The geographic distribution of the ACE II genotype: a novel finding

Angiotensin converting enzyme (ACE) gene polymorphism insertion (I) or deletion (D) has been widely studied in different populations, and linked to various functional effects and associated with common diseases. The purpose of the present study was to investigate the relationship between the ACE I/D frequency in different populations and geographic location; ACE I/D allele frequency in the Lebanese population and ACE II genotype contribution to the geographic trend were also identified. Five hundred and seventy healthy volunteers were recruited from the Lebanese population. Genomic DNA was extracted from buccal cells, and amplified by polymerase chain reaction; products were then identified by gel electrophoresis. The frequencies of the different ACE I/D genotypes were determined and tested for Hardy-Weinberg equilibrium (HWE). To assess the relationship between ACE I/D frequency and geographic location, and to identify how the Lebanese population contributes to the geographic trend in ACE I/D frequencies, Eurasian population samples and Asians were incorporated in the analyses from the literature. The frequency of the I allele in the Lebanese population was 27% and the corresponding II genotype was at a frequency of 7.37% (in HWE; P=0.979). The ACE I allele and genotype frequencies show an association with longitude, with frequencies increasing eastwards and westwards from the Middle East.     

Cellular organisation in meiotic and early post-meiotic rice anthers

We have used fluorescent, confocal laser and transmission electron microscopy (TEM) to examine cellular organisations, including callose (1,3-beta-glucan) behaviour, in meiotic and early post-meiotic rice anthers. These features are critical for pollen formation and provide information to better understand pollen sterility caused by abiotic stress in rice and other monocotyledonous species. Among organelles during meiosis, abundant plastids, mitochondria and nuclei of the anther cells show distinctive features. Chloroplasts in the endothecium store starch and indicate a potential for photosynthetic activity. During meiosis, the middle layer cells are markedly compressed and at the tetrad stage are either vacuolated or filled with degenerating electron-opaque organelles. Viable mitochondria, stained with Rhodamine 123, are seen in the endothecium and tapetum, but the mitochondria in the middle layer are not stained during meiosis. The radial walls of the tapetum are disorganised and degenerating, indicating the formation of a syncytium; pro-orbicules are located at the locular walls at the tetrad stage. Immunohistochemical studies show that the sporogenous cells are entirely enveloped by a thick callosic layer at early meiosis. Cell plate callose was assembled in a plane between the dyad cells. In the tetrads, however, callose formed only at the centre, showing that the tetrad microspores are not enveloped but separated by callose walls. Thick, undulating electron-opaque walls around the tetrads indicate the beginning of exinous microspore wall differentiation.     

Enrichment strategies for laboratory animals from the viewpoint of clinical veterinary behavioral medicine: emphasis on cats on dogs

Behavioral wellness has become a recent focus for the care of laboratory animals, farm and zoo animals, and pets. Behavioral enrichment issues for these groups are more similar than dissimilar, and each group can learn from the other. The emphasis on overall enhancement for laboratory dogs and cats in this review includes an emphasis on behavioral enrichment. Understanding the range of behaviors, behavioral choices, and cognitive stimulation that cats and dogs exhibit under non-laboratory conditions can increase the ability of investigators to predict which enrichments are likely to be the most successful in the laboratory. Many of the enrichment strategies described are surprisingly straightforward and inexpensive to implement.     

