Molecular Cloning, Expression, and Pharmacological Characterization of humEAA1, a Human Kainate Receptor Subunit

Abstract: Kainate is a potent neuroexcitatory agent; its neurotoxicity is thought to be mediated by an ionotropic receptor with a nanomolar affinity for kainate. In this report, we describe the cloning of a cDNA encoding a human glutamate ionotropic receptor subunit protein from a human hippocampal library. This cDNA, termed humEAA1, is most closely related to rat and human cDNAs for kainate receptor proteins and, when expressed in COS or Chinese hamster ovary cells, is associated with high-affinity kainate receptor binding. We have successfully established cell lines stably expressing humEAA1. This is the first report of establishment of stable cell lines expressing a glutamate receptor subunit. The relative potency of compounds for displacing [³H] kainate binding of humEAA1 receptors expressed in these stable cell lines was kainate > quisqualate > domoate > L-glutamate > ( RS )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid > dihydro-kainate > 6, 7-dinitroquinoxaline-2, 3-dione > 6-cyano-7-nitroquinoxaline-2, 3-dione. Homooligomeric expression of humEAA1 does not appear to elicit ligand-gated ion channel activity. Nevertheless, the molecular structure and pharmacological characterization of high-affinity kainate binding of the humEAA1 expressed in the stable cell line (ppEAA1–16) suggest that the humEAA1 is a subunit protein of a human kainate receptor complex.     

Genetic and phylogenetic analyses of human T-cell lymphotropic virus type I variants from Melanesians with an without spastic myelopathy

Molecular variants of human T-cell lymphotropic virus type I (HTLV-I) have been isolated recently from lifelong residents of remote Melanesian populations, including a Solomon Islander with tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) or HTLV-I myeloneuropathy. To clarify the genetic heterogeneity and molecular epidemiology of disease-associated strains of HTLV-I, we enzymatically amplified, then directly sequenced representative regions of thegag, pol, env, andpX genes of HTLV-I strains from Melanesians with and without TSP/HAM, and aligned and compared these sequences with those of HTLV-I strains from patients with TSP/HAM or adult T-cell leukemia/lymphoma and from asymptomatic carriers from widely separated and culturally disparate populations. Overall, the HTLV-I variant from the Solomon Islander with TSP/HAM, like HTLV-I strains from asymptomatically infected Melanesians, diverged by approx 7% from cosmopolitan HTLV-I strain. No disease-specific viral sequences were found. Gene phylogenies, as determined by the unweighted pair-goup method of assortment and by the maximum parsimony method, indicated that the Melanesian and cosmopolitan strains of HTLV-I have evolved along separate geographically dependent lineages, one comprised of HTLV-I strains from Papua New Guinea and the Solomon Islands, and the other composed of virus strains from Japan, India, the Caribbean, Polynesia, the Americas, and Africa. The total absence of nonhuman primates in Papua New Guinea and the Solomon Islands precludes any possibility that the Melanesian HTLV-I strains have evolved recently from the simian homolog of HTLV-I.     

“Macrophage” nitric oxide synthase is a glucocorticoid-inhibitable primary response gene in 3T3 cells

Both nitric oxide and prostaglandins are potent paracrine mediators of intercellular communication. An endotoxin-lipopolysaccharide (LPS) inducible form of nitric oxide synthase (mac-NOS) has recently been cloned from murine macrophages. An inducible prostaglandin synthase (TIS1O/PGS-2), cloned from 3T3 cells, is also induced in LPS-activated macrophage. Because of the wide range of ligands that induce primary response genes in 3T3 cells, the ease of studying chimeric promoter constructs in 3T3 cells, and the importance of both nitric oxide and prostaglandins as paracrine mediators, we examined expression of mac-NOS in 3T3 cells. Tetradecanoyl phorbol-13 acetate (TPA), forskolin, platelet-derived growth factor, fibroblast growth factor, and serum all induce mac-NOS expression in Swiss 3T3 cells. Thus the mac-NOS gene can respond to a far wider range of inducers than previously suspected. mac-NOS is a primary response gene; cycloheximide does not block induction. TPA-induced mac-NOS and TIS10/PGS-2 mRNA accumulation patterns are similar. LPS is a potent inducer of mac-NOS in Swiss 3T3 cells but cannot induce TIS10/PGS-2. In contrast, v-src expression induces TIS10/PGS-2 message, but not iNOS message in a BALB/c 3T3 cell line containing a temperature-sensitive v-src gene. Dexamethasone (DEX) prevents induction of TIS10/PGS-2, but not most other primary response genes. DEX also blocks mac-NOS induction in Swiss 3T3 cells. The inducible TIS10/PGS-2 and mac-NOS genes, responsible for the production of two distinct paracrine agents, appear to share many regulatory features in 3T3 cells. © 1993 Wiley-Liss, Inc.     

Meiotic recombination in an Irish family with beta-thalassaemia

Using the technique of allele-specific priming of the polymerase chain reaction (PCR), the C-T substitution in codon 39 was identified as the cause of -thalassaemia in an Irish family. Analysis of the restriction fragment length polymorphisms (RFLPs) in the -globin gene cluster established linkage of the -thalassaemia mutation to a particular -haplotype but indicated that a recombinational event had occurred in the paternal chromosome in the younger of two affected children. Non-paternity was excluded by DNA fingerprinting analysis with hypervariable minisatellite probes. This is the fourth case of recombination in the -globin gene cluster to be reported. The event has occurred 5 of the polymorphic RsaI site at position-550 bp upstream of the -globin gene mRNA Cap site, within the 9.1-kb region that has been shown to be a hot spot for recombination in the -globin gene cluster.     

