Intracellular trapping of cycloSal-pronucleotides by enzymatic cleavage

A new synthesis for cycloSal-pronucleotides bearing enzymatically cleavable triggers is presented. This trigger is introduced to trap the pronucleotide inside cells. The general concept and hydrolysis data in different media are discussed.     

Potential drivers of samango monkey (Cercopithecus albogularis) population subdivision in a highly fragmented mountain landscape in northern South Africa

Primates - Forests affected by fragmentation are at risk of losing their primate populations over the long term. The impact of fragmentation on primate populations has been studied in several...     

The denatured state of HIV-1 protease under native conditions

The denatured state of several proteins has been shown to display transient structures that are relevant for folding, stability, and aggregation. To detect them by nuclear magnetic resonance (NMR) spectroscopy, the denatured state must be stabilized by chemical agents or changes in temperature. This makes the environment different from that experienced in biologically relevant processes. Using high-resolution heteronuclear NMR spectroscopy, we have characterized several denatured states of a monomeric variant of HIV-1 protease, which is natively structured in water, induced by different concentrations of urea, guanidinium chloride, and acetic acid. We have extrapolated the chemical shifts and the relaxation parameters to the denaturant-free denatured state at native conditions, showing that they converge to the same values. Subsequently, we characterized the conformational properties of this biologically relevant denatured state under native conditions by advanced molecular dynamics simulations and validated the results by comparison to experimental data. We show that the denatured state of HIV-1 protease under native conditions displays rich patterns of transient native and non-native structures, which could be of relevance to its guidance through a complex folding process.     

Atg1 allows second-signaled autophagic cell death in Dictyostelium

We investigated the role of Atg1 in autophagic cell death (ACD) in a Dictyostelium monolayer model. The model is especially propitious, not only because of genetic tractability and absence of apoptosis machinery, but also because induction of ACD requires two successive exogenous signals, first the combination of starvation and cAMP, second the differentiation factor DIF-1. This enables one to analyze separately first-signal-induced autophagy and subsequent second-signal-induced ACD. We used mutants of atg1, a gene that plays an essential role in the initiation of autophagy. Upon starvation/cAMP, in contrast to parental cells, atg1 mutant cells showed irreversible lesions, clearly establishing a protective role for Atg1. Upon subsequent exposure to DIF-1 or to more ACD-specific second signals, starved parental cells progressed to ACD, but starved atg1 mutant cells did not, showing that Atg1 was required for ACD. Thus, in the same cells Atg1 was required in two apparently opposite ways, upon first-signaling for cell survival and upon second-signaling for ACD. Our findings strongly suggest that Atg1, thus presumably autophagy, protects the cells from starvation-induced cell death, allowing subsequent induction of ACD by the second signal. ACD is therefore not only "with" autophagy (since it showed signs of autophagy throughout), but is also "allowed by" autophagy. This does not exclude a role for autophagy also after second signaling. These results may account for discrepancies reported in the literature, encourage searches for second signals in different developmental models of ACD, and incite caution in autophagy-related therapeutic attempts.     

Stream ecosystem properties and processes along a temperature gradient

We surveyed macrophyte community structure and measured community metabolism and nutrient uptake along a temperature gradient (9.7–17.4°C) in four Icelandic streams influenced by geothermal heating. The study streams are part of the geothermal area in Hengill that is uniquely characterised by streams with comparable water chemistry despite the geothermal influence. Stream metabolism was studied applying the diurnal upstream–downstream dissolved oxygen change technique. Nutrient uptake was studied by adding solutions of nitrogen and phosphorus together with a conservative tracer. Rates of primary production (GPP) and uptake of nitrate–N and phosphate-P increased with increasing stream temperature. GPP was 20 times higher (up to 12.99 g O₂ m⁻² day⁻¹) and rates of nutrient uptake were up to 30-times higher (up to 22.99, 13.31 and 7.94 mg m⁻² h⁻¹ for ammonium, nitrate and phosphate, respectively) in the warmest streams compared with the coldest. Furthermore, macrophytes, when present, were strongly controlling ecosystem processes. Our study implies that temperature may affect stream ecosystem processes both directly (i.e. physiologically) and indirectly (i.e. by changing other structural parameters).     

