Complementation of α-thalassaemia in α-globin Knockout Mice with a 191 kb Transgene Containing the Human α-Globin Locus

alpha-thalassaemia is an inherited blood disorder caused by a decrease in the synthesis of alpha-globin due to mutations in one or both of the alpha-globin genes located on human chromosome 16. A 191 kb transgene derived from a sequenced bacterial artificial chromosome (BAC) clone carrying the human alpha-globin gene cluster, together with about 100 kb of sequence upstream of DNase1 hypersensitive site HS-40 and 30 kb downstream of the alpha1-globin gene, was introduced into fertilised mouse oocytes by pronuclear microinjection. Three transgenic founder mice were obtained. Analysis of one transmitting line by fluorescent in situ hybridisation and quantitative PCR demonstrated a single copy integration of the human alpha-globin transgene on chromosome 1. Analysis of haemoglobins from the peripheral blood by cellulose acetate electrophoresis and high performance liquid chromatography (HPLC) demonstrated synthesis of human alpha-globin to about 36% of the level of each mouse alpha-globin locus. Breeding of transgenic mice with mice heterozygous for a knockout (KO) deletion of both murine alpha-globin genes showed that the human alpha-globin locus restored haemoglobin levels and red cell distribution width to normal in double heterozygous mice and significantly normalised other haematological parameters. Interestingly the human transgene also induced a significant increase in red cell production and haematocrit above wild type values. This is the first report demonstrating complementation of a murine alpha-globin KO mutation by human alpha-globin gene expression from an intact human alpha-globin locus. The transgenic mouse model described in this report should be very useful for the study of human alpha-globin gene regulation and for the development of strategies to down regulate alpha-globin production as a means of ameliorating the severity of beta-thalassaemia.     

Characterization and modelling of the hydrophobic domain of a sunflower oleosin

The oleosins are a group of hydrophobic proteins present on the surface of oil bodies in seeds, where they are thought to prevent coalescence. They contain a central hydrophobic domain of 68-74 residues that is thought to form a loop into the triacylglycerol matrix of the oil body, but the conformation adopted by this sequence is uncertain. We have therefore expressed an oleosin cDNA from sunflower (Helianthus annuus L.) in Escherichia coli as a fusion with maltose-binding protein (MBP) and isolated a peptide corresponding to the hydrophobic domain by sequential digestion with factor Xa (to remove the MBP) followed by trypsin and Staphylococcus V8 protease to remove the N- and C-terminal domains of the oleosin. Circular dichroism spectroscopy of the peptide in two solvent systems chosen to mimic the environment within the oil body (trifluoroethanol and SDS) demonstrated high proportions of alpha-helical structure, with no beta-sheet. A model was therefore developed in which the domain forms an alpha-helical hairpin structure, the two helices being separated by a turn region. We consider that this model is consistent with our current knowledge of oleosin structure and properties.     

Activation of mitogen-activated protein kinase and NF-kappaB pathways by a Kaposi's sarcoma-associated herpesvirus K15 membrane protein

The K15 gene of Kaposi's sarcoma-associated herpesvirus (also known as human herpesvirus 8) consists of eight alternatively spliced exons and has been predicted to encode membrane proteins with a variable number of transmembrane regions and a common C-terminal cytoplasmic domain with putative binding sites for SH2 and SH3 domains, as well as for tumor necrosis factor receptor-associated factors. These features are reminiscent of the latent membrane proteins LMP-1 and LMP2A of Epstein-Barr virus and, more distantly, of the STP, Tip, and Tio proteins of the related gamma(2)-herpesviruses herpesvirus saimiri and herpesvirus ateles. These viral membrane proteins can activate a number of intracellular signaling pathways. We have therefore examined the abilities of different K15-encoded proteins to initiate intracellular signaling. We found that a 45-kDa K15 protein derived from all eight K15 exons and containing 12 predicted transmembrane domains in addition to the cytoplasmic domain activated the Ras/mitogen-activated protein kinase (MAPK) and NF-kappaB pathways, as well as (more weakly) the c-Jun N-terminal kinase/SAPK pathway. Activation of the MAPK and NF-kappaB pathways required phosphorylation of tyrosine residue 481 within a putative SH2-binding site (YEEVL). This motif was phosphorylated by the tyrosine kinases Src, Lck, Yes, Hck, and Fyn. The region containing the YEEVL motif interacted with tumor necrosis factor receptor-associated factor 2 (TRAF-2), and a dominant negative TRAF-2 mutant inhibited the K15-mediated activation of the Ras/MAPK pathway, suggesting the involvement of TRAF-2 in the initiation of these signaling routes. In contrast, several smaller K15 protein isoforms activated these pathways only weakly. All of the K15 isoforms tested were, however, localized in lipid rafts, suggesting that incorporation into lipid rafts is not sufficient to initiate signaling. Additional regions of K15, located presumably in exons 2 to 5, may therefore contribute to the activation of these pathways. These findings illustrate that the 45-kDa K15 protein engages pathways similar to LMP1, LMP2A, STP, Tip, and Tio but combines functional features that are separated between LMP1 and LMP2A or STP and Tip.     

