Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures

Neospora caninum, like Toxoplasma gondii, undergoes stage conversion in chronically infected animals, and forms tissue cysts which contain the slowly proliferating bradyzoite stage. These tissue cysts are delineated by a cyst wall, protect the parasite from physiological and immunological reactions on part of the host, and bradyzoites remain viable within an infected host for many years. However, unlike T. gondii, N. caninum bradyzoites have been difficult to obtain using in vitro culture techniques, and current protocols, based on those developed for T. gondii, have been shown to be not very efficient in promoting tachyzoite-to-bradyzoite stage conversion. We report here an alternative in vitro culture method to obtain stage conversion of N. caninum from the proliferative to the cystic stage by using the Nc-Liverpool isolate, murine epidermal keratinocytes as host cells, and continuous treatment of infected cultures with 70 microM sodium nitroprusside for up to 8 days. This treatment significantly reduced parasite proliferation as assessed by Neospora-specific quantitative real-time PCR. The expression of bradyzoite markers was analysed by immunofluorescence following 4 and 8 days of in vitro culture using antibodies directed against bradyzoite antigen 1, the mAbCC2, and the lectin Dolichos biflorus agglutinin. Expression of the tachyzoite-specific immunodominant antigen NcSAG1 and the tachyzoite antigen NcMIC1 was also assessed. Transmission electron microscopy revealed that the majority of parasitophorous vacuoles were in the process of forming a distinct cyst wall through accumulation of granular material at the periphery of the vacuole, and parasites exhibited the typical features of bradyzoites. These findings demonstrate the usefulness of this culture technique as a promising way to study tachyzoite-to-bradyzoite stage conversion in N. caninum in vitro.     

Mechanisms underpinning community stability along a latitudinal gradient: Insights from a niche-based approach

At large scales, the mechanisms underpinning stability in natural communities may vary in importance due to changes in species composition, mean abundance, and species richness. Here we link species characteristics (niche positions) and community characteristics (richness and abundance) to evaluate the importance of stability mechanisms in 156 butterfly communities monitored across three European countries and spanning five bioclimatic regions. We construct niche-based hierarchical structural Bayesian models to explain first differences in abundance, population stability, and species richness between the countries, and then explore how these factors impact community stability both directly and indirectly (via synchrony and population stability). Species richness was partially explained by the position of a site relative to the niches of the species pool, and species near the centre of their niche had higher average population stability. The differences in mean abundance, population stability, and species richness then influenced how much variation in community stability they explained across the countries. We found, using variance partitioning, that community stability in Finnish communities was most influenced by community abundance, whereas this aspect was unimportant in Spain with species synchrony explaining most variation; the UK was somewhat intermediate with both factors explaining variation. Across all countries, the diversity–stability relationship was indirect with species richness reducing synchrony which increased community stability, with no direct effects of species richness. Our results suggest that in natural communities, biogeographical variation observed in key drivers of stability, such as population abundance and species richness, leads to community stability being limited by different factors and that this can partially be explained due to the niche characteristics of the European butterfly assemblage.     

Studies on the ferrochelatase activity of mitochondria and submitochondrial particles with special reference to the regulatory function of the mitochondrial inner membrane

The question addressed in the title was examined by measuring fluorescence emission spectra and light-induced fluorescence-yield changes of chloroplasts which had been frozen to ?196 °C rapidly, as very thin samples adsorbed into substrates which were plunged directly into liquid nitrogen, or slowly by the cooling action of liquid nitrogen through the wall of the cuvette. Contrary to previous reports, we found that the rate of cooling had no influence on the shape of the emission spectrum, the extent of the variable fluorescence or the fraction of the absorbed quanta which are delivered initially to Photosystem I.     

Analysis of neuronal and glial phenotypes in brains of mice deficient in leukemia inhibitory factor