Axis formation--beta-catenin catches a Wnt

In this issue of Cell, the Heasman group implicates Wnt11 as a component of the canonical Wnt signaling pathway that specifies Xenopus laevis axis formation (Tao et al., 2005). This important work not only identifies a long-sought-after dorsalizing factor but also highlights the pivotal role of extracellular cofactors in specifying the activation of either canonical or noncanonical Wnt pathways.     

Effect of leukemia inhibitory factor (LIF) on in vitro produced bovine embryos and their outgrowth colonies

In vitro produced (IVP) bovine embryos were subjected to in vitro culture with or without 1000 U/ml human recombinant leukemia inhibitory factor (LIF) added to the culture medium from Days 5 to 8 post insemination (p.i.). Resulting blastocysts were subsequently plated intact on mouse feeder cells in a medium with or without LIF. Significantly more embryos reached the hatched blastocyst stage, and the number of blastocysts with excellent morphology was significantly higher, when LIF was omitted. At Day 8 p.i., total cell count (TCC) and inner cell mass (ICM) cell count was significantly higher in embryos cultured without LIF. In embryos cultured with LIF, cytoplasmic vesicles and lipid droplets were abundant and a decreased expression of both Oct4 and laminin could be observed. Initial hypoblast formation was revealed in almost 1/3 of the LIF-cultured blastocysts whereas this feature was evident in 2/3 of the blastocysts cultured in the absence of LIF. Overall, almost 60% of the blastocysts cultured without LIF formed outgrowth colonies (OCs) when plated on feeders, whereas this phenomenon was only observed in 30% of the blastocysts cultured in the presence of LIF. A tendency for retaining a tightly packed central growth of putative ICM-derived cells was observed, when attachment to the feeder layer was initiated close to the embryonic pole of the blastocyst. At Day 8 of outgrowth culture, approximately 20% of the colonies contained a central core of putative ICM-derived cells appearing large enough for mechanical isolation and further subculture. Immunohistochemical labeling for Oct4 revealed staining of both trophectodermal and ICM-derived cells. The presence of LIF in the outgrowth culture medium did not have any apparent effect on the plating efficiency or colony type. In conclusion, LIF had an adverse effect on in vitro embryonic development when added to the culture medium in the period from Days 5 to 8 p.i., whereas it had no apparent effect on the OCs subsequently formed from such embryos.     

Lamin A/C-dependent localization of Nesprin-2, a giant scaffolder at the nuclear envelope

The vertebrate proteins Nesprin-1 and Nesprin-2 (also referred to as Enaptin and NUANCE) together with ANC-1 of Caenorhabditis elegans and MSP-300 of Drosophila melanogaster belong to a novel family of alpha-actinin type actin-binding proteins residing at the nuclear membrane. Using biochemical techniques, we demonstrate that Nesprin-2 binds directly to emerin and the C-terminal common region of lamin A/C. Selective disruption of the lamin A/C network in COS7 cells, using a dominant negative lamin B mutant, resulted in the redistribution of Nesprin-2. Furthermore, using lamin A/C knockout fibroblasts we show that lamin A/C is necessary for the nuclear envelope localization of Nesprin-2. In normal skin where lamin A/C is differentially expressed, strong Nesprin-2 expression was found in all epidermal layers, including the basal layer where only lamin C is present. This indicates that lamin C is sufficient for proper Nesprin-2 localization at the nuclear envelope. Expression of dominant negative Nesprin-2 constructs and knockdown studies in COS7 cells revealed that the presence of Nesprin-2 at the nuclear envelope is necessary for the proper localization of emerin. Our data imply a scaffolding function of Nesprin-2 at the nuclear membrane and suggest a potential involvement of this multi-isomeric protein in human disease.