Protein O-Glucosyltransferase 1 (POGLUT1) Promotes Mouse Gastrulation through Modification of the Apical Polarity Protein CRUMBS2

Crumbs family proteins are apical transmembrane proteins with ancient roles in cell polarity. Mouse Crumbs2 mutants arrest at midgestation with abnormal neural plate morphology and a deficit of mesoderm caused by defects in gastrulation. We identified an ENU-induced mutation, wsnp, that phenocopies the Crumbs2 null phenotype. We show that wsnp is a null allele of Protein O-glucosyltransferase 1 (Poglut1), which encodes an enzyme previously shown to add O-glucose to EGF repeats in the extracellular domain of Drosophila and mammalian Notch, but the role of POGLUT1 in mammalian gastrulation has not been investigated. As predicted, we find that POGLUT1 is essential for Notch signaling in the early mouse embryo. However, the loss of mouse POGLUT1 causes an earlier and more dramatic phenotype than does the loss of activity of the Notch pathway, indicating that POGLUT1 has additional biologically relevant substrates. Using mass spectrometry, we show that POGLUT1 modifies EGF repeats in the extracellular domain of full-length mouse CRUMBS2. CRUMBS2 that lacks the O-glucose modification fails to be enriched on the apical plasma membrane and instead accumulates in the endoplasmic reticulum. The data demonstrate that CRUMBS2 is the target of POGLUT1 for the gastrulation epithelial-to-mesenchymal transitions (EMT) and that all activity of CRUMBS2 depends on modification by POGLUT1. Mutations in human POGLUT1 cause Dowling-Degos Disease, POGLUT1 is overexpressed in a variety of tumor cells, and mutations in the EGF repeats of human CRUMBS proteins are associated with human congenital nephrosis, retinitis pigmentosa and retinal degeneration, suggesting that O-glucosylation of CRUMBS proteins has broad roles in human health.     

Lack of Association of a Spontaneous Mutation of the Chrm2 Gene with Behavioral and Physiologic Phenotypic Differences in Inbred Mice

The nucleotide substitution C797T in the Chrm2 gene causes substitution of leucine for proline at position 266 (P266L) of the CHRM2 protein. Because Chrm2 codes for the type 2 muscarinic receptor, this mutation could influence physiologic and behavioral phenotypes of mice. Chrm2 mRNA was not differentially expressed in 2 brain regions with high cholinergic innervation in a mouse strain that does (BALB/cByJ) or does not (C57BL/6J) have the mutation. In addition, strains of mice with and without the C797T point mutation in Chrm2 did not differ significantly in muscarinic binding properties. Variation across strains was detected in terms of acoustic startle, prepulse inhibition, and the physiologic effects of the muscarinic agonist oxotremorine. However, interstrain differences in these measures did not correlate with the presence of the mutation. Although we were unable to associate a measurable phenotype with the Chrm2 mutation, assessment of the mutation on other genetic backgrounds or in the context of other traits might reveal differential effects. Therefore, despite our negative findings, evaluation of characteristics that involve muscarinic function should be undertaken with caution when comparing mice with different alleles of the Chrm2 gene.Abbreviations: M2R, type 2 muscarinic receptor; NMS, N-methylscopolamine; OXO, oxotremorine; PPI, prepulse inhibition; RI, recombinant inbredAcetylcholine, a crucial neurotransmitter in both the central and peripheral nervous systems, acts through 2 major types of receptors: muscarinic and nicotinic. Muscarinic acetylcholine receptors are members of the superfamily of G protein-coupled receptors.¹⁷mRNA and protein for the type 2 muscarinic receptor (M2) are present in many peripheral and central sites in the nervous system and peripheral target organs. M2R mediates a complex combination of postsynaptic and presynaptic events in noncholinergic and cholinergic neurons, respectively.⁹The M2R is encoded by the gene Chrm2. The proline at position 266 and surrounding residues of the Chrm2 gene are relatively conserved across several species, including human, rat, mouse, and swine (http://www.ncbi.nlm.nih.gov/). However, a nucleo­tide substitution (C797T) has been identified in several strains of inbred mice (Mouse Genome Informatics SNP query for Chrm2; http://www.informatics.jax.org/searches). This nucleotide substitution results in an amino acid substitution, P266L, in the protein. Proline is the only amino acid that contains a secondary amino group and forms tertiary peptide bonds. Because of this attribute, substitution of leucine for proline could cause alloste­ric alterations in proteins, with potential structural or functional consequences.Allosteric modulation is a recognized regulatory mechanism of muscarinic receptors.^17,21 For example, introduction of a point mutation (Asn to Tyr) at position 410 (the junction of transmembrane domain 6 and the 3rd intracellular loop) of the human M2R generated a constitutively active receptor with altered receptor–G-protein coupling in response to agonist administration.²³ Single-nucleotide polymorphisms in the human Chrm2 gene are implicated in responses to visual stimuli requiring attention, working memory, and response selection.^8,15,16 In addition, a common Chrm2 polymorphism has been associated with major depression in women in some studies^6,35 but not others.⁵ Furthermore, Chrm2 has been implicated in nicotine addiction; Chrm2 single-nucleotide polymorphisms may be associated with the general possibility of becoming addicted, personality traits that predispose the person to becoming addicted, or altered regulation of cholinergic systems that affect the smoker''s response to nicotine and its addictive properties.²²These reports suggest that mouse strains that bear the Chrm2 mutation, as compared with strains that do not, potentially provide a unique model for exploring mechanisms by which Chrm2 variants may affect cholinergic mechanisms and associated physiologic processes in both brain and the periphery. We report here on studies conducted to determine whether the C797T Chrm2 mutation confers a detectable phenotypic difference in M2R-related processes in mice.     

