Detection of procathepsin D in rat milk

The presence of procathepsin D, a zymogen of the soluble lysosomal aspartic proteinase cathepsin D, was detected in rat milk using Western blot analysis and assay of proteolytic activity in acidic buffers. No other forms of cathepsin D were found. Two different polyclonal anti-procathepsin D antibodies were used for immunochemical detection of procathepsin D. Both antibodies we found to recognize rat procathepsin D. Proteolytic activity in acidic buffers was detected using a fluorogenic substrate specific for cathepsin D and was abolished by pepstatin A, a specific inhibitor of aspartic proteinases. This study represents third demonstration of presence of procathepsin D in mammal breast milk. Potential sources and physiological functions are discussed.     

Dispersal patterns of endemic alpine butterflies with contrasting population structures:<Emphasis Type="Italic"> Erebia epiphron</Emphasis> and<Emphasis Type="Italic"> E. sudetica</Emphasis>

We studied population sizes and mobility of Erebia epiphron and Erebia sudetica, two high mountain butterflies forming endemic subspecies in the Hrubý Jeseník Mountains, Czech Republic. E. epiphron formed two continuous populations containing 100,000 and 4,500 individuals on alpine grasslands. The butterflies moved freely within their habitats, but movements between the two populations were highly unlikely. E. sudetica formed a system of colonies at timberline sites on valley headwalls and in forest clearings. Two such colonies studied in detail contained 4,500 and 450 adults and were interconnected by limited dispersal. The negative exponential function and the sigmoid function (this assumes flat decrease of movements over short distances) were superior to the inverse power function in fitting mobility data for both species. For E. sudetica, the functions describing movements within a habitat differed significantly from total movements, suggesting different behaviours of dispersing individuals. The habitats of E. epiphron are uniform and highly isolated, favouring free within-habitat mobility but prohibiting leaving their boundaries. The habitats of E. sudetica are diverse and disturbance-dependent; leaving such habitats is less risky, and a source-sink model may explain the persistence of the species in the mountains.     

Dynamics of herpes simplex virus capsid maturation visualized by time-lapse cryo-electron microscopy

The capsid of the herpes simplex virus initially assembles as a procapsid that matures through a massive conformational change of its 182 MDa surface shell. This transition, which stabilizes the fragile procapsid, is facilitated by the viral protease that releases the interaction between the shell and the underlying scaffold; however, protease-deficient procapsids mature slowly in vitro. To study procapsid maturation as a time-resolved process, we monitored this reaction by cryo-electron microscopy (cryo-EM). The resulting images were sorted into 17 distinct classes, and three-dimensional density maps were calculated for each. When arranged in a chronological series, these maps yielded molecular movies of procapsid maturation. A single major switching event takes place at stages 8-9, preceded by relatively subtle adjustments in the pattern of interactions and followed by similarly small 'aftershocks'. The primary mechanism underlying maturation is relative rotations of domains of VP5, the major capsid protein.     

Modulation of PKCdelta tyrosine phosphorylation and activity in salivary and PC-12 cells by Src kinases

Protein kinase C (PKC) delta becomes tyrosine phosphorylated in rat parotid acinar cells exposed to muscarinic and substance P receptor agonists, which initiate fluid secretion in this salivary cell. Here we examine the signaling components of PKCdelta tyrosine phosphorylation and effects of phosphorylation on PKCdelta activity. Carbachol- and substance P-promoted increases in PKCdelta tyrosine phosphorylation were blocked by inhibiting phospholipase C (PLC) but not by blocking intracellular Ca2+ concentration elevation, suggesting that diacylglycerol, rather than D-myo-inositol 1,4,5-trisphosphate production, positively modulated this phosphorylation. Stimuli-dependent increases in PKCdelta activity in parotid and PC-12 cells were blocked in vivo by inhibitors of Src tyrosine kinases. Dephosphorylation of tyrosine residues by PTP1B, a protein tyrosine phosphatase, reduced the enhanced PKCdelta activity. Lipid cofactors modified the tyrosine phosphorylation-dependent PKCdelta activation. Two PKCdelta regulatory sites (Thr-505 and Ser-662) were constitutively phosphorylated in unstimulated parotid cells, and these phosphorylations were not altered by stimuli that increased PKCdelta tyrosine phosphorylation. These results demonstrate that PKCdelta activity is positively modulated by tyrosine phosphorylation in parotid and PC-12 cells and suggest that PLC-dependent effects of secretagogues on salivary cells involve Src-related kinases.     

