A requirement for microglial TLR4 in leukocyte recruitment into brain in response to lipopolysaccharide

To study the mechanisms involved in leukocyte recruitment induced by local bacterial infection within the CNS, we used intravital microscopy to visualize the interaction between leukocytes and the microvasculature in the brain. First, we showed that intracerebroventricular injection of LPS could cause significant rolling and adhesion of leukocytes in the brain postcapillary venules of wild-type mice, while negligible recruitment was observed in TLR4-deficient C57BL/10ScCr mice and CD14 knockout mice, suggesting recruitment is mediated by TLR4/CD14-bearing cells. Moreover, we observed reduced but not complete inhibition of recruitment in MyD88 knockout mice, indicating both MyD88-dependent and -independent pathways are involved. The leukocyte recruitment responses in chimeric mice with TLR4-positive microglia and endothelium, but TLR4-negative leukocytes, were comparable to normal wild-type mice, suggesting either endothelium or microglia play a crucial role in the induction of leukocyte recruitment. LPS injection induced both microglial and endothelial activation in the CNS. Furthermore, minocycline, an effective inhibitor of microglial activation, completely blocked the rolling and adhesion of leukocytes in the brain and blocked TNF-alpha production in response to LPS in vivo. Minocycline did not affect activation of endothelium by LPS in vitro. TNFR p55/p75 double knockout mice also exhibited significant reductions in both rolling and adhesion in response to LPS, indicating TNF-alpha signaling is critical for the leukocyte recruitment. Our results identify a TLR4 detection system within the blood-brain barrier. The microglia play the role of sentinel cells detecting LPS thereby inducing endothelial activation and leading to efficient leukocyte recruitment to the CNS.     

Proteins associated with the exon junction complex also control the alternative splicing of apoptotic regulators

Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-x(S) splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level.     

Analysis of the trap gene provides evidence for the role of elevation and vector abundance in the genetic diversity of Plasmodium relictum in Hawaii

ABSTRACT: BACKGROUND: The avian disease system in Hawaii offers an ideal opportunity to investigate host-pathogen interactions in a natural setting. Previous studies have recognized only a single mitochondrial lineage of avian malaria (Plasmodium relictum) in the Hawaiian Islands, but cloning and sequencing of nuclear genes suggest a higher degree of genetic diversity. METHODS: In order to evaluate genetic diversity of P. relictum at the population level and further understand host-parasite interactions, a modified single-base extension (SBE) method was used to explore spatial and temporal distribution patterns of single nucleotide polymorphisms (SNPs) in the thrombospondin-related anonymous protein (trap) gene of P. relictum infections from 121 hatch-year amakihi (Hemignathus virens) on the east side of Hawaii Island. RESULTS: Rare alleles and mixed infections were documented at three of eight SNP loci; this is the first documentation of genetically diverse infections of P. relictum at the population level in Hawaii. Logistic regression revealed that the likelihood of infection with a rare allele increased at low-elevation, but decreased as mosquito capture rates increased. The inverse relationship between vector capture rates and probability of infection with a rare allele is unexpected given current theories of epidemiology developed in human malarias. CONCLUSIONS: The results of this study suggest that pathogen diversity in Hawaii may be driven by a complex interaction of factors including transmission rates, host immune pressures, and parasite-parasite competition.     

Cuticular hydrocarbons on elytra of the Diaprepes root weevil Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae)

Abstract 1 External gland openings and associated structures on the elytra of teneral and mature Diaprepes root weevils, Diaprepes abbreviatus (L.), were elucidated by scanning electron microscopy (SEM). 2 There were clear differences between teneral, callow adults and fully mature adults. In the field, teneral adults remain in the pupal chamber in the soil until sclerotization of the cuticle is complete or nearly so. 3 Phenotypic variation of the elytra in this species consists of varying patterns and coloration of scaled intervals between a variable number of raised ridges devoid of scales. In addition to being thinner and lighter in colour than fully mature adults, the elytra of teneral adults were devoid of waxy hydrocarbon secretions. 4 External gland openings at the base of each scale were observed on teneral elytra and mature elytra washed with methylene chloride. 5 SEM evidence to document the production of waxy filaments by these glands and partial characterization of these by gas chromatography and mass spectrometry are presented.     

Investigation on the induction of 14-3-3 in white spruce

 A cDNA encoding a 14-3-3 protein was isolated from white spruce. The corresponding polypeptide contains several motifs that are conserved in this type of protein and is predicted to be 260 amino acids in length. Multiple banding in Southern blot analysis suggests that the gene encoding this cDNA is, in fact, part of a small family of genes. Wounding and chitosan treatment of spruce plants followed by Northern blot analysis indicates that these stimuli caused the accumulation of 14-3-3 mRNA. In addition, cell suspension cultures treated with methyl jasmonate showed up-regulation of 14-3-3-encoding mRNA. Chitosan and methyl jasmonate are both signalling molecules in the activation of plant defense response genes. Therefore, our results suggest a possible role for this 14-3-3 protein in the pathogen defense response of coniferous trees. Received: 13 December 1999 / Revision received: 21 August 2000 / Accepted: 22 August 2000     

Defining subregions of Hensen's node essential for caudalward movement, midline development and cell survival.

