Characterization of low-glycosylated forms of soluble human urokinase receptor expressed in Drosophila Schneider 2 cells after deletion of glycosylation-sites

The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein that is thought to play an active role during cancer cell invasion and metastasis. We have expressed a truncated soluble form of human uPAR using its native signal peptide in stably transfected Drosophila Schneider 2 (S2) cells. This recombinant product, denoted suPAR (residues 1–283), is secreted in high quantities in serum-free medium and can be isolated in very high purity. Characterization by SDS–PAGE and mass spectrometry reveals that suPAR produced in this system carries a uniform glycosylation composed of biantennary carbohydrates. In contrast, suPAR produced in stably transfected Chinese hamster ovary (CHO) cells carries predominantly complex-type glycosylation and exhibits in addition a site-specific microheterogeneity of the individual N-linked carbohydrates. Measurement of binding kinetics for the interaction with uPA by surface plasmon resonance reveals that S2-produced suPAR exhibits binding properties similar to those of suPAR produced by CHO cells. By site-directed mutagenesis we have furthermore removed the five potential N-linked glycosylation-sites either individually or in various combinations and studied the effect thereof on secretion and ligand-binding. Only suPAR completely deprived of N-linked glycosylation exhibits an impaired level of secretion. All the other mutants showed comparable secretion levels and retained the ligand-binding properties of suPAR-wt. In conclusion, stable expression of suPAR in Drosophila S2 cells offers a convenient and attractive method for the large scale production of homogeneous preparations of several uPAR mutants, which may be required for future attempts to solve the three-dimensional structure of uPAR by X-ray crystallography.     

Phylogenetic relationships within the Mysidae (Crustacea, Peracarida, Mysida) based on nuclear 18S ribosomal RNA sequences

Species of the order Mysida (Crustacea, Peracarida) are shrimp-like animals that occur in vast numbers in coastal regions of the world. The order Mysida comprises 1,053 species and 165 genera. The present study covers 25 species of the well-defined Mysidae, the most speciose family within the order Mysida. 18S rRNA sequence analysis confirms that the subfamily Siriellinae is monophyletic. On the other hand the subfamily Gastrosaccinae is paraphyletic and the subfamily Mysinae, represented in this study by the tribes Mysini and Leptomysini, consistently resolves into three independent clades, and hence is clearly not monophyletic. The tribe Mysini is not monophyletic either, and forms two clades of which one appears to be closely related to the Leptomysini. Our results are concordant with a number of morphological differences urging a taxonomic revision of the Mysidae.     

The phylogenetic position of the zokors (Myospalacinae) and comments on the families of muroids (Rodentia)

Recent molecular studies have concluded that the genus Myospalax evolved from within the rodent subfamily Cricetinae. This conclusion was tested using the complete sequences from the mitochondrial 12S rRNA and cytochrome b genes. Based on our analyses, Myospalax appears to be sister to a clade containing the subfamilies Spalacinae and Rhizomyinae, and all three of these lineages appear to be basal to the superfamily Muroidea. Based on the position of these three lineages, we suggest that they be placed in a distinct family, the Spalacidae, rather than subsumed as subfamilies in the family Muridae. Finally, our analyses suggest that the earlier placement of Myospalax as a member of the Cricetinae is the result of a single misidentified specimen, which was not a Myospalax.     

Widespread polyphyly among Alopiinae snail genera: when phylogeny mirrors biogeography more closely than morphology

Consider a group of species that is evenly divided by an easily identifiable complex morphological character. Most biologists would assume that this character should provide better phylogenetic information than, say, the spatial distribution of these species over a fairly continuous 500-km radius area. Paradoxically, this is not the case among terrestrial snail genera in the clausiliid subfamily Alopiinae. Phylogenetic analysis using the nuclear markers ITS1/ITS2 and mitochondrial markers COI/12S reveals widespread homoplasy in the clausilial apparatus (a complex aperture-closing mechanism), and concomitant extensive polyphyly among Carinigera, Isabellaria, and Sericata. In contrast, phylogenetic relationships as revealed by molecular data are closely congruent with biogeography at a relatively small scale. A combination of extremely low vagility and extremely high morphological convergence has conspired to produce this unexpected result. Implications as to the function of the clausilial apparatus are discussed.     

Molecular phylogeny of Demospongiae: implications for classification and scenarios of character evolution

An analysis of the phylogenetic relationships of the 13 orders of Demospongiae, based on 18S and C1, D1 and C2 domains of 28S rRNA (for, respectively, 26 and 32 taxa) has been performed. The class Demospongiae as traditionally defined is not found to be monophyletic. Instead, a clade comprising all demosponges except Homoscleromorpha is well-supported, and we define phylogenetically the name Demospongiae in this more restricted sense to preclude the possibility of drastic alterations of the meaning of Demospongiae in the future, depending on the position of Homoscleromorpha. Within this clade Demospongiae s.s., ceractinomorphs and tetractinomorphs are polyphyletic, implying homoplastic evolution of characters such as reproductive strategies (viviparity vs. oviparity) and skeleton architecture (reticulate vs. radiate). The topology derived from our molecular data provides a basis for proposing a new classification of Demospongiae s.s., and suggests a reverse polarity of some characters, with respect to traditional conceptions: viviparity, presence of monaxon spicules and of spongin appear to be ancestral, whereas oviparity, and presence of tetraxon spicules appear as derived characters.     

