TRPV1 receptors and nasal trigeminal chemesthesis

The trigeminal nerve responds to a wide variety of irritants. Trigeminal nerve fibers express several receptors that respond to chemicals, including TRPV1 (vanilloid) receptors, acid-sensing ion channels, P2X (purinergic) receptors, and nicotinic acetylcholine receptors. In order to assess whether TRPV1 plays a role in responses to a broad array of substances, TRPV1 (along with green fluorescent protein) was expressed in human embyonic kidney cells (HEK) 293t cells which were then stimulated with diverse trigeminal irritants. Calcium imaging was used to measure responses to capsaicin, amyl acetate, cyclohexanone, acetic acid, toluene, benzaldehyde, (-)-nicotine, (R)-(+)-limonene, (R)-(-)-carvone, and (S)-(+)-carvone. Three irritants (acetic acid and the 2 carvones) stimulated nontransfected controls. Two irritants (capsaicin and cyclohexanone) stimulated only transfected cells. The response could be eliminated with capsazepine, a TRPV1 blocker. The 5 remaining irritants were nonstimulatory in both nontransfected and transfected cells. Because all the compounds tested on HEK cells elicited neural responses from the ethmoid branch of the trigeminal nerve in rats, the 5 nonstimulatory compounds must do so by a non-TRPV1 receptor. These results suggest that TRPV1 serves as a receptor for both cyclohexanone and capsaicin in trigeminal nerve endings.     

Ectomycorrhizal species associated with Pinus radiata in New Zealand including novel associations determined by molecular analysis

Ectomycorrhizal (ECM) associates of the exotic plantation species Pinus radiata were investigated above and below ground over two years in the North Island of New Zealand. ECM species were identified using morphological and molecular (restriction fragment length polymorphism and DNA sequencing) analysis. Eighteen ECM species were observed fruiting above ground; 19 ECM species were identified below ground. In the above ground study, Wilcoxina mikolae, Rhizopogon pseudoroseolus and Inocybe sindonia were noted for the first time as ECM associates of P. radiata in New Zealand. Below ground, the species W. mikolae, R. pseudoroseolus, Rhizopogon luteorubescens, Pseudotomentella sp., Pseudotomentella tristis and Tomentella sp. were found as new associates of P. radiata in New Zealand. Additionally, six ECM types were found that could not be identified with molecular analysis. The putative ECM taxa Tricholoma pessundatum, Laccaria laccata and Hebeloma crustuliniforme were examined by molecular analysis, and species identifications were proposed to be changed to Tricholoma sp., L. laccata and Hebeloma sp. for specimens associated with P. radiata in New Zealand. The species identity of I. sindonia, previously unidentified to species level, was determined with direct sequencing.     

Fine-Scale Survey of X Chromosome Copy Number Variants and Indels Underlying Intellectual Disability

Copy number variants and indels in 251 families with evidence of X-linked intellectual disability (XLID) were investigated by array comparative genomic hybridization on a high-density oligonucleotide X chromosome array platform. We identified pathogenic copy number variants in 10% of families, with mutations ranging from 2 kb to 11 Mb in size. The challenge of assessing causality was facilitated by prior knowledge of XLID-associated genes and the ability to test for cosegregation of variants with disease through extended pedigrees. Fine-scale analysis of rare variants in XLID families leads us to propose four additional genes, PTCHD1, WDR13, FAAH2, and GSPT2, as candidates for XLID causation and the identification of further deletions and duplications affecting X chromosome genes but without apparent disease consequences. Breakpoints of pathogenic variants were characterized to provide insight into the underlying mutational mechanisms and indicated a predominance of mitotic rather than meiotic events. By effectively bridging the gap between karyotype-level investigations and X chromosome exon resequencing, this study informs discussion of alternative mutational mechanisms, such as noncoding variants and non-X-linked disease, which might explain the shortfall of mutation yield in the well-characterized International Genetics of Learning Disability (IGOLD) cohort, where currently disease remains unexplained in two-thirds of families.     

Diploid and polyploid cytotype distribution in Melampodium cinereum and M. leucanthum (Asteraceae, Heliantheae)

Previous chromosomal studies within Melampodium (Asteraceae, Heliantheae) of Mexico and Central America have documented chromosome numbers n = 9, 10, 11, 12, 18, 20, 23, 25 ± 1, 27, 30, and 33. Some species also have been shown to exhibit infra- and interpopulational polyploidy. The presence of cytotype mixtures is especially pronounced in the white-rayed complex, which occurs in the southwestern United States and adjacent Mexico. This group includes M. cinereum (n = 10 and 20), M. leucanthum (n = 10 and 20), and M. argophyllum (n = 30). Cytotype distribution has been newly analyzed in 415 plants from 152 populations and added to existing data from 185 plants from 113 populations, yielding information from a total of 600 individuals from 265 populations. Within M. cinereum and M. leucanthum are parapatric distributions of cytotypes, with tetraploids centered in the eastern and diploids in the western portions of their ranges. Tetraploids are most likely of autopolyploid origin, forming recurrently, with adaptations that allow colonization and establishment in new ecological regions. Contact zones are relatively narrow and only two triploid individuals have been detected. The tetraploid cytotypes probably extended eastward into central and southern Texas to the natural barriers at the edge of the Edward's Plateau in M. leucanthum and the low sandy plains in M. cinereum. The hexaploid M. argophyllum is interpreted as a relict surviving in the low mountains of northern Mexico; it may be an allopolyploid of hybrid origin between ancestors of the evolutionary lines that eventually yielded M. cinereum and M. leucanthum.     