Combinations of PARP Inhibitors with Temozolomide Drive PARP1 Trapping and Apoptosis in Ewing’s Sarcoma

Ewing’s sarcoma is a malignant pediatric bone tumor with a poor prognosis for patients with metastatic or recurrent disease. Ewing’s sarcoma cells are acutely hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibition and this is being evaluated in clinical trials, although the mechanism of hypersensitivity has not been directly addressed. PARP inhibitors have efficacy in tumors with BRCA1/2 mutations, which confer deficiency in DNA double-strand break (DSB) repair by homologous recombination (HR). This drives dependence on PARP1/2 due to their function in DNA single-strand break (SSB) repair. PARP inhibitors are also cytotoxic through inhibiting PARP1/2 auto-PARylation, blocking PARP1/2 release from substrate DNA. Here, we show that PARP inhibitor sensitivity in Ewing’s sarcoma cells is not through an apparent defect in DNA repair by HR, but through hypersensitivity to trapped PARP1-DNA complexes. This drives accumulation of DNA damage during replication, ultimately leading to apoptosis. We also show that the activity of PARP inhibitors is potentiated by temozolomide in Ewing’s sarcoma cells and is associated with enhanced trapping of PARP1-DNA complexes. Furthermore, through mining of large-scale drug sensitivity datasets, we identify a subset of glioma, neuroblastoma and melanoma cell lines as hypersensitive to the combination of temozolomide and PARP inhibition, potentially identifying new avenues for therapeutic intervention. These data provide insights into the anti-cancer activity of PARP inhibitors with implications for the design of treatment for Ewing’s sarcoma patients with PARP inhibitors.     

Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats

Background

Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.

Results

This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.

Conclusions

Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage.     

Habitat use of the endangered butterfly Euphydryas maturna and forestry in Central Europe

The knowledge of ecological requirements of declining butterflies of European woodlands remains limited, which hinders conservation management of their localities. This also applies for continentally threatened scarce fritillary Euphydryas maturna . On the basis of the largest data set on its habitat use ever collected in Central Europe, we analyse habitat requirements of its populations in Austria (A), the Czech Republic (Cz) and Germany (D). All studied populations inhabit open-canopy sites within woodlands, but larval survival decreases under full sun and preferred sites are relatively humid and sheltered. Nests of pre-hibernation larvae occur at terminal branches of Fraxinus excelsior , 1.5–3 m above the ground. Pre-hibernation mortality reaches 70% (Cz, D). Another limiting factor is quality of woodland vegetation: post-hibernation larvae consume a wide range of herbs and shrubs, and adult distribution is linked to nectar availability. The butterfly thus depends on highly heterogeneous early successional stages of deciduous woods, historically maintained by coppicing (Cz, D) and forest pasture (A). Restoration of these traditional methods offers the only chance for survival of E. maturna in Central Europe, and the butterfly may become a flagship for other threatened organisms of open-canopy woodlands.     

Allosteric signaling and a nuclear exit strategy: binding of UL25/UL17 heterodimers to DNA-Filled HSV-1 capsids

UL25 and UL17 are two essential minor capsid proteins of HSV-1, implicated in DNA packaging and capsid maturation. We used cryo-electron microscopy to examine their binding to capsids, whose architecture observes T = 16 icosahedral geometry. C-capsids (mature DNA-filled capsids) have an elongated two-domain molecule present at a unique, vertex-adjacent site that is not seen at other quasiequivalent sites or on unfilled capsids. Using SDS-PAGE and mass spectrometry to analyze wild-type capsids, UL25 null capsids, and denaturant-extracted capsids, we conclude that (1) the C-capsid-specific component is a heterodimer of UL25 and UL17, and (2) capsids have additional populations of UL25 and UL17 that are invisible in reconstructions because of sparsity and/or disorder. We infer that binding of the ordered population reflects structural changes induced on the outer surface as pressure builds up inside the capsid during DNA packaging. Its binding may signal that the C-capsid is ready to exit the nucleus.