Cyclic AMP-dependent protein kinase controls energy interconversion during the catalytic cycle of the yeast copper-ATPase

The pathogenesis of human Menkes and Wilson diseases depends on alterations in copper transport. Some reports suggest that intracellular traffic of copper might be regulated by kinase-mediated phosphorylation. However, there is no evidence showing the influence of kinase-related processes in coupled ATP hydrolysis/copper transport cycles. Here, we show that cyclic AMP-dependent protein kinase (PKA) regulates Ccc2p, the yeast Cu(I)-ATPase, with PKA-mediated phosphorylation of a conserved serine (Ser258) being crucial for catalysis. Long-range intramolecular communication between Ser258 and Asp627 (at the catalytic site) modulates the key pumping event: the conversion of the high-energy to the low-energy phosphorylated intermediate associated with copper release.     

The 'P-usher', a novel protein transporter involved in fimbrial assembly and TpsA secretion

We identified a new bacterial transporter, the Pseudomonas aeruginosa CupB3 protein, which is an outer membrane usher involved in pili assembly. In CupB3, the usher domain has fused during evolution with a POTRA (polypeptide-transport-associated)-like domain found in TpsB transporters of two-partner secretion systems. In TpsBs, the POTRA captures the TpsA passenger, which is then transported across the outer membrane through the TpsB beta-barrel. We named CupB3 a 'P-usher' for POTRA-like domain-containing usher. We showed that CupB3 assembles CupB1 fimbrial subunits into pili and secretes CupB5, a TpsA-like protein. The CupB3 usher domain has the function of a TpsB beta-barrel in CupB5 translocation. We revealed that the POTRA-like domain is neither essential for CupB1 fimbriae assembly nor for cell surface exposition of CupB5, but is crucial to coordinate bona fide transport of CupB1 and CupB5 through the usher domain. The P-usher defines a novel transport pathway involving a molecular machine made with old spare parts.     

A note on the time budget and social behaviour of densely housed horses: A case study in Arab breeding mares

We observed a high-density herd (200 mares/ha) of 44 Arab breeding mares, while in a bare paddock in Tunisia. Twenty-minute animal focal samples and scan sampling were used to determine the time budget of the mares during the period from 9 a.m. to 3 p.m. and study their social behaviour. The data obtained reveal restricted behavioural repertoires with missing behaviour like rolling, allogrooming and lying down; unusual time budgets with a high frequency of locomotion that constitutes the most frequent activity (27.9 ± 19.47%) of the mares. Social interactions were restricted to agonistic interactions but despite the high stocking density, aggressions were not that frequent among mares.     

Use of Low-Molecular�CWeight Heparin to Decrease Mortality in Mice after Intracardiac Injection of Tumor Cells