Actin associated with plasmodesmata

Summary We have used several methods to localise actin associated with plasmodesmata. In meristematic plant material fixed in 0.1% glutaraldehyde/1% paraformaldehyde and embedded in LR White resin, actin was localised (in TEM using 5 nm gold-labelled secondary antibody to C₄ anti-actin primary antibody) in the neck region by the plasma membrane and endoplasmic reticulum, and also down the length of the plasmodesma, deep in the cell wall. When the chemical fixation was replaced by rapid freezing in liquid propane (without cryoprotectants) and substitution in acetone, the plasmodesmata were labelled in similar positions, but with less background label on sections. While only 8–20% of plasmodesmata were labelled, the label was 10 to 100 fold denser over plasmodesmata than over the surrounding wall indicating specific association with plasmodesmata. We presume the apparent extracellular location of some label was due to the size of the antibodies between the site of attachment and the observed position of the gold particle. Gold label was found in similar locations in material fixed in 3% paraformaldehyde, infiltrated with sucrose, frozen, sectioned (10–12 m thick), then labelled with antibodies before resin embedding. Furthermore, cell walls in epidermal peels stained with rhodamine-phalloidin showed localised patches of fluorescence, presumably at the site of plasmodesmata (or primary pit-fields), which were connected on either side to fluorescent strands of actin in the cytoplasm. Suspension cultured cells ofNicotiana plumbaginifolia similarly stained showed very faint, narrow fluorescent strands crossing the walls of sister cells, which may indicate actin associated with individual plasmodesmata, shown in TEM to be sparsely distributed in these walls. In addition, the neck regions of cytochalasin-treated plasmodesmata were greatly enlarged and lacked the normal extracellular ring of particles. We propose that actin associated with plasmodesmata stabilizes the neck region and possibly also the cytoplasmic sleeve, and may be actively involved in regulating cell-to-cell transport.Abbreviations BSA bovine serum albumin - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline - Pipes piperazine-N,N-bis(2-ethanesulphonic acid) - Mes 2(N-morpholino)ethanesulfonic acid - SDS sodium dodecyl sulphate - Tris tris-(hydroxymethyl)aminomethane     

Novel N-terminal and Lysine Methyltransferases That Target Translation Elongation Factor 1A in Yeast and Human