Transfer of Nosema locustae (Microsporidia) to Antonospora locustae n. comb. based on molecular and ultrastructural data

Nosema locustae is a microsporidian parasite of grasshopper pests that is used as a biological control agent, and is one of the emerging model systems for microsporidia. Due largely to its diplokaryotic nuclei, N. locustae has been classified in the genus Nosema, a large genus with members that infect a wide variety of insects. However, some molecular studies have cast doubt on the validity of certain Nosema species, and on the taxonomic position of N. locustae. To clarify the affinities of this important insect parasite we sequenced part of the rRNA operon of N. locustae and conducted a phylogenetic analysis using the complete small subunit rRNA gene. Nosema locustae is only distantly related to the nominotypic N. bombycis, and is instead closely related to Antonospora scoticae, a recently described parasite of bees. We examined the ultrastructure of mature N. locustae spores, and found the spore wall to differ from true Nosema species in having a multi-layered exospore resembling that of Antonospora (one of the distinguishing features of that genus). Based on both molecular and morphological evidence, therefore, we propose transferring N. locustae to the genus Antonospora, as Antonospora locustae n. comb.     

Tolerance to Diazepam-Induced Motor Impairment: A Study with GABAA Receptor α6 Subunit Knockout Mice

Development of tolerance to motor-impairing effects of repeated administration of moderate diazepam doses (5.0–7.5 mg/kg; three times daily PO 3 weeks) was compared between mice deficient in the cerebellar granule cell–restricted GABA_A receptor 6 subunit and their wild-type controls. The 6–/– mice were more impaired by the initial challenge doses of diazepam (5 or 10 mg/kg) than their controls, but acquired partial tolerance by the second tests with the same doses 4–7 days later. Chronic treatment produced complete tolerance in both mouse lines. Ligand autoradiography revealed a significant reduction in baseline benzodiazepine and chloride channel site-bindings in various regions of the 6–/– brains, but the chronic diazepam treatment did not consistently alter baseline or benzodiazepine site agonist and inverse agonist-modulated binding in the 6–/– and wildtype mice. The results indicate that tolerance to motor-impairing actions of diazepam is independent of the diazepam-insensitive 6 subunit-containing receptors, which rules out the possibility that tolerance emerges as an increase in structurally benzodiazepine-insensitive receptor population.     

Alterations in mRNA expression of ribosomal protein S9 in hydrogen peroxide-treated neurotumor cells and in rat hippocampus after transient ischemia

This study was designed to isolate new genes related to apoptosis in rat pheochromocytoma (PC12) cells treated with hydrogen peroxide (H₂O₂), and to characterize the roles of the genes using both in vitro and in vivo models of oxidative injury. cDNA libraries were prepared from H₂O₂-treated and -untreated PC12 cells, and a ribosomal protein S9 (RPS9) clone was isolated by a differential screening method. Increase of RPS9 expression in both H₂O₂-treated PC12 and neuroblastoma (Neuro-2A) cells was shown by Northern blot analysis. Viability of the antisense-transfected Neuro-2A (RPS9-AS) cells following H₂O₂ treatment was significantly reduced in a dose-dependent manner. In an in vivo model of transient forebrain ischemia, an increase in RPS9 expression was prominent by 1 day postischemia in the granule cell layer neurons of the dentate gyrus. Both activation of caspase-3 and significant recovery of viability following pretreatment with cycloheximide were shown in RPS9-AS cells treated with H₂O₂. These data suggest that RPS9 plays a protective role in oxidative injury of neuronal cells.     

Antioxidant Effect of Phenelzine on MPP+-Induced Cell Viability Loss in Differentiated PC12 Cells

Phenelzine, deprenyl, and antioxidants (SOD, catalase, ascorbate, or rutin) reduced the loss of cell viability in differentiated PC12 cells treated with 250 M MPP⁺, whereas N-acetylcysteine and dithiothreitol did not inhibit cell death. Phenelzine reduced the condensation and fragmentation of nuclei caused by MPP⁺ in PC12 cells. Phenelzine and deprenyl prevented the MPP⁺-induced decrease in mitochondrial membrane potential, cytochrome c release, formation of reactive oxygen species, and depletion of GSH in PC12 cells. Phenelzine revealed a scavenging action on hydrogen peroxide and reduced the hydrogen peroxide–induced cell death in PC12 cells, whereas deprenyl did not depress the cytotoxic effect of hydrogen peroxide. Both compounds reduced the iron and EDTA-mediated degradation of 2-deoxy-d-ribose degradation. The results suggest that phenelzine attenuates the MPP⁺-induced viability loss in PC12 cells by reducing the alteration of mitochondrial membrane permeability that seems to be mediated by oxidative stress.