Chromosomal abnormalities in Day-6, in vitro-produced pig embryos

A cytogenetic study was undertaken to quantify, by chromosomal karyotyping, the incidence and type of chromosomal abnormalities present in Day-6 in vitro-produced (IVP) porcine embryos. Morphologically normal Day-6 blastocysts (n=318) were fixed and grouped into six classes according to the number of total cells (from < or =20 to 61-70). Of 248 embryos suitable for analysis, 97 (39.1%) displayed chromosomal abnormalities. The abnormalities included haploidy (9.3%), polyploidy (71.1%) and mixoploidy (19.6%). Within polyploid embryos, triploidy and tetraploidy showed the highest incidence (56.5 and 27.5%, respectively); among mixoploid embryos, diploid-triploid embryos (2n/3n) were prevalent (36.8%). Overall, the mean cell number was 34.3 +/- 12.1 and the mitotic index was 8.6 +/- 6.1. Chromosomally abnormal embryos had fewer (P<0.01) total cells compared to normal (2n) embryos (31.8 +/- 1.3 versus 35.9 +/- 1.0). In addition, the incidence of polyploidy decreased as the number of cells increased, while that of mixoploidy did not differ. These data indicate that polyploidy affects a large percentage of IVP porcine embryos capable of developing to blastocysts and the incidence of chromosomal abnormalities is much higher than that reported previously in in vivo embryos in this species. Given the ability of morphologically normal embryos with an abnormal chromosome complement to undergo preimplantation development in vitro, and the inability to identify blastocysts with abnormal karyotype without cytogenetic analysis, careful consideration should be given to factors affecting ploidy of IVP embryos, especially the incidence of polyspermic fertilization, when evaluating criteria of a porcine in vitro embryo production scheme.     

A central role of the BK potassium channel in behavioral responses to ethanol in C. elegans

The activities of many neuronal proteins are modulated by ethanol, but the fundamental mechanisms underlying behavioral effects of ethanol remain unclear. To identify mechanisms responsible for intoxication, we screened for Caenorhabditis elegans mutants with altered behavioral responses to ethanol. We found that slo-1 mutants, which were previously recognized as having slightly uncoordinated movement, are highly resistant to ethanol in two behavioral assays. Numerous loss-of-function slo-1 alleles emerged from our screens, indicating that slo-1 has a central role in ethanol responses. slo-1 encodes the BK potassium channel. Electrophysiological analysis shows that ethanol activates the channel in vivo, which would inhibit neuronal activity. Moreover, behaviors of slo-1 gain-of-function mutants resemble those of ethanol-intoxicated animals. These results demonstrate that selective activation of BK channels is responsible for acute intoxicating effects of ethanol in C. elegans. BK channel activation may explain a variety of behavioral responses to ethanol in invertebrate and vertebrate systems.     

Natural variation in the npr-1 gene modifies ethanol responses of wild strains of C. elegans

Variation in the acute response to ethanol between individuals has a significant impact on determining susceptibility to alcoholism. The degree to which genetics contributes to this variation is of great interest. Here we show that allelic variation that alters the functional level of NPR-1, a neuropeptide Y (NPY) receptor-like protein, can account for natural variation in the acute response to ethanol in wild strains of Caenorhabditis elegans. NPR-1 negatively regulates the development of acute tolerance to ethanol, a neuroadaptive process that compensates for effects of ethanol. Furthermore, dynamic changes in the NPR-1 pathway provide a mechanism for ethanol tolerance in C. elegans. This suggests an explanation for the conserved function of NPY-related pathways in ethanol responses across diverse species. Moreover, these data indicate that genetic variation in the level of NPR-1 function determines much of the phenotypic variation in adaptive behavioral responses to ethanol that are observed in natural populations.     