Eukaryotic elongation factor 1A (eEF1A) is an essential, highly methylated protein that facilitates translational elongation by delivering aminoacyl-tRNAs to ribosomes. Here, we report a new eukaryotic protein N-terminal methyltransferase, Saccharomyces cerevisiae YLR285W, which methylates eEF1A at a previously undescribed high-stoichiometry N-terminal site and the adjacent lysine. Deletion of YLR285W resulted in the loss of N-terminal and lysine methylation in vivo, whereas overexpression of YLR285W resulted in an increase of methylation at these sites. This was confirmed by in vitro methylation of eEF1A by recombinant YLR285W. Accordingly, we name YLR285W as elongation factor methyltransferase 7 (Efm7). This enzyme is a new type of eukaryotic N-terminal methyltransferase as, unlike the three other known eukaryotic N-terminal methyltransferases, its substrate does not have an N-terminal [A/P/S]-P-K motif. We show that the N-terminal methylation of eEF1A is also present in human; this conservation over a large evolutionary distance suggests it to be of functional importance. This study also reports that the trimethylation of Lys⁷⁹ in eEF1A is conserved from yeast to human. The methyltransferase responsible for Lys⁷⁹ methylation of human eEF1A is shown to be N6AMT2, previously documented as a putative N(6)-adenine-specific DNA methyltransferase. It is the direct ortholog of the recently described yeast Efm5, and we show that Efm5 and N6AMT2 can methylate eEF1A from either species in vitro. We therefore rename N6AMT2 as eEF1A-KMT1. Including the present work, yeast eEF1A is now documented to be methylated by five different methyltransferases, making it one of the few eukaryotic proteins to be extensively methylated by independent enzymes. This implies more extensive regulation of eEF1A by this posttranslational modification than previously appreciated.Protein methylation is emerging as one of the most prominent posttranslational modifications in the eukaryotic cell (1). Often showing high evolutionary conservation, it is increasingly recognized for its role in modulating protein–protein interactions (2). Indeed, it has been documented in protein interaction codes (3), such as those of the histones and p53 (4, 5), where it shows interplay with modifications such as acetylation and phosphorylation. Despite this, there remains a paucity of understanding of the enzymes that catalyze protein methylation. Many of the known methyltransferases target histones. However, many other methyltransferases have been discovered recently that act on nonhistone proteins (6).While protein methylation predominantly occurs on lysine and arginine residues, it is also known to occur on glutamine, asparagine, glutamate, histidine, cysteine, and the N- and C termini of proteins. Although the presence of N-terminal methylation on numerous proteins has been known for decades (7), the first enzymes responsible for this methylation have only recently been discovered (8, 9). The Saccharomyces cerevisiae protein Tae1 and its human ortholog N-terminal methyltransferase 1 (NTMT1) catalyze N-terminal methylation of proteins with an N-terminal [A/P/S]-P-K motif (after methionine removal). Yet there is evidence that these enzymes may recognize a more general N-terminal motif (10). Human NTMT2 is a monomethyltransferase that methylates the same substrates as NTMT1 and may prime substrate proteins with monomethylation to assist subsequent trimethylation by NTMT1 (11).The biological function of N-terminal methylation on some proteins has been recently revealed. For example, N-terminal methylation of regulator of chromatin condensation protein 1 (RCC1) is known to affect its binding to chromatin and thereby the correct chromosomal segregation during mitosis (12, 13), and N-terminal methylation of DNA damage-binding protein 2 (DDB2) is important for its role in UV-damaged DNA repair (14). Interestingly, there is evidence of interplay between N-terminal methylation and other posttranslational modifications (15), suggesting that, like lysine and arginine methylation, it may be incorporated into protein interaction codes (3). N-terminal methylation therefore appears to be a modification of functional importance in the cell.Eukaryotic elongation factor 1A (eEF1A), and its bacterial ortholog EF-Tu, is an essential translation elongation factor that is found in all living organisms. Its canonical function is in facilitating delivery of aminoacyl-tRNAs to the ribosome; however, it is also known to have a role in many other cellular functions, such as actin bundling, nuclear export, and proteasomal degradation (16). A number of methyltransferases have been discovered in both S. cerevisiae and human that target translation elongation factors. In yeast, four of these elongation factor methyltransferases (EFMs) act on eEF1A, namely Efm1, Efm4, Efm5, and Efm6, generating monomethylated Lys³⁰, dimethylated Lys³¹⁶, trimethylated Lys⁷⁹, and monomethylated Lys³⁹⁰, respectively (17–19). Human METTL10 is the ortholog of Efm4 in that it trimethylates eEF1A at Lys³¹⁸, which is equivalent to Lys³¹⁶ in yeast (20). Interestingly, eukaryotic elongation factor 2 (eEF2) is also methylated by a number of lysine methyltransferases. In yeast, Efm2 and Efm3 act on eEF2, generating dimethylated Lys⁶¹³ and trimethylated Lys⁵⁰⁹, respectively (21–24). Human eEF2-KMT is the ortholog of Efm3 in that it trimethylates eEF2 at Lys⁵²⁵, which is equivalent to Lys⁵⁰⁹ in yeast eEF2 (23).Here, we report the N-terminal methylation of eEF1A in S. cerevisiae and the identification of the methyltransferase that catalyzes this event. Using parallel reaction monitoring and MS/MS/MS (MS3), we unambiguously localize the modification to the N-terminal glycine and show it is conserved in the human cell. We also show that YLR285W, which we rename elongation factor methyltransferase 7 (Efm7), is responsible for this modification in yeast, as well as dimethylation at the adjacent lysine. We also characterize the methyltransferases responsible for methylation of lysine 79 in eEF1A. Human N6AMT2 is shown to be the ortholog of yeast Efm5 through its capacity to methylate yeast and human eEF1A at Lys⁷⁹ in vitro. We therefore rename N6AMT2 as eEF1A-KMT1.     

Prolactin stimulates precursor cells in the adult mouse hippocampus

In the search for ways to combat degenerative neurological disorders, neurogenesis-stimulating factors are proving to be a promising area of research. In this study, we show that the hormonal factor prolactin (PRL) can activate a pool of latent precursor cells in the adult mouse hippocampus. Using an in vitro neurosphere assay, we found that the addition of exogenous PRL to primary adult hippocampal cells resulted in an approximate 50% increase in neurosphere number. In addition, direct infusion of PRL into the adult dentate gyrus also resulted in a significant increase in neurosphere number. Together these data indicate that exogenous PRL can increase hippocampal precursor numbers both in vitro and in vivo. Conversely, PRL null mice showed a significant reduction (approximately 80%) in the number of hippocampal-derived neurospheres. Interestingly, no deficit in precursor proliferation was observed in vivo, indicating that in this situation other niche factors can compensate for a loss in PRL. The PRL loss resulted in learning and memory deficits in the PRL null mice, as indicated by significant deficits in the standard behavioral tests requiring input from the hippocampus. This behavioral deficit was rescued by direct infusion of recombinant PRL into the hippocampus, indicating that a lack of PRL in the adult mouse hippocampus can be correlated with impaired learning and memory.