Population dynamics, distribution, and growth rate of tilapia (Oreochromis mossambicus) in the Salton Sea, California, with notes on bairdiella (Bairdiella icistia) and orangemouth corvina (Cynoscion xanthulus)

The Salton Sea is a highly saline lake that has long supported sportfishery and large populations of fish-eating birds. A study was initiated in 1999 to assess the status of orangemouth corvina (Cynoscion xanthulus), bairdiella (Bairdiella icistia) and tilapia (Oreochromis mossambicus × O. urolepis). Multimesh (50 × 2 m) gillnets were set at nine stations in 1999, ten stations in 2000 and six stations in 2002. These stations were sampled every two months in 1999, every three months in 2000 and once in 2002. O. mossambicus was the most abundant of the four species, with a maximum mean catch per unit effort (CPUE) 13.8 kg net⁻¹ h⁻¹ or 29.9 fish net⁻¹ h⁻¹ being observed at the river mouth stations in August 1999. From spring to summer, tilapia CPUE increased at nearshore and river mouth stations and decreased at pelagic stations, apparently reflecting migration away from midlake areas in response to anoxia or hypoxia caused by periodic springtime overturn events in deep waters. Tilapia catches in nearshore, river mouth and pelagic habitats were 83 and 60% males in 1999 and 2000, respectively. Tilapia catches in rivers in August 1999 averaged only 6% male. During 1999–2000, the tilapia population consisted essentially of only the 1995 and 2000 year classes. Harsh conditions at the Salton Sea have led to erratic reproduction and survival rates and unstable age structures for its resident fishes. Massive parasite infestations of fry and physiological stressors such as anoxia, high sulfide levels, high salinity and high and low temperatures are potential causes of the irregular recruitment and periodic dieoffs of tilapia. The abundance of all fish species declined over the years of study. Between 1999 and 2002, the late summer mean CPUEs for tilapia, bairdiella and orangemouth corvina at four nearshore stations dropped from 16 fish to 0.02 fish, from 4.7 fish net to 0.23 fish, and from 0.08 fish to 0.02 fish, respectively. During 2000–2003, parallel declines occurred in estimated numbers of adult fish involved in mass mortality events at the Sea. The boom-and-bust dynamics of tilapia and other fish populations in the Sea have major consequences for fish-eating bird populations, for other components of the ecosystem, and for the recreational value of the lake. Guest Editor: John M. Melack Saline Waters and their Biota     

Eosinophils and IL-4 Support Nematode Growth Coincident with an Innate Response to Tissue Injury

It has become increasingly clear that the functions of eosinophils extend beyond host defense and allergy to metabolism and tissue regeneration. These influences have strong potential to be relevant in worm infections in which eosinophils are prominent and parasites rely on the host for nutrients to support growth or reproduction. The aim of this study was to investigate the mechanism underlying the observation that eosinophils promote growth of Trichinella spiralis larvae in skeletal muscle. Our results indicate that IL-4 and eosinophils are necessary for normal larval growth and that eosinophils from IL-4 competent mice are sufficient to support growth. The eosinophil-mediated effect operates in the absence of adaptive immunity. Following invasion by newborn larvae, host gene expression in skeletal muscle was compatible with a regenerative response and a shift in the source of energy in infected tissue. The presence of eosinophils suppressed local inflammation while also influencing nutrient homeostasis in muscle. Redistribution of glucose transporter 4 (GLUT4) and phosphorylation of Akt were observed in nurse cells, consistent with enhancement of glucose uptake and glycogen storage by larvae that is known to occur. The data are consistent with a mechanism in which eosinophils promote larval growth by an IL-4 dependent mechanism that limits local interferon-driven responses that otherwise alter nutrient metabolism in infected muscle. Our findings document a novel interaction between parasite and host in which worms have evolved a strategy to co-opt an innate host cell response in a way that facilitates their growth.     

