NMR resolved multiple anesthetic binding sites in the TM domains of the α4β2 nAChR

The α4β2 nicotinic acetylcholine receptor (nAChR) has significant roles in nervous system function and disease. It is also a molecular target of general anesthetics. Anesthetics inhibit the α4β2 nAChR at clinically relevant concentrations, but their binding sites in α4β2 remain unclear. The recently determined NMR structures of the α4β2 nAChR transmembrane (TM) domains provide valuable frameworks for identifying the binding sites. In this study, we performed solution NMR experiments on the α4β2 TM domains in the absence and presence of halothane and ketamine. Both anesthetics were found in an intra-subunit cavity near the extracellular end of the β2 transmembrane helices, homologous to a common anesthetic binding site observed in X-ray structures of anesthetic-bound GLIC (Nury et al., [32]). Halothane, but not ketamine, was also found in cavities adjacent to the common anesthetic site at the interface of α4 and β2. In addition, both anesthetics bound to cavities near the ion selectivity filter at the intracellular end of the TM domains. Anesthetic binding induced profound changes in protein conformational exchanges. A number of residues, close to or remote from the binding sites, showed resonance signal splitting from single to double peaks, signifying that anesthetics decreased conformation exchange rates. It was also evident that anesthetics shifted population of two conformations. Altogether, the study comprehensively resolved anesthetic binding sites in the α4β2 nAChR. Furthermore, the study provided compelling experimental evidence of anesthetic-induced changes in protein dynamics, especially near regions of the hydrophobic gate and ion selectivity filter that directly regulate channel functions.     

Human nerve stem cells in vitro

The growth characteristics of a fraction of the human embryonic brain Nest-positive cells cultivated in vitro are the subject of the study. Our data demonstrate a probable increase in the content of Nest-positive cells in the cell population from 44.1 to 62.5% as a response to the presence of EGF and bFGF in a growth medium supplemented with heat-inactivated serum. Further differentiation in the cell culture led to a decrease in the number of Nest-positive cells. Retinoic acid-based stimulation of neural induction causes a decrease in the content of these cells in the population by 14.1%. The results of this study will allow for the obtaining of a nerve cell population that could be more effective as a graft material suitable for therapeutic use.     

Ultrastructural study of the male gamete of Pleurogonius truncatus Prudhoe, 1944 (Platyhelminthes, Digenea, Pronocephalidae) parasite of Eretmochelys imbricata (Linnaeus, 1766)

In Pronocephaloidea, the spermatozoa of only two species have been studied today. Because of this, we present in this work data concerning to a third specie, Pleurogonius truncatus Prudhoe, 1944. The mature spermatozoon of P. truncatus possesses two axonemes with the 9+"1" pattern typical of Trepaxonemata, mitochondrion, nucleus, parallel cortical microtubules, spinelike bodies, cytoplasmic expansion and an external ornamentation of the plasma membrane. A particularity of the spermatozoon of P. truncatus is in the ultrastructure of the anterior spermatozoon extremity with only cortical microtubules and ornamentation of the plasma membrane. This type of anterior extremity has never been described until today in Pronocephaloidea. On the other hand, the ultrastructure of the posterior extremity of the spermatozoon confirms that already described in Pronocephalidae.     

Amplification and Temporal Filtering during Gradient Sensing by Nerve Growth Cones Probed with a Microfluidic Assay

Nerve growth cones (GCs) are chemical sensors that convert graded extracellular cues into oriented axonal motion. To ensure a sensitive and robust response to directional signals in complex and dynamic chemical landscapes, GCs are presumably able to amplify and filter external information. How these processing tasks are performed remains however poorly known. Here, we probe the signal-processing capabilities of single GCs during γ-Aminobutyric acid (GABA) directional sensing with a shear-free microfluidic assay that enables systematic measurements of the GC output response to variable input gradients. By measuring at the single molecule level the polarization of GABA_A chemoreceptors at the GC membrane, as a function of the external GABA gradient, we find that GCs act as i), signal amplifiers over a narrow range of concentrations, and ii), low-pass temporal filters with a cutoff frequency independent of stimuli conditions. With computational modeling, we determine that these systems-level properties arise at a molecular level from the saturable occupancy response and the lateral dynamics of GABA_A receptors.     

Isoform and splice-variant specific functions of dynamin-2 revealed by analysis of conditional knock-out cells

Dynamin (Dyn) is a multifunctional GTPase implicated in several cellular events, including endocytosis, intracellular trafficking, cell signaling, and cytokinesis. The mammalian genome encodes three isoforms, Dyn1, Dyn2, and Dyn3, and several splice variants of each, leading to the suggestion that distinct isoforms and/or distinct splice variants might mediate distinct cellular functions. We generated a conditional Dyn2 KO cell line and performed knockout and reconstitution experiments to explore the isoform- and splice variant specific cellular functions of ubiquitously expressed Dyn2. We find that Dyn2 is required for clathrin-mediated endocytosis (CME), p75 export from the Golgi, and PDGF-stimulated macropinocytosis and cytokinesis, but not for other endocytic pathways. Surprisingly, CME and p75 exocytosis were efficiently rescued by reintroduction of Dyn2, but not Dyn1, suggesting that these two isoforms function differentially in vesicular trafficking in nonneuronal cells. Both isoforms rescued macropinocytosis and cytokinesis, suggesting that dynamin function in these processes might be mechanistically distinct from its role in CME. Although all four Dyn2 splice variants could equally restore CME, Dyn2ba and -bb were more effective at restoring p75 exocytosis. This splice variant specificity correlated with their differential targeting to the Golgi. These studies reveal isoform and splice-variant specific functions for Dyn2.     

