuclear complexes, showing fluxional behaviour and frequent exchange of the coordinated ML₂ fragments. Thus, reactions of [M₂(μ-OMe)₂(cod)₂] (cod = 1,5-cyclooctadiene) with HOnapy and HOMe₂napy yield the compounds of the general formula [M(μ-OR₂napy) (cod)]_n (M = Ir, R = Me (1a, 1b, H (2); M = Rh, R = Me (3a, 3b). They crystallise as inconvertible yellow (a) and purple/orange (b) forms and also show a puzzling behaviour in solution. X-ray diffraction studies on both forms (3a, 3b) and spectroscopic data reveal that the yellow forms are mononuclear complexes whilst the dark-coloured crystals contain dinuclear complexes. In solution, the nuclearity of the complexes depends on the solvent. In addition both types of complexes are fluxional. The mixed-ligand complexes [M₂(μ-OMe₂napy)₂(CO)₂(cod)] M = Ir (5), Rh (6) have been isolated and characterised; they are found to be intermediates in the synthesis of the trinuclear complexes [M₃(μ₃-OMe₂napy)₂(CO)₂(cod)₂]⁺ M = Rh (8), Ir (9). Reactions of [IrCl(CO)₂(NH₂-p-tolyl] with the complexes [Rh(μ-OR₂napy)(diolefin)]_n followed by addition of a poor donor anion is a general one-pot synthesis for the hetertrinuclear complexes [Rh₂Ir(μ₃-OR₂napy)₂(CO)₂(diolefin)₂]⁺ (R=Me, DIOLEFIN = cod (10), tetrafluorobenzo-barrelene (tfbb) (11), 2,5-norbornadiene (nbd) (12); R=H, DIOLEFIN=cod (13)). This synthesis follows a stepwise mechanism from the mononuclear to the trinuclear complexes in which mixed-ligand heterodinuclear complexes are involved as intermediates of the type [(diolefin)Rh(μ-OMe₂napy)₂Ir(CO)₂]. Heteronuclear complexes which possess the core [RhIr₂]³⁺, such as [RhIr₂(μ₃-OR₂napy)₂(CO)₂(cod)₂]BF₄ (R=Me (14), H (15)), result from the reaction of 1 or 2 with [Rh(CO)₂S_x]⁺ (S = solvent). The trinuclear complexes undergo two chemically reversible one-electron oxidation processes. The chemical oxidation of 10, 14 and 9 with silver salts gives the mixed-valence trinuclear radicals [Rh₂Ir(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (16), [RhIr₂(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (17) and [Ir₃(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (18), which have been isolated as the perchlorate and tetrafluoroborate salts. The EPR spectrum of 16 indicates that the unpaired electron is essentially in an orbital delocalised on the metals. The molecular structures of the complexes 3a, 3b, 6, 10b and 16a are described. Crystals of 3a are triclinic, P-1, with a = 9.7393(2), b = 14.0148(4), c = 16.0607(4) Å, α = 88.122(3), β = 83.924(3), γ = 87.038(3)°, Z = 4; 3b crystallises in the Pna2_i orthorhhombic space group, with a = 16.7541(3), B = 11.7500(8), c = 17.7508(7) Å, Z = 4; complex 6 is packed in the monoclinic space group P2_i/c, a = 9.6371(1), b = 11.8054(4), c = 27.2010(9) Å, β = 90.556(4)°, Z = 4; crystals of 10b are monoclinic, P2₁/n, with a = 17.546(7), b = 13.232(6), c = 17.437(8) Å, β = 106.18(1)°, Z = 4; crystals of 16a are triclinic, P-1, with a = 10.318(4), b = 12.562(6), C = 19.308(8) Å, α = 92.12(8), β = 97.65(9), γ = 90.68(5)°, Z = 2. The five different structures show the coordination versatility of the OMe₂napy molecule as ligand, which behaves as a N,N′-chelating (3a), bidentate N,O-donor (3b, 6), or as a tridentate N,N′,O-donor bridging ligand (10b, 16a).     

The Tau/A152T mutation,a risk factor for frontotemporal‐spectrum disorders,leads to NR2B receptor‐mediated excitotoxicity

We report on a novel transgenic mouse model expressing human full‐length Tau with the Tau mutation A152T (hTau^AT), a risk factor for FTD‐spectrum disorders including PSP and CBD. Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis‐sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short‐ or long‐term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage‐gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau^AT causes excitotoxicity mediated by NR2B‐containing NMDA receptors due to enhanced extracellular glutamate.     

Red-shifted light-harvesting system of freshwater eukaryotic alga Trachydiscus minutus (Eustigmatophyta,Stramenopila)

Photosynthesis Research - Survival of phototrophic organisms depends on their ability to collect and convert enough light energy to support their metabolism. Phototrophs can extend their absorption...     

