Differential expression and function of ABCG1 and ABCG4 during development and aging

ABCG1 and ABCG4 are highly homologous members of the ATP binding cassette (ABC) transporter family that regulate cellular cholesterol homeostasis. In adult mice, ABCG1 is known to be expressed in numerous cell types and tissues, whereas ABCG4 expression is limited to the central nervous system (CNS). Here, we show significant differences in expression of these two transporters during development. Examination of β-galactosidase-stained tissue sections from Abcg1^−/−LacZ and Abcg4^−/−LacZ knockin mice shows that ABCG4 is highly but transiently expressed both in hematopoietic cells and in enterocytes during development. In contrast, ABCG1 is expressed in macrophages and in endothelial cells of both embryonic and adult liver. We also show that ABCG1 and ABCG4 are both expressed as early as E12.5 in the embryonic eye and developing CNS. Loss of both ABCG1 and ABCG4 results in accumulation in the retina and/or brain of oxysterols, in altered expression of liver X receptor and sterol-regulatory element binding protein-2 target genes, and in a stress response gene. Finally, behavioral tests show that Abcg4^−/− mice have a general deficit in associative fear memory. Together, these data indicate that loss of ABCG1 and/or ABCG4 from the CNS results in changes in metabolic pathways and in behavior.     

Endemic Seed Plant Species from Hainan Island: A Checklist

Global conservation of plant biodiversity on tropical islands is a major priority, as approximately one third of all endangered plant species are insular endemics. Checklists can be an important first step in determining conservation priorities on islands. Hainan, the largest island in the Indo-Burma Biodiversity Hotspot, and therefore an international focus for conservation, has the most extensive and best preserved tropical forests in China. In this study we enumerate the endemic seed plants of Hainan Island. The checklist was prepared by consulting: (1) several bibliographic/taxonomic data base resources, (2) relevant taxonomic treatments and floras, and (3) plant taxonomists who are actively working with Chinese plants. The checklist also contains information concerning conservation status, the occurrence of Hainan endemics in four protected areas on the island, and available molecular phylogenies. An additional checklist of the species that were until recently thought to be endemic to Hainan, but are no longer considered to be, is also presented. In a separate paper in this issue of Botanical Review the patterns of endemism on Hainan Island are discussed.     

Mechano-electrical Transduction: New Insights into Old Ideas

The gating-spring theory of hair cell mechanotransduction channel activation was first postulated over twenty years ago. The basic tenets of this hypothesis have been reaffirmed in hair cells from both auditory and vestibular systems and across species. In fact, the basic findings have been reproduced in every hair cell type tested. A great deal of information regarding the structural, mechanical, molecular and biophysical properties of the sensory hair bundle and the mechanotransducer channel has accumulated over the past twenty years. The goal of this review is to investigate new data, using the gating spring hypothesis as the framework for discussion. Mechanisms of channel gating are presented in reference to the need for a molecular gating spring or for tethering to the intra- or extracellular compartments. Dynamics of the sensory hair bundle and the presence of motor proteins are discussed in reference to passive contributions of the hair bundle to gating compliance. And finally, the molecular identity of the channel is discussed in reference to known intrinsic properties of the native transducer channel.     

MICROTUBULE ORGANIZATION 1 regulates structure and function of microtubule arrays during mitosis and cytokinesis in the Arabidopsis root