Insights from the analysis of conserved motifs and permitted amino acid exchanges in the human, the fly and the worm GPCR clusters

G-protein coupled receptors (GPCRs) belong to biologically important and functionally diverse and largest super family of membrane proteins. GPCRs retain a characteristic membrane topology of seven alpha helices with three intracellular, three extracellular loops and flanking N' and C' terminal residues. Subtle differences do exist in the helix boundaries (TM-domain), loop lengths, sequence features such as conserved motifs, and substituting amino acid patterns and their physiochemical properties amongst these sequences (clusters) at intra-genomic and inter-genomic level (please re-phrase into 2 statements for clarity). In the current study, we employ prediction of helix boundaries and scores derived from amino acid substitution exchange matrices to identify the conserved amino acid residues (motifs) as consensus in aligned set of homologous GPCR sequences. Co-clustered GPCRs from human and other genomes, organized as 32 clusters, were employed to study the amino acid conservation patterns and species-specific or cluster-specific motifs. Critical analysis on sequence composition and properties provide clues to connect functional relevance within and across genome for vast practical applications such as design of mutations and understanding of disease-causing genetic abnormalities.     

Development and validation of functional marker targeting an InDel in the major rice blast disease resistance gene Pi54 (Pik h )

Rice blast is one of the most devastating diseases affecting the rice crop throughout the world. In molecular breeding for host plant resistance, functional markers are very useful for enhancing the precision and accuracy in marker-assisted selection (MAS) of target gene(s) with minimum effort, time and cost. Pi54 (which was earlier known as Pik ^h ) is one of the major blast resistance genes and has been observed to show resistance against many isolates of the blast pathogen in India. The gene has been cloned through map-based strategy and encodes a nucleotide-binding site?Cleucine-rich repeat (NBS?CLRR) domain-containing protein. In the present study, we carried out allele mining for this gene and identified a 144-bp insertion/deletion (InDel) polymorphism in the exonic region of the gene. A PCR-based co-dominant molecular marker targeting this InDel, named Pi54 MAS, was developed. Pi54 MAS was observed to perfectly co-segregate with blast resistance in a mapping population with no recombinants. Validation of this marker in 105 genotypes which are either susceptible or resistant to rice blast disease showed that the marker is polymorphic in most of the resistant?Csusceptible genotype combinations and is more accurate than the earlier reported markers for Pi54. Hence this functional, co-dominant marker is suggested for routine deployment in MAS of Pi54 in breeding programs.     

Tolerance of Bt corn (MON 810) to maize stem borer, Chilo partellus (Lepidoptera: Pyralidae)

Transgenic corn (MON 810), expressing the Bacillus thuringiensis (Bt) protein, Cry1Ab, was evaluated under greenhouse conditions for its tolerance to the maize stem borer, Chilo partellus. Bt corn (MON 810) provided effective protection against the stem borer even under a high level of larval infestation in the greenhouse. The observed tolerance is examined and discussed in the light of the susceptibility of C. partellus to the Cry1Ab protein in laboratory bioassays. The implications of the tissue concentrations of Cry1Ab in MON 810, and baseline susceptibility recorded in the current study, for insect-resistance management are discussed.     

A two-state homology model of the hERG K+ channel: application to ligand binding

Homology models based on available K+ channel structures have been used to construct a multiple state representation of the hERG cardiac K+ channel. These states are used to capture the flexibility of the channel. We show that this flexibility is essential in order to correctly model the binding affinity of a set of diverse ligands. Using this multiple state approach, a binding affinity model was constructed for set of known hERG channel binders. The predicted pIC50s are in good agreement with experiment (RMSD: 0.56 kcal/mol). In addition, these calculations provide structures for the bound ligands that are consistent with published mutation studies. These computed ligand bound complex structures can be used to guide synthesis of analogs with reduced hERG liability.     

