Pituitary adenylate cyclase-activating polypeptide is up-regulated in cortical pyramidal cells after focal ischemia and protects neurons from mild hypoxic/ischemic damage

The protective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) in stroke models is poorly understood. We studied patterns of PACAP, vasoactive intestinal peptide, and the PACAP-selective receptor PAC1 after middle cerebral artery occlusion and neuroprotection by PACAP in cortical cultures exposed to oxygen/glucose deprivation (OGD). Within hours, focal ischemia caused a massive, NMDA receptor (NMDAR)-dependent up-regulation of PACAP in cortical pyramidal cells. PACAP expression dropped below the control level after 2 days and was normalized after 4 days. Vasoactive intestinal peptide expression was regulated oppositely to that of PACAP. PAC1 mRNA showed ubiquitous expression in neurons and astrocytes with minor changes after ischemia. In cultured cortical neurons PACAP27 strongly activated Erk1/2 at low and p38 MAP kinase at higher nanomolar concentrations via PAC1. In astrocyte cultures, effects of PACAP27 on Erk1/2 and p38 were weak. During OGD, neurons showed severely reduced Erk1/2 activity and dephosphorylation of Erk1/2-regulated Ser112 of pro-apoptotic Bad. PACAP27 stimulation counteracted Erk1/2 inactivation and Bad dephosphorylation during short-term OGD but was ineffective after expanded OGD. Consistently, PACAP27 caused MEK-dependent neuroprotection during mild but not severe hypoxic/ischemic stress. While PACAP27 protected neurons at 1–5 nmol/L, full PAC1 activation by 100 nmol/L PACAP exaggerated hypoxic/ischemic damage. PACAP27 stimulation of astrocytes increased the production of Akt-activating factors and conferred ischemic tolerance to neurons. Thus, ischemia-induced PACAP may act via neuronal and astroglial PAC1. PACAP confers protection to ischemic neurons by maintaining Erk1/2 signaling via neuronal PAC1 and by increasing neuroprotective factor production via astroglial PAC1.     

Seasonal and wastewater stream variation of trace organic compounds in a dairy processing plant aerobic bioreactor

Bioreactors are often an integral part of dairy factory efforts to reduce the biological oxygen demand of their wastewater. In this study, infeed, mixed liquor and supernatant samples of an aerobic bioreactor used by a dairy factory in South-Eastern Australia were analyzed for nutrients and organic compounds using gas chromatography-mass spectrometry and physicochemical analyses. Despite different concentrations of organic inputs into the bioreactor, nutrients and trace organic compounds were reduced significantly (i.e. average concentration of trace organic compounds: infeed = 1681 μg/L; mixed liquor = 257 μg/L; supernatant = 23 μg/L). However, during one sampling period the bioreactor was adversely affected by the organic loading. Trace organic compounds in the samples were predominantly fatty acids associated with animal products. The analyses suggest that it is possible to trace a disruptive input (i.e. infeed with high organic carbon concentrations) into an aerobic bioreactor by measuring concentrations of fatty acids or ammonia.     

Orchard pollination in Capitol Reef National Park,Utah, USA. Honey bees or native bees?

Capitol Reef National Park in central Utah, USA surrounds 22 managed fruit orchards started over a century ago by Mormon pioneers. Honey bees are imported for pollination, although the area in which the Park is embedded has over 700 species of native bees, many of which are potential orchard pollinators. We studied the visitation of native bees to apple, pear, apricot, and sweet cherry over 2 years. Thirty species of bees visited the flowers but, except for pear flowers, most were uncommon compared to honey bees. Evidence that honey bees prevented native bees from foraging on orchard crop flowers was equivocal: generally, honey bee and native bee visitation rates to the flowers were not negatively correlated, nor were native bee visitation rates positively correlated with distance of orchards from honey bee hives. Conversely, competition was tentatively suggested by much larger numbers of honey bees than natives on the flowers of apples, apricots and cherry; and by the large increase of native bees on pears, where honey bee numbers were low. At least one-third of the native bee species visiting the flowers are potential pollinators, including cavity-nesting species such as Osmia lignaria propinqua, currently managed for small orchard pollination in the US, plus several fossorial species, including one rosaceous flower specialist (Andrena milwaukiensis). We suggest that gradual withdrawal of honey bees from the Park would help conserve native bee populations without decreasing orchard crop productivity, and would serve as a demonstration of the commercial value of native pollinators.     

