Developmental analysis of the cell recognition mechanism in the ciliate Euplotes raikovi.

Euplotes raikovi, like other ciliates, passes through a postconjugal immaturity, operatively identified by an apparent cell inability to form mating pairs under experimental conditions that are the same as those used for inducing mating at maturity. In cells homozygous for the gene mat-2, which controls the pheromone Er-2, Er-2 mRNA synthesis and mature Er-2 secretion were shown to start from the very beginning of the life cycle and continue throughout immaturity, although to extents estimated to be 5- to 10-fold lower than at maturity. In addition, experiments of 125I-Er-2 binding and crosslinking provided evidence that autocrine pheromone-binding sites, showing values of the dissociation constant of the order of 10(-9) M, are on the surface of immature cells. The number of these sites per cell was estimated to increase from less than 10(6) per cell of 5-7 fissions of age, to about 16 x 10(6) at maturity. These results were taken to suggest that a pheromone-receptor production is stimulated during immaturity by autocrine pheromone binding to cells and that this production might be essential for the development of a pheromone-receptor density high enough to transform the cell from "immature" to "adult," that is competent to respond as well to pheromones of conspecific, genetically different cells.     

Mating-Type Polymorphic Variation in Euplotes minuta (Ciliophora: Hypotrichida)1

An investigation was carried out to probe into the mating-type structure of a local population of the marine ciliate, Euplotes minuta. From this population, nine different mating types belonging to a unique set were isolated. The nine type-representative wild stocks analyzed were found to be heterozygous at the mating-type (mat) locus and provided, together with their sexual progeny, a total of 15 pure mating types. In E. minuta, the high-multiple nature of the basic mating system controlled by a series of peck-order alleles at a single locus should be considered a virtual certainty. The relationships among the genetic economies of the similar bottom-dwelling marine ciliates of the genus Euplotes, the E. vannus-crassus-minuta group, are discussed.     

Neurobiological Correlates of Alpha-Tocopherol Antiepileptogenic Effects and MicroRNA Expression Modulation in a Rat Model of Kainate-Induced Seizures

Seizure-triggered maladaptive neural plasticity and neuroinflammation occur during the latent period as a key underlying event in epilepsy chronicization. Previously, we showed that α-tocopherol (α-T) reduces hippocampal neuroglial activation and neurodegeneration in the rat model of kainic acid (KA)-induced status epilepticus (SE). These findings allowed us to postulate an antiepileptogenic potential for α-T in hippocampal excitotoxicity, in line with clinical evidence showing that α-T improves seizure control in drug-resistant patients. To explore neurobiological correlates of the α-T antiepileptogenic role, rats were injected with such vitamin during the latent period starting right after KA-induced SE, and the effects on circuitry excitability, neuroinflammation, neuronal death, and microRNA (miRNA) expression were investigated in the hippocampus. Results show that in α-T-treated epileptic rats, (1) the number of population spikes elicited by pyramidal neurons, as well as the latency to the onset of epileptiform-like network activity recover to control levels; (2) neuronal death is almost prevented; (3) down-regulation of claudin, a blood–brain barrier protein, is fully reversed; (4) neuroinflammation processes are quenched (as indicated by the decrease of TNF-α, IL-1β, GFAP, IBA-1, and increase of IL-6); (5) miR-146a, miR-124, and miR-126 expression is coherently modulated in hippocampus and serum by α-T. These findings support the potential of a timely intervention with α-T in clinical management of SE to reduce epileptogenesis, thus preventing chronic epilepsy development. In addition, we suggest that the analysis of miRNA levels in serum could provide clinicians with a tool to evaluate disease evolution and the efficacy of α-T therapy in SE.     

Purification to apparent homogeneity of the mating pheromone of mat-1 homozygous Euplotes raikovi

Mating type-specific mating pheromones autonomously released into the environment control cell-cell union in conjugation of the marine ciliate Euplotes raikovi. The mating pheromone, termed euplomone r-1, of cells of the homozygous mating type determined by the mat-1/mat-1 genotype was purified by means of a three-stage purification procedure which provides a yield of 79%. Starting from 10 liters of supernatant, 3.3 mg of euplomone r-1 were regularly recovered. Euplomone r-1 was identified with a protein showing a molecular weight of 12,000, an isoelectric point of 3.7, and unusually high contents of cysteine (15%) and tyrosine (5.8%). It appeared active at a concentration of 3.4 +/- 0.5 X 10(-12) M. Carbohydrate and a small colored compound, not yet identified, occurred in association with partially purified euplomone r-1 samples, whereas they were not detected in samples of the final product of purification. An acidic shift was apparently capable of destroying the carbohydrate/euplomone r-1 association, suggesting that it does not involve covalent bonds.     

