Condensin is required for nonhistone protein assembly and structural integrity of vertebrate mitotic chromosomes

The dramatic condensation of chromosomes that occurs during mitosis is widely thought to be largely controlled by a protein complex termed condensin. Here, we describe a conditional knockout of the condensin subunit ScII/SMC2 in chicken DT40 cells. In cells lacking this condensin subunit, chromosome condensation is delayed, but ultimately reaches near-normal levels. However, these chromosomes are structurally compromised. Kinetochores appear normal, but the localization of nonhistone proteins such as topoisomerase II and INCENP is aberrant. Both proteins also fail to partition into the chromosome scaffold fraction, which appears to be largely missing in the absence of condensin. Furthermore, the chromosomes lack structural integrity, as defined by an assay that tests the stability of the chromosomal higher-order structure. Thus, a major function of condensin is to promote the correct association of nonhistone proteins with mitotic chromosomes, and this is essential for establishment of a robust chromosome structure.     

Systemic and microvascular responses to hemorrhagic shock and resuscitation with Hb vesicles

A phospholipid vesicle encapsulating hemoglobin (Hb vesicle, HbV) has been developed to provide O(2)-carrying capacity to plasma expanders. Its ability to restore systemic and microcirculatory conditions after hemorrhagic shock was evaluated in the dorsal skinfold window preparation of conscious hamsters. The HbV was suspended in 8% human serum albumin (HSA) at Hb concentrations of 3.8 g/dl [HbV(3.8)/HSA] and 7.6 g/dl [HbV(7.6)/HSA]. Shock was induced by 50% blood withdrawal, and mean arterial pressure (MAP) at 40 mmHg was maintained for 1 h by the additional blood withdrawal. The hamsters receiving either HbV(3.8)/HSA or HbV(7.6)/HSA suspensions restored MAP to 93 +/- 14 and 93 +/- 10 mmHg, respectively, similar with those receiving the shed blood (98 +/- 13 mmHg), which were significantly higher by comparison with resuscitation with HSA alone (62 +/- 12 mmHg). Only the HSA group tended to maintain hyperventilation and negative base excess after the resuscitation. Subcutaneous microvascular blood flow reduced to approximately 10-20% of baseline during shock, and reinfusion of shed blood restored blood flow to approximately 60-80% of baseline, an effect primarily due to the sustained constriction of small arteries A(0) (diameter 143 +/- 29 microm). The HbV(3.8)/HSA group had significantly better microvascular blood flow recovery and nonsignificantly better tissue oxygenation than of the HSA group. The recovery of base excess and improved tissue oxygenation appears to be primarily due to the increased oxygen-carrying capacity of HbV fluid resuscitation.     

Epithelial innate immunity. A novel antimicrobial peptide with antiparasitic activity in the blood-sucking insect Stomoxys calcitrans

The gut epithelium is an essential interface in insects that transmit parasites. We investigated the role that local innate immunity might have on vector competence, taking Stomoxys calcitrans as a model. S. calcitrans is sympatric with tsetse flies, feeds on many of the same vertebrate hosts, and is thus regularly exposed to the trypanosomes that cause African sleeping sickness and nagana. Despite this, S. calcitrans is not a cyclical vector of these trypanosomes. Trypanosomes develop exclusively in the lumen of digestive organs, and so epithelial immune mechanisms, and in particular antimicrobial peptides (AMPs), may be the prime determinants of the fate of an infection. To investigate why S. calcitrans is not a cyclical vector of trypanosomes, we have looked in its midgut for AMPs with trypanolytic activity. We have identified a new AMP of 42 amino acids, which we named stomoxyn, constitutively expressed and secreted exclusively in the anterior midgut of S. calcitrans. It displays an amphipathic helical structure and exhibits a broad activity spectrum affecting the growth of microorganisms. Interestingly, this AMP exhibits trypanolytic activity to Trypanosoma brucei rhodesiense. We argue that stomoxyn may help to explain why S. calcitrans is not a vector of trypanosomes causing African sleeping sickness and nagana.     

Ligand binding of a ribosome-displayed protein detected in solution at the single molecule level by fluorescence correlation spectroscopy

Interaction of a single-chain antibody fragment (scFv) with its cognate antigen while still attached to the ribosome was studied by fluorescence correlation spectroscopy (FCS). In experiments with purified scFv, FCS was capable of resolving the difference in diffusion time between free and antibody-bound labelled antigen. Ribosome-displayed antibody fragments generated by in vitro translation, in which neither the protein nor the mRNA leaves the ribosome owing to the absence of a stop codon and stabilizing buffer conditions, could be shown to specifically bind the antigen. The antibody-antigen interaction was specific, as shown by inhibition or displacement with unlabelled antigen and by control experiments with a non-cognate antibody fragment.     

Extract screening by HPLC coupled to MS-MS,NMR, and CD: a dimeric and three monomeric naphthylisoquinoline alkaloids from Ancistrocladus griffithii

Three new monomeric naphthylisoquinoline alkaloids, ancistrogriffines A, B, and C, and the first dimer of a 7,8'-coupled naphthylisoquinoline, ancistrogriffithine A, have been detected by phytochemical online screening of plant extracts of Ancistrocladus griffithii, using the analytical 'triad' HPLC-MS/MS, HPLC-NMR, and HPLC-CD. Ancistrogriffithine A, as well as ancistrogriffines A and C, were structurally completely assigned (including the absolute configuration) right from the extract, without previous isolation. Furthermore, two related, but known alkaloids, ancistrocladine and hamatine, were identified. Except for ancistrogriffine B, which occurs in trace quantities only, all new alkaloids were then isolated preparatively and the initial assignments were fully confirmed by conventional offline methods. Of particular interest is the constitutionally and configurationally unprecedented structure of ancistrogriffithine A, which is simultaneously the first dimeric naphthylisoquinoline alkaloid from an Asian Ancistrocladus species. Ancistrogriffithine A and ancistrogriffine A are active against Plasmodium falciparum. Furthermore, the latter compound shows good activity against Leishmania donovani. The results demonstrate the ability of modern online methods like HPLC-NMR, -MS/MS, and -CD to serve as powerful tools for the reliable structural elucidation of even complex structures of trace compounds in crude biological matrices.     

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