Intracardiac injection of human tumor cells into anesthetized nude mice is an established model of bone metastasis. However, intracardiac injection of some human tumor cell lines cause acute neurologic signs and high mortality, making some potentially relevant tumor cell lines unusable for investigation. We showed that intracardiac injection of tumor cells can induce a hypercoagulable state leading to platelet consumption and thromboemboli formation and that pretreatment with intravenous injection of low-molecular–weight heparin (LMWH; enoxaparin) blocks this state. In addition, intravenous injection of enoxaparin before intracardiac injection with 2 different small-cell lung carcinoma lines, H1975 and H2126, dramatically decreased mouse mortality while still generating bone metastases. Therefore, reduction of mortality by pretreatment with LMWH increases the types of cells that can be studied in this metastasis model and decreases the number of animals used.Abbreviations: APTT, activated partial thromboplastin time; BLI, bioluminescent imaging; CBC, complete blood count; DIC, disseminated intravascular coagulation; H1975luc, H1975 cell line tagged with lucerifase–green fluorescent protein; H2126luc, H2126 cell line tagged with lucerifase–green fluorescent protein; LMWH, low-molecular–weight heparin; PT, prothrombin time; UFH, unfractionated heparinResearch using animal models mimicking the metastasis of human tumors to bone is critical for the development of cancer therapeutics. Bone metastases are present in almost all people who die of cancer and are more likely to occur with breast, prostate, lung, kidney, and thyroid cancers.^1,24 In patients with advanced breast and prostate cancers, much of the tumor burden at the time of death will be found in bone.²⁰ The pattern of bone metastases can range from purely destructive (osteolytic) to mostly osteoblastic (bone-forming) lesions. Osteolysis is accompanied by pain, bone fragility, and increased susceptibility to pathologic fracture. In osteolytic metastasis, a 2-way interaction between tumor cells and osteoclasts in the bone microenvironment leading to continued osteolysis and tumor growth is suspected.²⁰ Current therapies for bone metastases, such as bisphosphonates, are directed at inhibiting bone resorption, but other therapies are in development that specifically target tumor cell or osteoclast factors involved in the 2-way cycle between tumor growth and osteolysis.¹⁸Bone metastasis is rare in mouse models of spontaneous mammary and prostate carcinomas, experimentally implanted animal tumor models (such as syngeneic and xenograft tumors), and chemical or transgenic induction of mammary and prostate carcinomas. To increase the frequency of bone metastases, injection techniques using either orthotopic tumor cell injection into mammary glands or prostate or intracardiac injection of human tumor cell lines into the left ventricle of nude mice have been developed.^5,14,25,31 In contrast to the late stage, low incidence of metastasis after orthotopic injection, intracardiac injection of human tumor cell lines results in much higher rates of bone metastasis at an early stage in the disease, with osteolytic metastases to the metaphyses of long bones.^6,23 Development of osteolytic lesions in this model can be monitored by various methods, including radiography and, more recently, in vivo bioluminescent imaging (BLI) using lucerifase-tagged tumor cells. Bioluminescent imaging detects micrometastatic lesions and allows for serial in vivo monitoring of bone metastases.^9-11 After a BLI study, bone metastases can be assessed histologically, with tumor foci typically seen in the femur or tibia.Bone metastasis models using the intracardiac tumor injection technique have been primarily focused on a few breast (for example, MDA-MB-231) and prostate models (for example, PC3), but additional models of other tumors that interact with bone (especially lung carcinomas) need to be developed.^24,30 Intracardiac injection of some nonsmall cell lung carcinoma tumor cell lines have led to stroke-like clinical signs, including head tilt, spinning, and failure to recover from anesthesia after intracardiac injection.¹⁵ We postulated that the stroke-like clinical signs and mortality were due to thromboembolism formation immediately after intracardiac tumor cell injection.Tumor cells have procoagulant activity. Procoagulants, such as tissue factor, may be increased on the surface of or secreted into the blood by cancer cells, leading to changes in the clotting cascade.¹³ Approximately 15% of all cancer patients are affected by thromboembolic disease, including superficial and deep-vein thrombosis, arterial thrombosis and embolism, pulmonary emboli, and thrombosis of venous access devices.^12,13 Anticoagulant treatments used clinically to prevent thrombi and thromboemboli include warfarin, unfractionated heparin (UFH), and low-molecular–weight heparins (LMWH), such as enoxaparin (Lovenox, Sanofi Aventis, Bridgewater, NJ) and dalteparin (Fragmin, Pfizer, New York, NY). LMWHs are prepared through chemical, hydrolytic, or enzymatic degradation of unfractionated heparin.¹³ Both UFH and LMWH exert their anticoagulant effects by binding to antithrombin and causing a confrontational change. This change increases the interaction of antithrombin with thrombin (IIa) and activated factors X (Xa) and IX (IXa), leading to inhibition of clotting.^8,28LMWHs decrease the formation of thromboembolism and subsequent mortality in several murine models of thromboembolism and disseminated intravascular coagulation (DIC). In the murine model of thrombin-induced thromboembolism, massive deposition of intravascular fibrin—mainly within the pulmonary arteries—causes death within 5 minutes after thrombin injection.^16,22 Both UFH and LMWH inhibit thrombin and prevent mortality in this model, but bleeding times and activated partial prothrombin time (APPT) are less prolonged with LMWH.¹⁶ LMWH is also effective in preventing murine DIC in a lipopolysaccharide model, in which mice given 2 injections of lipopolysaccharide develop DIC, multiple organ failure, and die. Mice given LMWH before lipopolysaccharide administration have fewer lung and liver microthrombi and greater survival than do mice not given LMWH.^26,27Here, we evaluated the use of LMWH in mice to prevent morbidity and mortality associated with intracardiac injection of human tumor cell lines. We determined that thromboembolism occurred in intracardiac tumor-challenged mice and that LMWH blocked thromboembolism. We also determined the effect of LMWH on animal survival and subsequent development of bone metastasis in this mouse model.     