Early degradation of paternal mitochondria in domestic pig (Sus scrofa) is prevented by selective proteasomal inhibitors lactacystin and MG132

Ubiquitin-dependent proteolysis has been implicated in the recognition and selective elimination of paternal mitochondria and mitochondrial DNA (mtDNA) after fertilization in mammals. Initial evidence suggests that this process is contributed to by lysosomal degradation of the ubiquitinated sperm mitochondrial membrane proteins. The present study examined the role of the proteasome-dependent protein degradation pathway of the ubiquitin system, as opposed to lysosomal proteolysis of the ubiquitinated proteins, in the regulation of sperm mitochondrion elimination after fertilization. Boar spermatozoa prelabeled with vital fluorescent mitochondrial probes MitoTracker were used to trace the degradation of paternal mitochondria after in vitro fertilization (IVF) of porcine oocytes. The degradation of sperm mitochondria in the cytoplasm of fertilized oocytes started very rapidly, i.e., within 12-20 h after insemination. Four stages of paternal mitochondrial degradation were distinguished, ranging from an intact mitochondrial sheath (type 1) to complete degradation (type 4). At 27-30 h postinsemination, 96% of zygotes contained the partially (type 3) or completely (type 4) degraded sperm mitochondria. Highly specific peptide inhibitors of the ubiquitin-proteasome pathway, lactacystin (10 and 100 microM) and MG132 (10 microM), efficiently blocked the degradation of the sperm mitochondria inside the fertilized egg when applied 6 h after insemination. Using 10 microM MG132, only 13.6% of fertilized oocytes screened 27-30 h after IVF displayed type 3 sperm mitochondria, and there was no incidence of type 4, completely degraded mitochondria. Although lactacystin is not a reversible agent, the effect of MG132 was fully reversible: zygotes transferred to regular culture medium after 24 h of culture with 10 microM MG132 resumed development and degraded sperm mitochondria within the next cell cycle. Surprisingly, penetration of the zona pellucida (ZP) was also inhibited by MG-132 and lactacystin when the inhibitors were added at insemination. Altogether, these data provide the first evidence of the participation of proteasomes in the control of mammalian mitochondrial inheritance and suggest a new role of the ubiquitin-proteasome pathway in mammalian fertilization.     

Minimal methylated substrate and extended substrate range of Escherichia coli AlkB protein,a 1-methyladenine-DNA dioxygenase

The Escherichia coli AlkB protein, and two human homologs ABH2 and ABH3, directly demethylate 1-methyladenine and 3-methylcytosine in DNA. They couple Fe(II)-dependent oxidative demethylation of these damaged bases to decarboxylation of alpha-ketoglutarate. Here, we have determined the kinetic parameters for AlkB oxidation of 1-methyladenine in poly(dA), short oligodeoxyribonucleotides, nucleotides, and nucleoside triphosphates. Methylated poly(dA) was the preferred AlkB substrate of those tested. The oligonucleotide trimer d(Tp1meApT) and even 5'-phosphorylated 1-me-dAMP were relatively efficiently demethylated, and competed with methylated poly(dA) for AlkB activity. A polynucleotide structure was clearly not essential for AlkB to repair 1-methyladenine effectively, but a nucleotide 5' phosphate group was required. Consequently, 1-me-dAMP(5') was identified as the minimal effective AlkB substrate. The nucleoside triphosphate, 1-me-dATP, was inefficiently but actively demethylated by AlkB; a reaction with 1-me-ATP was even slower. E. coli DNA polymerase I Klenow fragment could employ 1-me-dATP as a precursor for DNA synthesis in vitro, suggesting that demethylation of alkylated deoxynucleoside triphosphates by AlkB could have biological significance. Although the human enzymes, ABH2 and ABH3, demethylated 1-methyladenine residues in poly(dA), they were inefficient with shorter substrates. Thus, ABH3 had very low activity on the trimer, d(Tp1meApT), whereas no activity was detected with ABH2. AlkB is known to repair methyl and ethyl adducts in DNA; to extend this substrate range, AlkB was shown to reduce the toxic effects of DNA damaging agents that generate hydroxyethyl, propyl, and hydroxypropyl adducts.     

Permanent expression of a cyclin B homologue in the cell cycle of the dinoflagellate Karenia brevis

The eukaryotic cell cycle is driven by a set of cyclin-dependent kinases associated with their regulatory partners, the cyclins, which confer activity, substrate specificities and proper localization of the kinase activity. We describe the cell cycle of Karenia brevis and provide evidence for the presence of a cyclin B homologue in this dinoflagellate using two antibodies with different specificities. This cyclin B homologue has an unusual behavior, since its expression is permanent and it has a cytoplasmic location throughout the cell cycle. There is no evidence for translocation to the nucleus during mitosis. However, it appears also to be specifically bound to the nucleolus throughout the cell cycle. The permanent expression and the cytoplasmic localization during mitosis of this cyclin B homologue is similar to p56, a cyclin B homologue previously described in a different species of dinoflagellate, Crypthecodinium cohnii. Here we discuss this unusual behavior of the cyclin B homologue in dinoflagellates, its relationship to the unusual characteristics of dinomitosis, and its potential implications regarding the evolution of cell cycle regulation among eukaryotes.