Differential Histopathology and Chemokine Gene Expression in Lung Tissues following Respiratory Syncytial Virus (RSV) Challenge of Formalin-Inactivated RSV- or BBG2Na-Immunized Mice

A BALB/c mouse model of enhanced pulmonary pathology following vaccination with formalin-inactivated alum-adsorbed respiratory syncytial virus (FI-RSV) and live RSV challenge was used to determine the type and kinetics of histopathologic lesions induced and chemokine gene expression profiles in lung tissues. These data were compared and contrasted with data generated following primary and/or secondary RSV infection or RSV challenge following vaccination with a promising subunit vaccine, BBG2Na. Severe peribronchiolitis and perivascularitis coupled with alveolitis and interstitial inflammation were the hallmarks of lesions in the lungs of FI-RSV-primed mice, with peak histopathology evident on days 5 and 9. In contrast, primary RSV infection resulted in no discernible lesions, while challenge of RSV-primed mice resulted in rare but mild peribronchiolitis and perivascularitis, with no evidence of alveolitis or interstitial inflammation. Importantly, mice vaccinated with a broad dose range (20 to 0.02 microg) of a clinical formulation of BBG2Na in aluminium phosphate demonstrated histopathology similar to that observed in secondary RSV infection. At the molecular level, FI-RSV priming was characterized by a rapid and strong up-regulation of eotaxin and monocyte chemotactic protein 3 (MCP-3) relative gene expression (potent lymphocyte and eosinophil chemoattractants) that was sustained through late time points, early but intermittent up-regulation of GRO/melanoma growth stimulatory activity gene and inducible protein 10 gene expression, while macrophage inflammatory protein 2 (MIP-2) and especially MCP-1 were up-regulated only at late time points. By comparison, primary RSV infection or BBG2Na priming resulted in considerably lower eotaxin and MCP-3 gene expression increases postchallenge, while expression of lymphocyte or monocyte chemoattractant chemokine genes (MIP-1beta, MCP-1, and MIP-2) were of higher magnitude and kinetics at early, but not late, time points. Our combined histopathologic and chemokine gene expression data provide a basis for differentiating between aberrant FI-RSV-induced immune responses and normal responses associated with RSV infection in the mouse model. Consequently, our data suggest that BBG2Na may constitute a safe RSV subunit vaccine for use in seronegative infants.     

Study of asparagine 353 in aminopeptidase A: characterization of a novel motif (GXMEN) implicated in exopeptidase specificity of monozinc aminopeptidases.

Aminopeptidase A (EC 3.4.11.7, APA) is a 160 kDa membrane-bound zinc enzyme that contains the HEXXH consensus sequence found in members of the zinc metalloprotease family, the zincins. In addition, the monozinc aminopeptidases are characterized by another conserved motif, GXMEN, the glutamate residue of which has been shown to be implicated in the exopeptidase specificity of aminopeptidase A [Vazeux G. (1998) Biochem. J. 334, 407-413]. In carboxypeptidase A (EC 3.4.17.1, CPA), the exopeptidase specificity is conferred by an arginine residue (Arg-145) and an asparagine residue (Asn-144). Thus, we hypothesized that Asn-353 of the GXMEN motif in APA plays a similar role to Asn-144 in CPA and contributes to the exopeptidase specificity of APA. We investigated the functional role of Asn-353 in APA by substituting this residue with a glutamine (Gln-353), an alanine (Ala-353) or an aspartate (Asp-353) residue by site-directed mutagenesis. Expression of wild-type and mutated APAs revealed that Gln-353 and Ala-353 are similarly routed and glycosylated to the wild-type APA, whereas Asp-353 is trapped intracellularly and partially glycosylated. Kinetic studies, using alpha-L-glutamyl-beta-naphthylamide (GluNA) as a substrate showed that the K(m) values of the mutants Gln-353 and Ala-353 were increased 11- and 8-fold, respectively, whereas the k(cat) values were decreased (2-fold) resulting in a 24- and 14-fold reduction in cleavage efficiency. When alpha-L-aspartyl-beta-naphthylamide or angiotensin II were used as substrates, the mutations had a greater effect on k(cat), leading to a similar decrease in cleavage efficiencies as that observed with GluNA. We then measured the inhibitory potencies of several classes of inhibitors, glutamate thiol, glutamine thiol and two isomers (L- or D-) of glutamate phosphonate to explore the functional role of Asn-353. The data indicate that Asn-353 is critical for the integrity and catalytic activity of APA. This residue is involved in substrate binding via interactions with the free N-terminal part and with the P1 carboxylate side chain of the substrate. In conclusion, Asn-353 of the GXMEN motif, together with Glu-352, contributes to the exopeptidase specificity of APA and plays an equivalent role to Asn-144 in CPA.     