Seagrass ecosystem multifunctionality under the rise of a flagship marine megaherbivore

Large grazers (megaherbivores) have a profound impact on ecosystem functioning. However, how ecosystem multifunctionality is affected by changes in megaherbivore populations remains poorly understood. Understanding the total impact on ecosystem multifunctionality requires an integrative ecosystem approach, which is especially challenging to obtain in marine systems. We assessed the effects of experimentally simulated grazing intensity scenarios on ecosystem functions and multifunctionality in a tropical Caribbean seagrass ecosystem. As a model, we selected a key marine megaherbivore, the green turtle, whose ecological role is rapidly unfolding in numerous foraging areas where populations are recovering through conservation after centuries of decline, with an increase in recorded overgrazing episodes. To quantify the effects, we employed a novel integrated index of seagrass ecosystem multifunctionality based upon multiple, well-recognized measures of seagrass ecosystem functions that reflect ecosystem services. Experiments revealed that intermediate turtle grazing resulted in the highest rates of nutrient cycling and carbon storage, while sediment stabilization, decomposition rates, epifauna richness, and fish biomass are highest in the absence of turtle grazing. In contrast, intense grazing resulted in disproportionally large effects on ecosystem functions and a collapse of multifunctionality. These results imply that (i) the return of a megaherbivore can exert strong effects on coastal ecosystem functions and multifunctionality, (ii) conservation efforts that are skewed toward megaherbivores, but ignore their key drivers like predators or habitat, will likely result in overgrazing-induced loss of multifunctionality, and (iii) the multifunctionality index shows great potential as a quantitative tool to assess ecosystem performance. Considerable and rapid alterations in megaherbivore abundance (both through extinction and conservation) cause an imbalance in ecosystem functioning and substantially alter or even compromise ecosystem services that help to negate global change effects. An integrative ecosystem approach in environmental management is urgently required to protect and enhance ecosystem multifunctionality.     

Carryover effects from environmental change in early life: An overlooked driver of the amphibian extinction crisis?

Ecological carryover effects, or delayed effects of the environment on an organism's phenotype, are central predictors of individual fitness and a key issue in conservation biology. Climate change imposes increasingly variable environmental conditions that may be challenging to early life-history stages in animals with complex life histories, leading to detrimental physiological and fitness effects in later life. Yet, the latent nature of carryover effects, combined with the long temporal scales over which they can manifest, means that this phenomenon remains understudied and is often overlooked in short-term studies limited to single life-history stages. Herein, we review evidence for the physiological carryover effects induced by elevated ultraviolet radiation (UVR; 280–400 nm) as a potential contributor to recent amphibian population declines. UVR exposure causes a suite of molecular, cellular and physiological consequences known to underpin carryover effects in other taxa, but there is a lack of research linking embryonic and larval UVR exposures to fitness consequences post-metamorphosis in amphibians. We propose that the key impacts of UVR on disease-related amphibian declines are facilitated through carryover effects that bridge embryonic and larval UVR exposure with potential increased disease susceptibility post-metamorphosis. We conclude by identifying a practical direction for the study of ecological carryover effects in amphibians that could guide future ecological research in the broader field of conservation physiology. Only by addressing carryover effects can many of the mechanistic links between environmental change and population declines be elucidated.     

Loss of DNA repair mechanisms in cardiac myocytes induce dilated cardiomyopathy

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1^−/D mice). Ckmm-Cre^+/−;Ercc1^−/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre^+/−;Ercc1^−/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre^+/-;Ercc1^−/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre^+/−;Ercc1^−/fl and Ercc1^−/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.     

Molecular characterization of one of the maize polygalacturonase gene family members which are expressed during late pollen development

A gene exhibiting homology to the polygalacturonases of several species, including tomato and Oenothera, has been shown by RNA dot-blot analysis and in situ hybridization experiments to be expressed post-first microspore mitosis in maize. A 2.87 kbp section of the promoter fused to E. coli β-glucuronidase (uidA) coding sequence conferred the correct spatial and temporal expression in transgenic tobacco plants. However, low levels of expression were detected in other tissues, and in particular in the tissues surrounding the vascular branch points of leaf nodes. The maize polygalacturonase gene is one member of a highly conserved gene family. The lack of detectable expression in sporophytic tissues and the isolation of a number of related cDNAs from maize suggests that all expressed members of this family show the same spatial and temporal regulation.     

Nonparametric and semiparametric estimation with sequentially truncated survival data

In observational cohort studies with complex sampling schemes, truncation arises when the time to event of interest is observed only when it falls below or exceeds another random time, that is, the truncation time. In more complex settings, observation may require a particular ordering of event times; we refer to this as sequential truncation. Estimators of the event time distribution have been developed for simple left-truncated or right-truncated data. However, these estimators may be inconsistent under sequential truncation. We propose nonparametric and semiparametric maximum likelihood estimators for the distribution of the event time of interest in the presence of sequential truncation, under two truncation models. We show the equivalence of an inverse probability weighted estimator and a product limit estimator under one of these models. We study the large sample properties of the proposed estimators and derive their asymptotic variance estimators. We evaluate the proposed methods through simulation studies and apply the methods to an Alzheimer's disease study. We have developed an R package, seqTrun , for implementation of our method.