MICROTUBULE ORGANIZATION 1 (MOR1) is a plant member of the highly conserved MAP215/Dis1 family of microtubule-associated proteins. Prior studies with the temperature-sensitive mor1 mutants of Arabidopsis (Arabidopsis thaliana), which harbor single amino acid substitutions in an N-terminal HEAT repeat, proved that MOR1 regulates cortical microtubule organization and function. Here we demonstrate by use of live cell imaging and immunolabeling that the mor1-1 mutation generates specific defects in the microtubule arrays of dividing vegetative cells. Unlike the universal cortical microtubule disorganization in elongating mor1-1 cells, disruption of mitotic and cytokinetic microtubule arrays was not detected in all dividing cells. Nevertheless, quantitative analysis identified distinct defects in preprophase bands (PPBs), spindles, and phragmoplasts. In nearly one-half of dividing cells at the restrictive temperature of 30 degrees C, PPBs were not detected prior to spindle formation, and those that did form were often disrupted. mor1-1 spindles and phragmoplasts were short and abnormally organized and persisted for longer times than in wild-type cells. The reduced length of these arrays predicts that the component microtubule lengths are also reduced, suggesting that microtubule length is a critical determinant of spindle and phragmoplast structure, orientation, and function. Microtubule organizational defects led to aberrant chromosomal arrangements, misaligned or incomplete cell plates, and multinucleate cells. Antiserum raised against an N-terminal MOR1 sequence labeled the full length of microtubules in interphase arrays, PPBs, spindles, and phragmoplasts. Continued immunolabeling of the disorganized and short microtubules of mor1-1 at the restrictive temperature demonstrated that the mutant mor1-1(L174F) protein loses function without dissociating from microtubules, providing important insight into the mechanism by which MOR1 may regulate microtubule length.     

Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation

The angiopoietins Ang-1 and Ang-2 have been identified as ligands of the receptor tyrosine kinase Tie-2 (refs. 1,2). Paracrine Ang-1-mediated activation of Tie-2 acts as a regulator of vessel maturation and vascular quiescence. In turn, the antagonistic ligand Ang-2 acts by an autocrine mechanism and is stored in endothelial Weibel-Palade bodies from where it can be rapidly released upon stimulation. The rapid release of Ang-2 implies functions of the angiopoietin-Tie system beyond its established role during vascular morphogenesis as a regulator of rapid vascular responses. Here we show that mice deficient in Ang-2 (encoded by the gene Angpt2) cannot elicit an inflammatory response in thioglycollate-induced or Staphylococcus aureus-induced peritonitis, or in the dorsal skinfold chamber model. Recombinant Ang-2 restores the inflammation defect in Angpt2(-/-) mice. Intravital microscopy showed normal TNF-alpha-induced leukocyte rolling in the vasculature of Angpt2(-/-)mice, but rolling cells did not firmly adhere to activated endothelium. Cellular experiments showed that Ang-2 promotes adhesion by sensitizing endothelial cells toward TNF-alpha and modulating TNF-alpha-induced expression of endothelial cell adhesion molecules. Together, these findings identify Ang-2 as an autocrine regulator of endothelial cell inflammatory responses. Ang-2 thereby acts as a switch of vascular responsiveness exerting a permissive role for the activities of proinflammatory cytokines.     

Identification of a point mutation in an esterase gene in different populations of the southern cattle tick, Boophilus microplus

Two esterase cDNA sequences were obtained from susceptible and organophosphorus resistant strains of Boophilus microplus. Both sequences have a high degree of homology to carboxylesterase B. One gene has identical sequences in both strains and the other showed two point mutations. One mutation produces an amino acid substitution when the amino acid sequence is deduced, this mutation was detected in six different populations susceptible and resistant to insecticides, but a pyrethroid resistant strain was the only one that showed only the mutant allele. Identification of this mutation and the strong signal detected in southern blot with this strain, suggest that esterases are contributing to detoxification of pyrethroid compounds, as a resistant mechanism in Mexican strains of the southern cattle tick.     

Vpu mediates depletion of interferon regulatory factor 3 during HIV infection by a lysosome-dependent mechanism