A 191-kb genomic fragment containing the human α-globin locus can rescue α-thalassemic mice

A 191-kb human bacterial artificial chromosome (BAC) containing the human α-globin genomic locus was used to generate transgenic mice that express, exclusively, human α-globin (^huα-globin). Expression of ^huα-globin reaches a level of 36% of that of endogenous mouse α-globin (^muα-globin) on a heterozygous mouse α-thalassemia background (^muα-globin knockout, ^muα^+/−). Hemizygous transgenic mice carrying the ^huα-globin locus on a heterozygous knockout background (^huα^+/0, ^muα^++/−−) demonstrated complementation of most hematologic parameters. By crossing ^huα^+/0, ^muα^++/−− mice, we were able to generate mice entirely dependent on ^huα-globin synthesis. Breeding and fluorescent in situ hybridization studies demonstrate that only mice homozygous for the transgene were able to rescue embryonic lethal homozygous ^muα-globin knockout embryos (^muα^{−−/−−}). Adult rescued mice produce hemoglobin at levels similar to wild-type mice, with partial red cell complementation based on mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and red cell distribution width (RDW) measurements. Significant erythrocythemia above wild-type levels seems to be the main compensatory mechanism for the normalization of the hemoglobin levels in the rescued animals. Our studies demonstrate that the ^huα-globin locus in the 191-kb transgene contains all the necessary elements for the regulated expression of ^huα-globin in transgenic mice. This animal model should be valuable for studying the mechanisms regulating ^huα-globin production and for development of therapeutic strategies for β-thalassemia based on downregulation of α-globin expression. We dedicate this article to the memory of our valued friend and colleague Panayiotis A. Ioannou who passed away during the completion of this work     

Transgenic mice expressing the <Emphasis Type="Italic">Peripherin-EGFP</Emphasis> genomic reporter display intrinsic peripheral nervous system fluorescence

The development of homologous recombination methods for the precise modification of bacterial artificial chromosomes has allowed the introduction of disease causing mutations or fluorescent reporter genes into human loci for functional studies. We have introduced the EGFP gene into the human PRPH-1 locus to create the Peripherin-EGFP (hPRPH1-G) genomic reporter construct. The hPRPH1-G reporter was used to create transgenic mice with an intrinsically fluorescent peripheral nervous system (PNS). During development, hPRPH1-G expression was concomitant with the acquisition of neuronal cell fate and growing axons could be observed in whole embryo mounts. In the adult, sensory neurons were labeled in both the PNS and central nervous system, while motor neurons in the spinal cord had more limited expression. The fusion protein labeled long neuronal processes, highlighting the peripheral circuitry of hPRPH1-G transgenic mice to provide a useful resource for a range of neurobiological applications.     

KeR-EGI,a new index of gait quantification based on electromyography

PurposeTo define a new index of gait pathology in adults based on electromyographic data: the Ker-EGI for Kerpape-Rennes EMG-based Gait Index. The principle is similar to the one of Gait Deviation Index but using EMG profiles instead of joint angles. It first needs to build a database of healthy subjects gait to be able then to quantify the deviation of one peculiar patient’s gait from this typical behavior.MethodsNinety adults (59 healthy and 31 pathological) participated to this study. All pathological subjects had a diagnosis of central nervous system disorder. On each subject we collected the joint angles and the activation profile of seven muscles of each lower limb. Moreover, we recorded two videos (face and profile) of each patient to compute his/her Edinburgh Visual Gait Score (EVGS). Then for each patient, we computed the GGI (Gillette Gait Index), the GDI (Gait Deviation Index) and the Ker-EGI.ResultsCorrelation Ker-EGI and each of the three kinematical indices (GGI, GDI, EVGS) is fair to good (respectively R² = 0.62, 0.42, and 0.69).ConclusionKeR-EGI is a valid index to evaluate gait and is complementary to one of these three kinematical indices providing synthetic vision on patients’ motor control abilities.     

Anti-HIV-1 Activities of 1,3-Dioxolane Guanine and 2,6-Diaminopurine Dioxolane

Abstract

DXG and its prodrug DAPD have been demonstrated to be effective inhibitors of HIV-1 in various cells. The EC₅₀s for DXG were 0.032 μM in CBMCs and 0.05 μM in MT-4 cells, which were generally equipotent as 3TC. 3TC-resistant, but not AZT-resistant, HIV-1 had minimum diminished sensitivity to the compounds. Both DXG and DAPD were non-toxic to cells up to 500 μM.