Targeted deletion of RIC8A in mouse neural precursor cells interferes with the development of the brain,eyes, and muscles

Autosomal recessive disorders such as Fukuyama congenital muscular dystrophy, Walker–Warburg syndrome, and the muscle–eye–brain disease are characterized by defects in the development of patient's brain, eyes, and skeletal muscles. These syndromes are accompanied by brain malformations like type II lissencephaly in the cerebral cortex with characteristic overmigrations of neurons through the breaches of the pial basement membrane. The signaling pathways activated by laminin receptors, dystroglycan and integrins, control the integrity of the basement membrane, and their malfunctioning may underlie the pathologies found in the rise of defects reminiscent of these syndromes. Similar defects in corticogenesis and neuromuscular disorders were found in mice when RIC8A was specifically removed from neural precursor cells. RIC8A regulates a subset of G‐protein α subunits and in several model organisms, it has been reported to participate in the control of cell division, signaling, and migration. Here, we studied the role of RIC8A in the development of the brain, muscles, and eyes of the neural precursor‐specific conditional Ric8a knockout mice. The absence of RIC8A severely affected the attachment and positioning of radial glial processes, Cajal‐Retzius’ cells, and the arachnoid trabeculae, and these mice displayed additional defects in the lens, skeletal muscles, and heart development. All the discovered defects might be linked to aberrancies in cell adhesion and migration, suggesting that RIC8A has a crucial role in the regulation of cell–extracellular matrix interactions and that its removal leads to the phenotype characteristic to type II lissencephaly‐associated diseases. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 374–390, 2018     

Synthesis and biological activity of benzo-fused 7-deazaadenosine analogues. 5- and 6-substituted 4-amino- or 4-alkylpyrimido[4,5-b]indole ribonucleosides

Two series of new 4-aminopyrimido[4,5-b]indole ribonucleosides bearing phenyl or hetaryl group at position 5 or 6 have been prepared by Suzuki or Stille cross-coupling reactions employing X-Phos ligand with (het)arylboronic acids or stannanes. A series of 4-substituted nucleosides has been also prepared by Pd-catalyzed cross-couplings or nucleophilic substitution. Some of these compounds displayed moderate antiviral activities against HCV and dengue viruses.     

Agrobacterium bohemicum sp. nov. isolated from poppy seed wastes in central Bohemia

Two non-pathogenic strains R89-1 and R90^T isolated from poppy seed (Papaver somniferum L.) wastes were phenotypically and genotypically characterized. Multilocus sequence analysis (MLSA) was conducted with six genes (atpD, glnA, gyrB, recA, rpoB, 16S rRNA). The strains represented a new species which clustered with Agrobacterium rubi NBRC 13261^T and Agrobacterium skierniewicense Ch11^T type strains. MLSA was further accompanied by whole-genome phylogeny, in silico DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses for both strains. ANI and dDDH values were deep below the species delineation threshold. Phenotypic features of the novel strains unequivocally allowed their differentiation from all other Agrobacterium species. Unlike other agrobacteria, the strains were salt sensitive and were able to biotransform morphine alkaloids. The dominant cellular fatty acids are 18:1 w7c, 16:0 and 12:0 aldehyde/16:1 iso I/14:0 3OH summed in feature 2 and the major respiratory quinine is Q-10 (87%). The DNA G + C content is 56 mol%. Microbial community analysis indicated probable association with P. somniferum plant material. Altogether, these characteristics showed that strains R90^T and R89-1 represent a new species of the genus Agrobacterium which we propose to name Agrobacterium bohemicum. The type strain of A. bohemicum is R90^T (=CCM 8736^T = DSM 104667^T).     

Exploring Leptin Antagonism in Ophthalmic Cell Models

Background

Emerging evidence suggests that angiogenic and pro-inflammatory cytokine leptin might be implicated in ocular neovascularization. However, the potential of inhibiting leptin function in ophthalmic cells has never been explored. Here we assessed mitogenic, angiogenic, and signaling leptin activities in retinal and corneal endothelial cells and examined the capability of a specific leptin receptor (ObR) antagonist, Allo-aca, to inhibit these functions.

Methods and Results

The experiments were carried out in monkey retinal (RF/6A) and bovine corneal (BCE) endothelial cells. Leptin at 50-250 ng/mL stimulated the growth of both cell lines in a dose-dependent manner. The maximal mitogenic response (35±7 and 27±3% in RF6A and BCE cells, respectively) was noted at 24 h of 250 ng/mL leptin treatments. Leptin-dependent proliferation was reduced to base levels with 10 and 100 nM Allo-aca in BCE and RF6A cells, respectively. In both cell lines, leptin promoted angiogenic responses, with the maximal increase in tube formation (163±10 and 133±8% in RF6A and BCE cultures, respectively) observed under a 250 ng/mL leptin treatment for 3 h. Furthermore, in both cell lines 250 ng/mL leptin modulated the activity or expression of several signaling molecules involved in proliferation, inflammatory activity and angiogenesis, such as STAT3, Akt, and ERK1/2, COX2, and NFκB. In both cell lines, leptin-induced angiogenic and signaling responses were significantly inhibited with 100 nM Allo-aca. We also found that leptin increased its own mRNA and protein expression in both cell lines, and this autocrine effect was abolished by 100-250 nM Allo-aca.

Conclusions

Our data provide new insights into the role of leptin in ocular endothelial cells and represent the first original report on targeting ObR in ophthalmic cell models.     

Induction of mutations inSerratia marcescens by a proteosynthesis block

The formation of colour mutations ofSerratia marcescens by the action of chloramphenicol was studied. Pink variants were the commonest; the proportion of white variants was much smaller. Almost 100% mutations were formed in a two-day culture containing 100 μg. chloramphenicol/ml. Comparative experiments showed that the change in pigment formation was hereditary, i.e. that actual mutation, and not selection of chloramphenicol-resistant mutants, occurred. Mutation occurred both in strain 151 and strain HY. The resultant mutations remained constant throughout ten passages on normal nutrient medium. The minimum chloramphenicol concentration which produced an increase in the mutation frequency was 5 μg./ml. The combined effect of X-ray irradiation and chloramphenicol treatment somewhat stimulated the increase in the frequency of mutation as compared with cells which were only irradiated. The increase in the frequency of mutation was much slower on solid medium containing chloramphenicol.