HIV has evolved sophisticated mechanisms to avoid restriction by intracellular innate immune defenses that otherwise serve to control acute viral infection and virus dissemination. Innate defenses are triggered when pattern recognition receptor (PRR) proteins of the host cell engage pathogen-associated molecule patterns (PAMPs) present in viral products. Interferon regulatory factor 3 (IRF3) plays a central role in PRR signaling of innate immunity to drive the expression of type I interferon (IFN) and interferon-stimulated genes (ISGs), including a variety of HIV restriction factors, that serve to limit viral replication directly and/or program adaptive immunity. Productive infection of T cells by HIV is dependent upon the targeted proteolysis of IRF3 that occurs through a virus-directed mechanism that results in suppression of innate immune defenses. However, the mechanisms by which HIV controls innate immune signaling and IRF3 function are not defined. Here, we examined the innate immune response induced by HIV strains identified through their differential control of PRR signaling. We identified viruses that, unlike typical circulating HIV strains, lack the ability to degrade IRF3. Our studies show that IRF3 regulation maps specifically to the HIV accessory protein Vpu. We define a molecular interaction between Vpu and IRF3 that redirects IRF3 to the endolysosome for proteolytic degradation, thus allowing HIV to avoid the innate antiviral immune response. Our studies reveal that Vpu is an important IRF3 regulator that supports acute HIV infection through innate immune suppression. These observations define the Vpu-IRF3 interface as a novel target for therapeutic strategies aimed at enhancing the immune response to HIV.     

Control of innate immune signaling and membrane targeting by the Hepatitis C virus NS3/4A protease are governed by the NS3 helix α0

Hepatitis C virus (HCV) infection is sensed in the host cell by the cytosolic pathogen recognition receptor RIG-I. RIG-I signaling is propagated through its signaling adaptor protein MAVS to drive activation of innate immunity. However, HCV blocks RIG-I signaling through viral NS3/4A protease cleavage of MAVS on the mitochondrion-associated endoplasmic reticulum (ER) membrane (MAM). The multifunctional HCV NS3/4A serine protease is associated with intracellular membranes, including the MAM, through membrane-targeting domains within NS4A and also at the amphipathic helix α(0) of NS3. The serine protease domain of NS3 is required for both cleavage of MAVS, a tail-anchored membrane protein, and processing the HCV polyprotein. Here, we show that hydrophobic amino acids in the NS3 helix α(0) are required for selective cleavage of membrane-anchored portions of the HCV polyprotein and for cleavage of MAVS for control of RIG-I pathway signaling of innate immunity. Further, we found that the hydrophobic composition of NS3 helix α(0) is essential to establish HCV replication and infection. Alanine substitution of individual hydrophobic amino acids in the NS3 helix α(0) impaired HCV RNA replication in cells with a functional RIG-I pathway, but viral RNA replication was rescued in cells lacking RIG-I signaling. Therefore, the hydrophobic amphipathic helix α(0) of NS3 is required for NS3/4A control of RIG-I signaling and HCV replication by directing the membrane targeting of both viral and cellular substrates.     

Development of natural growth regimes for hatchery-reared steelhead to reduce residualism, fitness loss, and negative ecological interactions

Wild steelhead (Oncorhynchus mykiss) typically spend two or more years in freshwater before migrating to sea, but hatchery steelhead are almost ubiquitously released as yearlings. Their large size at release coupled with life history pathways that include both male and female maturation in freshwater present ecological risks different from those posed by hatchery populations of Pacific salmon. Yearling hatchery reared steelhead that fail to attain minimum thresholds for smoltification or exceed thresholds for male maturation tend to ‘residualize’ (i.e., remain in freshwater). Residuals pose ecological risks including size-biased interference competition and predation on juvenile salmon and trout. Three hatchery populations of steelhead in Hood Canal, WA were reared under growth regimes designed to produce a more natural age at smoltification (age-2) to aid in rebuilding their respective natural populations. Mean smolt sizes and size variability at age-2 were within the range of wild smolts for two of the three populations. The third population reared at a different facility under similar temperatures exhibited high growth rate variability and high male maturation rates (20% of all released fish). Experimentally comparing age-1 and age-2 smolt programs will help identify optimal rearing strategies to reduce the genetic risk of domestication selection and reduce residualism rates and associated negative ecological effects on natural populations. Investigations of Winthrop National Fish Hatchery summer-run steelhead will measure a) selection on correlated behavioral traits (‘behavioral syndromes’), b) degree of smoltification, c) changes in hormones that regulate gonad growth at key developmental stages, and d) conduct extensive post-release monitoring of fish reared under each growth regime.