Low diversity and high levels of population genetic structuring in introduced eastern mosquitofish (<Emphasis Type="Italic">Gambusia holbrooki</Emphasis>) in the greater Melbourne area,Australia

Eastern mosquitofish (Gambusia holbrooki) were introduced into Australia in 1925 and released to control mosquitoes. Gambusia holbrooki rapidly became invasive in recipient environments and now threaten native fauna. In this study, we used five polymorphic microsatellite loci and sequence from two mitochondrial genes, cytochrome b and cytochrome oxidase I, to evaluate genetic variation, colonisation and movement patterns of introduced G. holbrooki in the greater Melbourne area, and to assist in identifying the feasibility of local eradication. Microsatellite variation was consistently low within populations and there was evidence of bottleneck events for several populations. Populations displayed significant structuring associated with river basins rather than geographic distance, suggesting that habitat connectivity is important for dispersal. However, a few populations within river basins were more closely related to populations in other river basins than within their own basin, most likely reflecting a role of human-assisted dispersal in population establishment. Mitochondrial sequencing revealed only a single haplotype and suggested all populations were founded by individuals from a common source. These genetic data help delineate boundaries for local management strategies.     

Phylogeny of the Gadidae (sensu Svetovidov, 1948) based on their morphology and two mitochondrial genes

Although Codfishes are probably one of the most studied groups of all teleost fishes worldwide owing to their great importance to fisheries, their phylogeny and classification are still far from being firmly established. In this study, we present phylogenetic relationships of 19 out of 22 genera traditionally included in the Gadidae based on the analysis of entire cytochrome b and partial cytochrome oxidase I genes (1530 bp). Maximum Parsimony, Maximum Likelihood, and Bayesian analyses all recovered five main clades that correspond to traditionally recognized groupings within Gadoids. The same clades were recovered with MP analysis based on 30 morphological characters (collected from the literature). Given these findings, we propose a revised provisional classification of Gadoids: one suborder Gadoidei containing two families, the Merlucciidae (1 genus) and the Gadidae (21 genera) distributed into four subfamilies: the Gadinae (12 genera), the Lotinae (3 genera), the Gaidropsarinae (3 genera), and the Phycinae (3 genera). Lastly, nuclear inserts of mitochondrial DNA (Numts) were identified in two species, i.e., Gadiculus argenteus and Melanogrammus aeglefinus.     

Electron paramagnetic resonance studies on the high-salt form of bovine spleen purple acid phosphatase.

The EPR spectra of the high-salt form of reduced bovine spleen purple acid phosphatase (BSPAPr) and its complexes with inhibitory tetrahedral oxyanions, AMP, and fluorine have been examined in the 4-30 K temperature range. The EPR spectrum of the high-salt form of BAPAPr is identical to that previously reported for the low-salt form (Averill et al. (1987) J. Am. Chem. Soc. 109, 3760-3767), indicating that the substantial differences in conformation of the two forms result in undetectable alterations in the electronic structure of the binuclear iron center. Phosphate, AMP, and arsenate all result in broadened, highly anisotropic EPR spectra with decreased values of the antiferromagnetic coupling constant, -2J, while molybdate and tungstate produce a sharp axial or slightly rhombic spectrum, respectively, and fluoride produces an anomalous spectrum with an inverted g-tensor. These results are consistent with binding of the two classes of oxyanions (and AMP) to distinct sites at or near the binuclear iron center, while fluoride binds in yet a third mode. EPR spectra of the BSPAPr complex with molybdate show altered relaxation behavior in the presence of phosphate, consistent with a 50% decrease in the magnitude of -2J, suggesting that phosphate binds to the molybdate complex to produce a ternary complex analogous to that proposed for molybdate inhibition on the basis of kinetics studies.     

Phagocytosis of collagen fibrils by periosteal fibroblasts in long bone explants. Effect of concanavalin A