Leukemia inhibitory factor (LIF) can regulate the survival and differentiation of certain neurons and glial cells in culture. To determine the role of this cytokine in the central nervous system in vivo, we examined the brains of young and adult mice in which the LIF gene was disrupted. Immunohistochemical staining of neurons for choline acetyltransferase, tyrosine hydroxylase, serotonin, parvalbumin, calbindin, neuropeptide Y, vasoactive intestinal polypeptide, and calcitonin gene-related peptide revealed no significant differences between null mutant and wild-type (WT) brains. In contrast, analysis of glial phenotypes demonstrated striking deficits in the LIF-knockout brain. Staining with several anti-glial fibrillary acidic protein (GFAP) antibodies showed that the number of GFAP-positive cells in various regions of the hippocampus in the female mutant is much lower than in the WT. The null male hippocampus also displays a significant, though less marked deficit. The number of astrocytes in the mutant hippocampus, as determined by S-100 staining, is not, however, significantly different from WT. In addition, quantification of immunohistochemical staining of female, but not male, mutants reveals a significant deficit in myelin basic protein content in three brain regions, suggesting alterations in oligodendrocytes as well. Thus, while overall brain histology appears normal, the absence of LIF in vivo leads to specific, sexually dimorphic alterations in glial phenotype. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 509–524, 1998     

Borealin: a novel chromosomal passenger required for stability of the bipolar mitotic spindle

The chromosomal passenger complex of Aurora B kinase, INCENP, and Survivin has essential regulatory roles at centromeres and the central spindle in mitosis. Here, we describe Borealin, a novel member of the complex. Approximately half of Aurora B in mitotic cells is complexed with INCENP, Borealin, and Survivin; and Borealin binds Survivin and INCENP in vitro. A second complex contains Aurora B and INCENP, but no Borealin or Survivin. Depletion of Borealin by RNA interference delays mitotic progression and results in kinetochore-spindle misattachments and an increase in bipolar spindles associated with ectopic asters. The extra poles, which apparently form after chromosomes achieve a bipolar orientation, severely disrupt the partitioning of chromosomes in anaphase. Borealin depletion has little effect on histone H3 serine10 phosphorylation. These results implicate the chromosomal passenger holocomplex in the maintenance of spindle integrity and suggest that histone H3 serine10 phosphorylation is performed by an Aurora B-INCENP subcomplex.     

Serological cloning of cancer/testis antigens expressed in prostate cancer using cDNA phage surface display

Serological cloning of tumor-associated antigens (TAAs) using patient autoantibodies and tumor cDNA expression libraries (SEREX) has identified a wide array of tumor proteins eliciting B-cell responses in patients. However, alternative cloning strategies with the possibility of high throughput analysis of patient sera and tumor libraries may be of interest. We explored the pJuFo phage surface display system, allowing display of recombinant tumor proteins on the surface of M13 filamentous phage, for cloning of TAAs in prostate cancer (PC). Control experiments established that after a few rounds of selection on immobilized specific IgG, a high degree of enrichment of seroreactive clones was achieved. With an increasing number of selection rounds, a higher yield of positive clones was offset by an apparent loss of diversity in the repertoire of selected clones. Using autologous patient serum IgG in a combined biopanning and immunoscreening approach, we identified 13 different TAAs. Three of these (NY-ESO-1, Lage-1, and Xage-1) were known members of the cancer/testis family of TAAs, and one other protein had previously been isolated by SEREX in cancer types other than PC. Specific IgG responses against NY-ESO-1 were found in sera from 4/20 patients with hormone refractory PC, against Lage-1 in 3/20, and Xage-1 in 1/20. No reactivity against the remaining proteins was detected in other PC patients, and none of the TAAs reacted with serum from healthy subjects. The results demonstrate that phage surface display combined with postselection immunoscreening is suitable for cloning a diverse repertoire of TAAs from tumor tissue cDNA libraries. Furthermore, candidate TAAs for vaccine development of PC were identified.     

Novel components of human mitotic chromosomes identified by proteomic analysis of the chromosome scaffold fraction

Chromosomal nonhistone proteins have important roles in mitotic chromosome formation and dynamics. In order to identify novel abundant proteins with a potential involvement in these processes, we initiated a proteomic screen of the chromosome scaffold fraction. This screen identified 79 proteins, 30 of which had not previously been described as components of mitotic chromosomes. Furthermore, half of these proteins had no documented function. We analyzed the cell-cycle dependent distribution of three uncharacterized proteins by expressing them as green fluorescent protein (GFP) fusions and showed that they associate with mitotic chromosomes in vivo. One of the proteins, nuclear protein p30, is a novel component of the inner centromere. Over-expression experiments indicated that p30 may have an active role in the formation of centromeric heterochromatin.     