Conformational Studies on Two FtsZ Targeting Cyclic Peptides

International Journal of Peptide Research and Therapeutics - Two FtsZ targeting cyclic peptides 1 (Ac-[Orn-Leu-Met-Asp]-Ala-Phe-Arg-Ser-NH2) and 2 (Ac-Ser-Leu-Met-[Asp-Ala-Phe-Arg-Orn]-NH2) were...     

Evidence for Gene Duplication and Allelic Codominance (not Hierarchical Dominance) at the Mating‐Type Locus of the Ciliate,Euplotes crassus

The high‐multiple mating system of Euplotes crassus is known to be controlled by multiple alleles segregating at a single locus and manifesting relationships of hierarchical dominance, so that heterozygous cells would produce a single mating‐type substance (pheromone). In strain L‐2D, now known to be homozygous at the mating‐type locus, we previously identified two pheromones (Ec‐α and Ec‐1) characterized by significant variations in their amino acid sequences and structure of their macronuclear coding genes. In this study, pheromones and macronuclear coding genes have been analyzed in strain POR‐73 characterized by a heterozygous genotype and strong mating compatibility with L‐2D strain. It was found that POR‐73 cells contain three distinct pheromone coding genes and, accordingly, secrete three distinct pheromones. One pheromone revealed structural identity in amino acid sequence and macronuclear coding gene to the Ec‐α pheromone of L‐2D cells. The other two pheromones were shown to be new and were designated Ec‐2 and Ec‐3 to denote their structural homology with the Ec‐1 pheromone of L‐2D cells. We interpreted these results as evidence of a phenomenon of gene duplication at the E. crassus mating‐type locus, and lack of hierarchical dominance in the expression of the macronuclear pheromone genes in cells with heterozygous genotypes.     

Calcareous nannofossils and foraminifers herald the Messinian Salinity Crisis: The Pollenzo section (Alba, Cuneo; NW Italy)

During the Messinian, the Mediterranean area experienced fast and prominent paleoenvironmental changes, culminating in the so-called Messinian Salinity Crisis, with the deposition of the evaporitic series. This work investigates the micropaleontological assemblages in the pre-evaporitic sediments of the Sant’Agata Fossili Marls (SAF) of the Pollenzo section (Cuneo area, North Western Italy). A semiquantitative analysis is carried out on the upper part of the marly and pelitic sediments of the SAF underlying the first gypsum bed, ascribed to the Vena del Gesso Fm. (VDF). The studied interval belongs to the planktonic foraminifer Globorotalia conomiozea Zone and “non distinctive Zone” of Iaccarino and to the calcareous nannofossil MNN11b/c Zone of Raffi et al. (1998, 2003) ( [Raffi et al., 1998] and [Raffi et al., 2003]). Decrease of diversity and abundance of the foraminifer and calcareous nannofossil assemblages is recorded 12 m below the VDG and clearly reflects environmental stress. From bottom to top, six paleoecological events are recorded: (1) the first peak abundance of “small” Reticulofenestra and the last recovery (LR) of planktonic foraminifers; (2) the peak abundance of Pontosphaera japonica and the last recovery of warm water taxa Discoaster spp.; (3) the last recovery of benthic foraminifers; (4) the co-occurring peak abundances of Helicosphaera carteri and Sphenolithus abies, and the last recovery of warm water taxa Amaurolithus spp.; (5) the second peak of “small” Reticulofenestra; (6) the definitive disappearance of calcareous nannofossils. These paleoecological events describe a progressive isolation of the basin from the world ocean and increasingly stressed environment (LR planktonic foraminifers; LR Discoaster spp.), increasing dysoxic to anoxic conditions at the sea floor (LR benthic foraminifers), shallowing of the water column (peak of H. carteri), increasing salinity in surface waters (peak of S. abies), and enhanced nutrient concentration in surface waters (peak of “small” Reticulofenestra); these are related to paleoenvironmental changes predating gypsum deposition at Pollenzo and affecting the whole Mediterranean basin.     