Effects of melanin and manganese on DNA damage and repair in PC12-derived neurons

The mechanism of neurotoxicity produced by the interaction of melanin with manganese was investigated in PC12-derived neuronal cell cultures. The cells were incubated with melanin (25-500 microg/ml), MnCl2 (10 ng/ml-100 microg/ml) and a combination of both substances for 24 and 72 h. Incubation with either toxicant alone resulted in a minimal decrease in cell viability. The combination of melanin and manganese caused significant (up to 60%) decreases in viability of PC12 cells in a dose-dependent manner. Increases in oxidative DNA damage, indicated by levels of 8-hydroxy-2'deoxyguanosine (8-oxodG), was associated with decreased cell viability. Melanin alone, but not manganese alone, resulted in increased oxidative DNA damage. The maximal increase in 8-oxodG caused by melanin was about seven times higher than control after 24 h of exposure. The activity of the DNA repair enzyme, 8-oxoguanine DNA glycosylase (OGG1), was increased in cells incubated with single toxicants and their combinations for 24 h. On the third day of incubation with the toxicants, activity of OGG1 declined below control levels and cell viability significantly decreased. Melanin was observed to have an inhibitory effect on OGG1 activity. Study of the regulation of OGG1 activity in response to melanin and manganese may provide insights into the vulnerability of nigral neurons to oxidative stress in Parkinson's disease.     

Acanthostomum macroclemidis n. sp. (Digenea: Cryptogonimidae: Acanthostominae) from the alligator snapping turtle,Macroclemys temmincki

Acanthostomum macroclemidis n. sp. is described from specimens found in the intestine of an alligator snapping turtle Macroclemys temmincki from southern Mississippi. The most important diagnostic features of the new species are the general shape and proportions of the body, the position of the pharynx (relative length of the prepharynx and esophagus), the egg size, the relative length and position of the vitelline fields, and the number, shape, and size of the circumoral spines. The new species has a very elongated body (length-width ratio, 8.9-13.0:1), 26 circumoral spines, which are almost oval in shape, a long prepharynx and a very short (shorter than the pharynx) esophagus, a seminal receptacle situated between the ovary and the anterior testis, a uterus not extending posterior to the anterior margin of the ovary, a long-stemmed and short-armed excretory vesicle, and 2 anal openings. Some features of the external morphology, such as the suckers, circumoral spines, sensory papillae, tegumental spines, and morphology of the posterior end, are examined using scanning electron microscopy. A diagnosis differentiating A. macroclemidis n. sp. from some other acanthostomine digeneans is provided. Acanthostomum macroclemidis n. sp. is the first digenean reported from an alligator snapping turtle and represents the northernmost record of an acanthostomine from turtles.     

Synthesis, structural investigation and thermal stability of aqua magnesium(II) phthalocyanine adducts with 2-methoxyethylamine

Three new aqua magnesium phthalocyaninato complexes with 2-methoxyethylamine (MEA) in crystalline form have been obtained. The composition of the complexes depends on the crystallisation temperature. (MgPcH₂O)₂ · MEA (I) and (MgPcH₂O)₂ · 2MEA (II) were formed at about 170 °C and 80 °C, respectively, while room temperature (MgPcH₂O)₂ · 3MEA (III) was obtained. In all crystals the Mg atom is 4+1 coordinated, equatorially by four N-isoindole atoms of Pc ligand and axially by O atom of water molecule. The MgPc moiety is non-planar, the Mg(II) deviates by ∼0.5 Å from the N₄-isoindole plane towards the oxygen atom of water molecule. The MEA molecules in the crystals interact via hydrogen bonds with coordinated water molecules of MgPcH₂O. The arrangement of MgPcH₂O and 2-methoxyethylamine molecules is determined by O-H?N and O-H?O hydrogen bonds and by π-π interactions. The thermogravimetric analyses show characteristic steeps responsible for the loss of MEA molecules (at lower temperature) and water (at higher temperature) and finally all the complexes transform into β-MgPc. From among the complexes only complex II exhibits an intense near-IR absorption band in the solid-state, while spectra in MEA solution are identical for all the complexes.     

Development of a transformation system for gene knock-out in the flavinogenic yeast Pichia guilliermondii

Pichia guilliermondii is a representative of a yeast species, all of which over-synthesize riboflavin in response to iron deprivation. Molecular genetic studies in this yeast species have been hampered by a lack of strain-specific tools for gene manipulation. Stable P. guilliermondii ura3 mutants were selected on the basis of 5'-fluoroorotic acid resistance. Plasmid carrying Saccharomyces cerevisiae URA3 gene transformed the mutant strains to prototrophy with a low efficiency. Substitution of a single leucine codon CUG by another leucine codon CUC in the URA3 gene increased the efficiency of transformation 100 fold. Deletion cassettes for the RIB1 and RIB7 genes, coding for GTP cyclohydrolase and riboflavin synthase, respectively, were constructed using the modified URA3 gene and subsequently introduced into a P. guilliermondii ura3 strain. Site-specific integrants were identified by selection for the Rib(-) Ura(+) phenotype and confirmed by PCR analysis. Transformation of the P. guilliermondii ura3 strain was performed using electroporation, spheroplasting or lithium acetate treatment. Only the lithium acetate transformation procedure provided selection of uracil prototrophic, riboflavin deficient recombinant strains. Depending on the type of cassette, efficiency of site-specific integration was 0.1% and 3-12% in the case of the RIB1 and RIB7 genes, respectively. We suggest that the presence of the ARS element adjacent to the 3' end of the RIB1 gene significantly reduced the frequency of homologous recombination. Efficient gene deletion in P. guilliermondii can be achieved using the modified URA3 gene of S. cerevisiae flanked by 0.8-0.9 kb sequences homologous to the target gene.