Relief of microRNA-mediated translational repression in human cells subjected to stress

In metazoans, most microRNAs imperfectly base-pair with the 3' untranslated region (3'UTR) of target mRNAs and prevent protein accumulation by either repressing translation or inducing mRNA degradation. Examples of specific mRNAs undergoing microRNA-mediated repression are numerous, but whether the repression is a reversible process remains largely unknown. Here we show that cationic amino acid transporter 1 (CAT-1) mRNA and reporters bearing its 3'UTR can be relieved from the microRNA miR-122-induced inhibition in human hepatocarcinoma cells subjected to different stress conditions. The derepression of CAT-1 mRNA is accompanied by its release from cytoplasmic processing bodies and its recruitment to polysomes. The derepression requires binding of HuR, an AU-rich-element binding protein, to the 3'UTR of CAT-1 mRNA. We propose that proteins interacting with the 3'UTR will generally act as modifiers altering the potential of miRNAs to repress gene expression.     

Experimental assessment of plant seed retention times in fur of cattle and horse

Epizoochorous dispersal of plant seeds is an important long-distance dispersal mechanism. Yet little is known about retention times of seeds in animal furs and hence about potential dispersal distances of the seeds. Here, we used marked seeds of 12 plant species to determine seed depletion curves on Galloway cattle and Haflinger horse in three vegetation types (forest, tall herbage vegetation and meadow), in both dry and rainy weather conditions. In the long fur of Galloway cattle, seeds were retained significantly longer than in the short fur of Haflinger horse. In general, seed retention times were not considerably affected by the structure of the surrounding vegetation. The impact of the weather was negligible, only affecting the retention of some plant species. Negative exponential functions were fitted to the seed depletion curves. Using the parameters of curve estimations in the different conditions of animal species and vegetation structure, half-life seed retention times of up to 13 h for Galloway cattle and up to more than 4 h for Haflinger horse could be calculated, with corresponding potential half-life dispersal distances in the order of magnitude of tens of metres to a few kilometres. Different seed traits correlated with seed retention times in the long cattle fur and in the short horse fur, respectively.     

Tyramine fragment binding to BACE-1

Fragment screening revealed that tyramine binds to the active site of the Alzheimer's disease drug target BACE-1. Hit expansion by selection of compounds from the Roche compound library identified tyramine derivatives with improved binding affinities as monitored by surface plasmon resonance. X-ray structures show that the amine of the tyramine fragment hydrogen-bonds to the catalytic water molecule. Structure-guided ligand design led to the synthesis of further low molecular weight compounds that are starting points for chemical leads.     

Counter-intuitive developmental plasticity induced by host quality

Adaptation to different hosts plays a central role in the evolution of specialization and speciation in phytophagous insects and parasites, and our ability to experimentally rank hosts by their quality is critical to research to understand these processes. Here we provide a counter-intuitive example in which growth is faster on poor quality hosts. The leaf beetles Oreina elongata and Oreina cacaliae share their host plant with the rust Uromyces cacaliae. Larvae reared on infected Adenostyles alliariae show reduced growth rate, reduced maximum weight and longer development time. However, they normally respond adaptively to the rust's mid-season arrival. When switched during development from healthy to infected leaves, larvae accelerate growth and reduce development time, but pupate at lower body weight. In this novel plant-insect-fungus interaction, infection forms the cue to trade off life-history traits in order to complete development within the brief alpine summer. It represents a novel mode of developmental plasticity, which is likely to be found in other host-parasite systems whenever host quality deteriorates due to multiple infection or ageing. This phenotypic plasticity would modify competition after co-infection and the mutual selection imposed by hosts and parasites, and creates a paradoxical negative correlation between growth rate and environmental quality.     

PAP- and GLD-2-type poly(A) polymerases are required sequentially in cytoplasmic polyadenylation and oogenesis in Drosophila

Cytoplasmic polyadenylation has an essential role in activating maternal mRNA translation during early development. In vertebrates, the reaction requires CPEB, an RNA-binding protein and the poly(A) polymerase GLD-2. GLD-2-type poly(A) polymerases form a family clearly distinguishable from canonical poly(A) polymerases (PAPs). In Drosophila, canonical PAP is involved in cytoplasmic polyadenylation with Orb, the Drosophila CPEB, during mid-oogenesis. We show that the female germline GLD-2 is encoded by wispy. Wispy acts as a poly(A) polymerase in a tethering assay and in vivo for cytoplasmic polyadenylation of specific mRNA targets during late oogenesis and early embryogenesis. wispy function is required at the final stage of oogenesis for metaphase of meiosis I arrest and for progression beyond this stage. By contrast, canonical PAP acts with Orb for the earliest steps of oogenesis. Both Wispy and PAP interact with Orb genetically and physically in an ovarian complex. We conclude that two distinct poly(A) polymerases have a role in cytoplasmic polyadenylation in the female germline, each of them being specifically required for different steps of oogenesis.