Rab11 polarization of the Drosophila oocyte: a novel link between membrane trafficking, microtubule organization, and oskar mRNA localization and translation.

The Drosophila embryonic body plan is specified by asymmetries that arise in the oocyte during oogenesis. These asymmetries are apparent in the subcellular distribution of key mRNAs and proteins and in the organization of the microtubule cytoskeleton. We present evidence that the Drosophila oocyte also contains important asymmetries in its membrane trafficking pathways. Specifically, we show that alpha-adaptin and Rab11, which function critically in the endocytic pathways of all previously examined animal cells, are localized to neighboring compartments at the posterior pole of stage 8-10 oocytes. Rab11 and alpha-adaptin localization occurs in the absence of a polarized microtubule cytoskeleton, i.e. in grk null mutants, but is later reinforced and/or refined by Osk, the localization of which is microtubule dependent. Analyses of germline clones of a rab11 partial loss-of-function mutation reveal a requirement for Rab11 in endocytic recycling and in the organization of posterior membrane compartments. Such analyses also reveal a requirement for Rab11 in the organization of microtubule plus ends and osk mRNA localization and translation. We propose that microtubule plus ends and, possibly, translation factors for osk mRNA are anchored to posterior membrane compartments that are defined by Rab11-mediated trafficking and reinforced by Rab11-Osk interactions.     

Pathways of carbon cycling in marine surface waters: the fate of small-sized phytoplankton in the Northeast Water Polynya

The fate of small-sized phytoplankton (<5 µm) and pathwaysof carbon cycling in surface waters, i.e. recycling within orexport out of the mixed layer, were investigated in the NortheastWater (NEW) Polynya (77–81°N) from 23 May to 22 July1993. The sampling covered a wide range of ice, hydrographicand nutrient conditions. Chlorophyll a concentrations, phytoplanktonproduction rates and zooplankton abundances were determinedin the field, and potential rates of grazing by protozoa, copepodsand appendicularians were calculated from abundances, usingassumptions from the literature. To our knowledge, this is thefirst published attempt to assess concurrently the grazing ofthese three plankton groups in the Arctic. The production rateof small-sized phytoplankton was significantly higher in ice-freecompared with ice-covered areas, but the biomasses of small-sizedphytoplankton and zooplankton were not. Potential recycling,downward export and horizontal advection of phytoplankton werecalculated by resolving carbon budgets for the mixed layer.A large fraction of the small-sized phytoplankton produced insidethe polynya was advected horizontally to the ice-covered partof the NEW, where these algae were necessary to sustain theheterotrophic community. We conclude that the fate of small-sizedphytoplankton production was mostly recycling (>70%). Downwardexport would have occurred infrequently, as a result of intensegrazing by appendicularians. Size-differential pathways of carboncycling in planktonic food webs are discussed.     

High-performance liquid chromatographic method for the determination of di(2-ethylhexyl) phthalate in total parenteral nutrition and in plasma

A simple, rapid and sensitive reversed-phase high-performance liquid chromatographic method with UV detection was developed for the quantification of di(2-ethylhexyl) phthalate (DEHP) in parenteral nutrition admixtures containing fat emulsion and in plasma samples of children daily treated by total parenteral nutrition. The analyte and the internal standard, di-n-heptyl phthalate, were extracted twice using hexane and the organic layer separated and dried under nitrogen. The residues were reconstituted with acetonitrile and 20 μl was injected into a Waters Spherisorb C₁₈ column, the UV detector was set at 202 nm. The mobile phase was acetonitrile–aqueous buffer (triethylamine 0.08% adjusted to pH 2.8 with 1 M phosphoric acid) mixture (88:12, v/v) and it was pumped at 1 ml/min. Average recoveries were 97% or greater. This method was successfully used to investigate the amounts of DEHP which can leach from bags and tubing into fat emulsion and which could contaminate children under long-term parenteral nutrition. On the other hand, the circulating DEHP concentrations were estimated in four children under regular long-term parenteral nutrition.     