Hensen's node, also called the chordoneural hinge in the tail bud, is a group of cells that constitutes the organizer of the avian embryo and that expresses the gene HNF-3(&bgr;). During gastrulation and neurulation, it undergoes a rostral-to-caudal movement as the embryo elongates. Labeling of Hensen's node by the quail-chick chimera system has shown that, while moving caudally, Hensen's node leaves in its wake not only the notochord but also the floor plate and a longitudinal strand of dorsal endodermal cells. In this work, we demonstrate that the node can be divided into functionally distinct subregions. Caudalward migration of the node depends on the presence of the most posterior region, which is closely apposed to the anterior portion of the primitive streak as defined by expression of the T-box gene Ch-Tbx6L. We call this region the axial-paraxial hinge because it corresponds to the junction of the presumptive midline axial structures (notochord and floor plate) and the paraxial mesoderm. We propose that the axial-paraxial hinge is the equivalent of the neuroenteric canal of other vertebrates such as Xenopus. Blocking the caudal movement of Hensen's node at the 5- to 6-somite stage by removing the axial-paraxial hinge deprives the embryo of midline structures caudal to the brachial level, but does not prevent formation of the neural tube and mesoderm located posteriorly. However, the whole embryonic region generated posterior to the level of Hensen's node arrest undergoes widespread apoptosis within the next 24 hours. Hensen's node-derived structures (notochord and floor plate) thus appear to produce maintenance factor(s) that ensures the survival and further development of adjacent tissues.     

Nutrient couplings between on-site sewage disposal systems, groundwaters, and nearshore surface waters of the Florida Keys

We performed a one-year study to determine the effects of on-site sewage disposal systems (OSDS, septic tanks) on the nutrient relations of limestone groundwaters and nearshore surface waters of the Florida Keys. Monitor wells were installed on canal residences with OSDS and a control site in the Key Deer National Wildlife Refuge on Big Pine Key. Groundwater and surface water samples were collected monthly during 1987 and analyzed for concentrations of dissolved inorganic nitrogen (DIN = NO_f3/sup- + NO_f2/sup- + NH_4/su+), soluble reactive phosphate (SRP), temperature and salinity. Significant nutrient enrichment (up to 5000-fold) occurred in groundwaters contiguous to OSDS; DIN was enriched an average of 400-fold and SRP some 70-fold compared to control groundwaters. Ammonium was the dominant nitrogenous species and its concentration ranged from a low of 0.77 μM in control wells to 2.75 mM in OSDS-enriched groundwaters. Concentrations of nitrate plus nitrite were also highly enriched and ranged from 0.05 μM in control wells to 2.89 mM in enriched groundwaters. Relative to DIN, concentrations of SRP were low and ranged from 30 nM in control wells to 107 μM in enriched groundwaters. N : P ratios of enriched groundwaters were consistently > 100 and increased with increasing distance from the OSDS, suggesting significant, but incomplete, adsorption of SRP by subsurface flow through carbonate substrata. Nutrient concentrations of groundwaters also varied seasonally and were approximately two-fold higher during the winter (DIN = 1035 μM; SRP = 10.3 μM) compared to summer (DIN = 470 μM; SRP = 4.0 μM). In contrast, surface water nutrient concentrations were two-fold higher during the summer (DIN = 5.0 μM; SRP = 0.50 μM) compared to winter (DIN = 2.5 μM; SRP = 0.15 μM). Direct measurement of subsurface groundwater flow rate indicated that tides and increased groundwater recharge enhanced flow some two-fold and six-fold, respectively. Accordingly, the observed seasonal coupling of OSDS-derived nutrients from groundwaters to surface waters is maximum during summer because of seasonally maximum tides and increased hydraulic head during the summer wet season. The yearly average benthic flux of anthropogenic DIN into contiguous canal surface waters is 55 mmol m^-2 day^-1, a value some five-fold greater than the highest rate of benthic N-fixation measured in carbonate-rich tropical marine waters.     

cDNA sequence and deduced amino acid sequence of bovine oviductal fluid catalase

A bovine oviductal fluid catalase (OFC) which preferentially binds to the acrosome surface of some mammalian spermatozoa has recently been purified. The objectives of this study were to clone the OFC, obtain the full-length cDNA and protein sequence and determine which characteristics of the proteins are associated with the binding of the enzyme to sperm surface. Northern blot analysis revealed low levels of catalase mRNA in bovine oviducts and uterus compared to the liver and kidney. Screening of a cDNA library from the cow oviduct permit to obtain a full-length cDNA of 2282 bp, with an open reading frame of 1581 bp coding for a deduced protein of 526 amino acids (59 789 Da). The deduced protein contained four potential N-glycosylation sites and many potential O-glycosylation sites. The OFC protein exhibited high identity with catalase from other bovine tissues, likewise with catalases from human fibroblast and kidney, and with rat liver catalase. The homology of amino acid sequence of OFC with bovine liver catalase was about 99%. However the OFC posses an extended carboxyl terminus of 20 amino acids not present on the liver catalase. This result is supported by a lower mobility of the OFC compared to the liver catalase when both proteins are submitted on SDS-PAGE. Mol. Reprod. Dev. 51:265–273, 1998. © 1998 Wiley-Liss, Inc.