Cystic fibrosis transmembrane conductance regulator differentially regulates human and mouse epithelial sodium channels in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl- channel properties, regulates other ion channels. CFTR inhibits murine or rat epithelial Na+ channel (mENaC or rENaC) currents in many epithelial and non-epithelial cells, whereas murine or rat ENaC increases CFTR functional expression. These regulatory interactions are reproduced in Xenopus oocytes where both the open probability and surface expression of wild type CFTR Cl- channels are increased when CFTR is co-expressed with alphabetagamma mENaC, and conversely the activity of mENaC is inhibited after wild type CFTR activation. Using the Xenopus oocyte expression system, differences in functional regulatory interactions were observed when CFTR was co-expressed with either alphabetagamma mENaC or alphabetagamma human ENaC (hENaC). Co-expression of CFTR and alphabetagamma mENaC or hENaC resulted in an approximately 3-fold increase in CFTR Cl- current compared with oocytes expressing CFTR alone. Oocytes co-injected with both CFTR and mENaC or hENaC expressed an amiloride-sensitive whole cell current that was decreased compared with that observed with the injection of mENaC or hENaC alone before CFTR activation with forskolin/3-isobutyl-1-methylxanthine. CFTR activation resulted in a further 50% decrease in mENaC-mediated currents, an approximately 20% decrease in alpha-T663-hENaC-mediated currents, and essentially no change in alpha-A663-hENaC-mediated currents. Changes in ENaC functional expression correlated with ENaC surface expression by oocyte surface biotinylation experiments. Assessment of regulatory interactions between CFTR and chimeric mouse/human ENaCs suggest that the 20 C-terminal amino acid residues of alpha ENaC confer species specificity regarding ENaC inhibition by activated CFTR.     

Assessment of dominance hierarchy through urine scent marking and its chemical constituents in male blackbuck Antelope cervicapra, a critically endangered species

In ungulates the process of chemical communication by urinary scent marking has been directly related to reproductive dominance, territorial defense and proximity to resources. The differences in the frequency of urine marking and chemical composition of urine of males Antelope cervicapra before, during and after the dominance hierarchy period were assessed. The variations in the urine marking and its chemical profiles of dominant males (n = 9), bachelors (n = 5) and sub-adult males (n = 5) were compared to find out how the dominance hierarchy influences the confined blackbuck herd under semi-natural captive conditions. The frequency of urine marking is significantly higher (p < 0.001) in dominant males. Twenty-eight major constituents were identified in the urine of dominant males (before, during and after the dominance hierarchy period), bachelor and sub-adult males. Among these, three specific compounds namely, 3-hexanone (I), 6-methyl-5-hepten-2-one (II) and 4-methyl-3-heptanone (III) were seen only in dominant males urine during the dominance hierarchy period. Based on the behavioural observation and the unique chemical constituents in the urine, it is concluded that the dominant male scent odor suppresses aggression, scent marking, scent production and territorial patrolling activities of subordinate males, through which the dominant male establish their hierarchy and attains success in reproduction.     

1-Iodo-2methylundecane, a putative estrus-specific urinary chemo-signal of female mouse (Mus musculus)

The present study was designed to identify the urinary volatiles across various reproductive stages of female mice in order to detect estrus-specific chemical signal. The urine of adult female mice were extracted with dichloromethane (1:1 ratio, v/v) and analyzed by gas chromatography linked mass-spectrometry (GC-MS). Numerous compounds were identified during estrus cycle of female mice urine. Among these, the compounds, namely, isocroctylhydrazine, 4-methyl-2-heptanone and auzulene were specific to proestrus stage and the compounds, 1-H-cyclopop-e.auzulene, caryophyllene, copanene were specific to estrus stage. However, the compound, 1-iodo-2methyl undecane (1I2MU) was observed both in proestrus and estrus phases and was absent in all other phases. The volatile signal produced at the end of proestrus and the beginning of estrus phase appears to be behaviourally important in the attraction of males. Moreover, the behaviour assay revealed that the compound, 1I2MU, is involved in attracting the male mice. This result concludes that the 1-iodo-2methyl undecane is considered as a putative estrus-specific chemo-signal.     

Linkage disequilibria and haplotype structure of four SNPs of the interleukin 1 gene cluster in seven Asian Indian populations

Variation at four single nucleotide polymorphism (SNP) sites of the interleukin 1 (IL1) gene cluster was investigated among 280 unrelated individuals, representing 7 caste groups from the state of Karnataka, India, and one European American community of Boston, Massachusetts. Allele and haplotype frequencies, strength of linkage disequilibrium, and signatures of recombination varied considerably among populations. Variable community sizes and traditions of consanguinity may account for the observed variation.