Saisonaler Verlauf der Gesangsaktivit?t der Singdrossel(Turdus philomelos), mit Anmerkungen zum nachbrutzeitlichen Gesangsschub

Zusammenfassung Der Zürichbergwald ist eine 350 ha große, bewaldete Kuppe auf 480–680 m ü.M. am Rande der Stadt Zürich. Hier untersuchten wir von 1989 bis 1995 die jahreszeitliche Gesangsaktivität der Singdrossel mit zwei verschiedenen Ansätzen. 1989 und 1991–1995 zählte JH an 123 Tagen auf zwei festgelegten Strecken von 6,1 und 7,1 km die Sänger jeweils in der Stunde der abendlichen Dämmerung. Im gleichen Gebiet zählte RS 1990 an 46 Tagen auf einer 6,7 km langen Strecke am Morgen in der Stunde nach Sonnenaufgang. Ebenfalls 1990 sammelte RS Daten zur Brutbiologie.Die Gesangsaktivität definierten wir mit der Anzahl singender Männchen pro km. Im Jahresverlauf zeigen die Kurven der Morgen- und der Abendaktivität keinen Unterschied. Die Kurve ist deutlich dreiphasig: Ein erster Gesangsschub vom Eintreffen der Vögel bis zum 5. April, danach eine gesangsarme Zwischenzeit bis zum 15. Mai, gefolgt von einem zweiten Gesangsschub, welcher bis zum 5. Juli dauern kann. Allenfalls ist der erste Gesangsschub (stark zeitverschoben) mit dem Beginn der Erstbruten korreliert; die von vielen Singvögel bekannte, markante Gesangsaktivität unmittelbar vor der Eiablage gibt es nicht. Der zweite Gesangsschub ist in der Stärke mit dem ersten vergleichbar. Allerdings liegt er eindeutig am Ende der Brutzeit und kann nicht mit einem Brutparameter (Partnerfindung, Paarbindung, Balz, Brut-Stimulation) in Zusammenhang stehen. Die Funktion dieser nachbrutzeitlichen Gesangsaktivität scheint zukunftsbezogen zu sein. In Frage kommt gehäuftes Singen der Väter vor den Jungen zum Erlernen des Gesangs und des Dialekts, oder/und eine Revier-Voranzeige für die nänchste Brutsaison durch die als philopatrisch bekannten Männchen.

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Annual variation in singing activity of the Song Thrush(Turdus philomelos), with comments on high postbreeding song output

Summary This study was carried out at the Zürichbergwald, a forest east of Zurich (47°20N/08°30E). The study site is a wooded hill of 350 ha between 480 to 680 m asl, characterised by a BeechFagus silvatica forest with patches of SprucePicea abies on 25 % of its surface. The Zürichbergwald is a popular recreational area with moderate forestry exploitation. We did not differentiate acoustic registration from singing activity, and we considered the number of singing males per km to be a measure for singing activity. Two different approaches were applied: in 6 breeding seasons (1989 and 1991 to 1995) JH counted birds at sunset on a 6.1 or 7.1 km circuit (n=123). In 1990, the same was done by RS at dawn each morning on a zigzag track of 6.7 km (n=46). Also in 1990, RS sampled data on the breeding biology of the species.The annual cycle of morning and evening song activity was significantly correlated (Spearman's rank-test; p<0.001 comparing pentads, p=0.025 comparing half of months). Morning and evening revealed the same pattern: there was a first large peak of singing activity early in the year (earliest onset of singing 19 February 1989; latest 8 March 1993) until 5 April (phase I). A period of low song activity followed from 6 April to 15 May (phase II). The period from 16 May to (circa) 5 July was characterized by a second large peak (phase III). Each of the corresponding phases was comparable between morning and evening (Wilcoxon matching pairs; p>0.05). The analysis of evening data reveals that phase II differed from I and from III (p=0.05), but the last two did not differ significantly (Wilcoxon matching pairs; p>0.05).The day with the highest song activity fell in phase I twice (maximum 6.1 singing males/km, 2 April 1995) and 5 times in phase III (maximum 6.9 singing males/km, 23 May 1994).The date females first laid was determined for 53 out of 68 nests. The first brood started 25 March, the last 25 June 1990. Only 3 broods were initiated later than 5 June.The first peak of singing activity could be correlated with the (delayed) onset of breeding, but the second started at the end of the breeding season and persisted too long to be correlated with any breeding activity such as female attraction or stimulation, mate-guarding, etc. We postulate the high post-breeding song output to have several possible functions: Song instruction by father to offspring, or territory announcement for the next season.