In an attempt to determine whether phagocytosis of collagen by fibroblasts involves binding of the fibril to the plasma membrane, the effect of the lectin concanavalin A (Con A) was studied in an in vitro model system. Metacarpal bone rudiments from 19-day-old mouse fetuses were incubated with varying concentrations of the lectin. Quantitative electron microscopic analysis indicated that Con A caused a dose-related increase in the amount of phagocytosed collagen fibrils in periosteal fibroblasts, suggesting either an enhanced uptake or a decreased intracellular breakdown of fibrils. Since a Con A-inducible increase was not seen in the combined presence of both the lectin and the proteinase inhibitor leupeptin, which is known to inhibit the intracellular digestion of phagocytosed fibrillar collagen, it is unlikely that Con A stimulated phagocytosis. Based on the finding that Con A interfered with the digestion of a synthetic substrate by the collagenolytic lysosomal enzyme cathepsin B it is suggested that the augmentation of intracellular fibrillar collagen under the influence of the lectin was due to a decreased intracellular digestion. Since Con A did not inhibit the uptake of collagen fibrils by the fibroblasts it is concluded that Con A-inhibitable binding sites for collagen molecules are unlikely to be involved in phagocytosis of collagen fibrils by fibroblasts.     

Venturia inaequalis: the causal agent of apple scab

The fungus Venturia inaequalis infects members of the Maloideae, and causes the disease apple scab, the most important disease of apple worldwide. The early elucidation of the gene-for-gene relationship between V. inaequalis and its host Malus has intrigued plant pathologists ever since, with the identification of 17 resistance (R)-avirulence (Avr) gene pairings. The Avr gene products are presumably a subset of the total effector arsenal of V. inaequalis (predominantly proteins secreted in planta assumed to facilitate infection). The supposition that effectors from V. inaequalis act as suppressors of plant defence is supported by the ability of the pathogen to penetrate the cuticle and differentiate into large pseudoparenchymatous structures, termed stromata, in the subcuticular space, without the initiation of an effective plant defence response. If effectors can be identified that are essential for pathogenicity, the corresponding R genes will be durable and would add significant value to breeding programmes. An R gene cluster in Malus has been cloned, but no V. inaequalis effectors have been characterized at the molecular level. However, the identification of effectors is likely to be facilitated by the resolution of the whole genome sequence of V. inaequalis. TAXONOMY: Teleomorph: Venturia inaequalis Cooke (Wint.); Kingdom Fungi; Phylum Ascomycota; Subphylum Euascomycota; Class Dothideomycetes; Family Venturiaceae; genus Venturia; species inaequalis. Anamorph: Fusicladium pomi (Fr.) Lind or Spilocaea pomi (Fr.). LIFE CYCLE: V. inaequalis is a hemibiotroph and overwinters as pseudothecia (sexual fruiting bodies) following a phase of saprobic growth in fallen leaf tissues. The primary inoculum consists of ascospores, which germinate and penetrate the cuticle. Stromata are formed above the epidermal cells but do not penetrate them. Cell wall-degrading enzymes are only produced late in the infection cycle, raising the as yet unanswered question as to how V. inaequalis gains nutrients from the host. Conidia (secondary inoculum) arise from the upper surface of the stromata, and are produced throughout the growing season, initiating multiple rounds of infection. VENTURIA INAEQUALIS AS A MODEL PATHOGEN OF A WOODY HOST: V. inaequalis can be cultured and is amenable to crossing in vitro, enabling map-based cloning strategies. It can be transformed readily, and functional analyses can be conducted by gene silencing. Expressed sequence tag collections are available to aid in gene identification. These will be complemented by the whole genome sequence, which, in turn, will contribute to the comparative analysis of different races of V. inaequalis and plant pathogens within the Dothideomycetes.     

Plasmid DNA fermentation strain and process‐specific effects on vector yield,quality, and transgene expression

Industrial plasmid DNA manufacturing processes are needed to meet the quality, economy, and scale requirements projected for future commercial products. We report development of a modified plasmid fermentation copy number induction profile that increases gene vaccination/therapy vector yields up to 2,600 mg/L. We determined that, in contrast to recombinant protein production, secretion of the metabolic byproduct acetate into the media had only a minor negative effect on plasmid replication. We also investigated the impact of differences in epigenetic dcm methylase‐directed cytosine methylation on plasmid production, transgene expression, and immunogenicity. While Escherichia coli plasmid production yield and quality are unaffected, dcm− versions of CMV and CMV‐HTLV‐I R promoter plasmids had increased transgene expression in human cells. Surprisingly, despite improved expression, dcm− plasmid is less immunogenic. Our results demonstrate that it is critical to lock the plasmid methylation pattern (i.e., production strain) early in product development and that dcm− strains may be superior for gene therapy applications wherein reduced immunogenicity is desirable and for in vitro transient transfection applications such as AAV production where improved expression is beneficial. Biotechnol. Bioeng. 2011;108: 354–363. © 2010 Wiley Periodicals, Inc.