Contingent vs. noncontingent EMG feedback and hand temperature in relation to anxiety and locus of control

This study was designed to measure the effects of contingent and noncontingent EMG feedback on hand temperature, anxiety, and locus of control. Two groups of six subjects each were selected on the basis of high test-anxiety scores. The groups participated in a reverse design study in which Group 1 received five sessions of contingent EMG ffedback followed by five sessions of noncontingent feedback. Group 2 received noncontingent feedback followed by contingent feedback. Results indicate a significant order of treatment effect. Subjects who received contingent feedback first produced lower EMG readings, lower test-anxiety scores, and higher hand temperatures during noncontingent feedback sessions. Receiving noncontingent feedback first may actually have interfered with utilizing contingent feedback.     

Multiparasitism of stink bug eggs: competitive interactions between Ooencyrtus pityocampae and Trissolcus agriope

Females of Trissolcus agriope (Platygastridae) avoid host (Brachynema signatum) eggs parasitized by conspecifics or Ooencyrtus pityocampae (Encyrtidae), but females of the latter species will superparasitize and multiparasitize, although they mostly attack unparasitized eggs. Females of T. agriope were more efficient in the laboratory and parasitized more hosts in 24 and 48 h. In multiparasitized hosts, O. pityocampae was a superior larval competitor and could complete also development as a facultative hyperparasitoid. When females of both species foraged together, T. agriope parasitized significantly more than O. pityocampae, about 50 % in both cases, although O. pityocampae almost doubled its parasitism rate as the exposure period was lengthened from 24 to 48 h. When O. pityocampae followed T. agriope in sequential foraging bouts, the former species successfully parasitized more hosts than the latter. The advisability of co-releases of both species to improve biological control of first generation B. signatum in Iranian pistachio orchards is discussed.     

Over-production, purification and properties of the uridine-diphosphate-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate ligase from Escherichia coli

The UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase was over-produced and purified from two plasmid-harbouring strains of Escherichia coli. The first strain, E. coli JM83(pHE5), gave a 15-fold over-production relative to parental strain. The enzyme could be partially purified (8.8-fold) by ion-exchange chromatography. With the second strain, E. coli JM83(pMLD25), a very strong over-production was obtained, since the enzyme represented about 20% of the cytoplasmic proteins. Purification yielded 77% protein homogeneity. However, the enzymatic activity, which was very unstable, was lost during the purification procedure. Several properties of the enzyme were studied. The enzyme gave maximal activity around pH 8. The isoelectric point was 5.2. The activity was increased by potassium phosphate. Reverse and exchange reactions could be catalysed. The N-terminal sequence of the protein was determined and correlated with the nucleotide sequence of the murE gene. The actual initiation codon was assigned.     

Early Deficits in Glycolysis Are Specific to Striatal Neurons from a Rat Model of Huntington Disease

In Huntington disease (HD), there is increasing evidence for a link between mutant huntingtin expression, mitochondrial dysfunction, energetic deficits and neurodegeneration but the precise nature, causes and order of these events remain to be determined. In this work, our objective was to evaluate mitochondrial respiratory function in intact, non-permeabilized, neurons derived from a transgenic rat model for HD compared to their wild type littermates by measuring oxygen consumption rates and extracellular acidification rates. Although HD striatal neurons had similar respiratory capacity as those from their wild-type littermates when they were incubated in rich medium containing a supra-physiological glucose concentration (25 mM), pyruvate and amino acids, respiratory defects emerged when cells were incubated in media containing only a physiological cerebral level of glucose (2.5 mM). According to the concept that glucose is not the sole substrate used by the brain for neuronal energy production, we provide evidence that primary neurons can use lactate as well as pyruvate to fuel the mitochondrial respiratory chain. In contrast to glucose, we found no major deficits in HD striatal neurons’ capacity to use pyruvate as a respiratory substrate compared to wild type littermates. Additionally, we used extracellular acidification rates to confirm a reduction in anaerobic glycolysis in the same cells. Interestingly, the metabolic disturbances observed in striatal neurons were not seen in primary cortical neurons, a brain region affected in later stages of HD. In conclusion, our results argue for a dysfunction in glycolysis, which might precede any defects in the respiratory chain itself, and these are early events in the onset of disease.