Involvement of glutamate receptors of the NMDA type in the modulation of acetylcholine and glutamate overflow from the guinea pig ileum during in vitro hypoxia and hypoglycaemia

The involvement of NMDA glutamate receptors in the effects of glucose/oxygen deprivation (in vitro ischaemia) on spontaneous endogenous acetylcholine and glutamate overflow from the guinea pig ileum was studied. Neurotransmitter overflow was measured by HPLC. Deprivation of glucose in the medium slightly reduced acetylcholine overflow, and did not significantly influence glutamate overflow. During oxygen deprivation and glucose/oxygen deprivation, acetylcholine overflow augmented with a biphasic modality: an early peak was followed by a long lasting increase, whereas glutamate overflow increased with a rapid and sustained modality. The effects of glucose/oxygen deprivation on both acetylcholine and glutamate overflow were abolished after reperfusion with normal oxygenated medium. Acetylcholine and glutamate overflow induced by glucose/oxygen deprivation were significantly reduced in the absence of external Ca(2+) as well as by the addition of the mitochondrial Na(+)-Ca(2+) exchanger blocker, CGP 37157, and of the endoplasmic reticulum Ca(2+)/ATPase blocker, thapsigargin. +/-AP5, an NMDA receptor antagonist, and 5,7-diCl-kynurenic acid, an antagonist of the glycine site associated to NMDA receptor, markedly depressed glucose/oxygen deprivation-induced acetylcholine and glutamate overflow as well. Our results suggest that in vitro simulated ischaemia evokes acetylcholine and glutamate overflow from the guinea pig ileum, which is partly linked to an increase in intracellular Ca(2+) concentration dependent on both Ca(2+) influx from the extracellular space and Ca(2+) mobilization from the endoplasmic reticulum and mitochondrial stores. During glucose/oxygen deprivation, ionotropic glutamate receptors of the NMDA type exert both a positive feedback modulation of glutamate output and contribute to increased acetylcholine overflow.     

Molecular cloning and functional characterization of two isoforms of dermonecrotic toxin from Loxosceles intermedia (brown spider) venom gland

Brown spider (Genus Loxosceles) bites are normally associated with necrotic skin degeneration, gravitational spreading, massive inflammatory response at injured region, platelet aggregation causing thrombocytopenia and renal disturbances. Brown spider venom has a complex composition containing many different toxins, of which a well-studied component is the dermonecrotic toxin. This toxin alone may produce necrotic lesions, inflammatory response and platelet aggregation. Biochemically, dermonecrotic toxin belongs to a family of toxins with 30-35 kDa characterized as sphingomyelinase-D. Here, employing a cDNA library of Loxosceles intermedia venom gland, we cloned and expressed two recombinant isoforms of the dermonecrotic toxin LiRecDT2 (1062 bp cDNA) and LiRecDT3 (1007 bp cDNA) that encode for signal peptides and complete mature proteins. Phylogenetic tree analysis revealed a structural relationship for these toxins compared to other members of family. Recombinant molecules were expressed as N-terminal His-tag fusion proteins in Escherichia coli and were purified to homogeneity from cell lysates by Ni(2+) chelating chromatography, resulting in proteins of 33.8 kDa for LiRecDT2 and 34.0 kDa for LiRecDT3. Additional evidence for related toxins containing sequence/epitopes identity comes from antigenic cross-reactivity using antibodies against crude venom toxins and antibodies raised with a purified dermonecrotic toxin. Recombinant toxins showed differential functionality in rabbits: LiRecDT2 caused a macroscopic lesion with gravitational spreading upon intradermal injection, while LiRecDT3 evoked transient swelling and erythema upon injection site. Light microscopic analysis of skin biopsies revealed edema, a collection of inflammatory cells in and around blood vessels and a proteinaceous network at the dermis. Moreover, differential functionality for recombinant toxins was also demonstrated by a high sphingomyelinase activity for LiRecDT2 and low activity for LiRecDT3 as well as greater in vitro platelet aggregation and blood vessel permeability induced by LiRecDT2 and residual activity for LiRecDT3. Cloning and expression of two recombinant dermonecrotic toxins demonstrate an intraspecific family of homologous toxins that act in synergism for deleterious activities of the venom and open possibilities for biotechnological applications for recombinant toxins as research tools for understanding the inflammatory response, vascular integrity and platelet aggregation modulators.