Trafficking of STEVOR to the Maurer's clefts in Plasmodium falciparum-infected erythrocytes

The human malarial parasite Plasmodium falciparum exports proteins to destinations within its host erythrocyte, including cytosol, surface and membranous profiles of parasite origin termed Maurer's clefts. Although several of these exported proteins are determinants of pathology and virulence, the mechanisms and trafficking signals underpinning protein export are largely uncharacterized-particularly for exported transmembrane proteins. Here, we have investigated the signals mediating trafficking of STEVOR, a family of transmembrane proteins located at the Maurer's clefts and believed to play a role in antigenic variation. Our data show that, apart from a signal sequence, a minimum of two addition signals are required. This includes a host cell targeting signal for export to the host erythrocyte and a transmembrane domain for final sorting to Maurer's clefts. Biochemical studies indicate that STEVOR traverses the secretory pathway as an integral membrane protein. Our data suggest general principles for transport of transmembrane proteins to the Maurer's clefts and provide new insights into protein sorting and trafficking processes in P. falciparum.     

Sorting nexin 17 facilitates LRP recycling in the early endosome

The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multiligand endocytic receptor and a member of the LDL receptor family. Here we show that sorting nexin 17 (Snx 17) is part of the cellular sorting machinery that regulates cell surface levels of LRP by promoting its recycling. While the phox (PX) domain of Snx 17 interacts with phosphatidylinositol-3-phosphate for membrane association, the FERM domain and the carboxyl-terminal region participate in LRP binding. Immunoelectron microscopy shows that the membrane-bound fraction of Snx 17 is localized to the limiting membrane and recycling tubules of early endosomes. The NPxY motif, proximal to the plasma membrane in the LRP cytoplasmic tail, is identified as the Snx 17-binding motif. Functional mutation of this motif did not interfere with LRP endocytosis, but decreased LRP recycling from endosomes, resulting in increased lysosomal degradation. Similar effects are found after knockdown of endogenous Snx 17 expression by short interfering RNA. We conclude that Snx 17 binds to a motif in the LRP tail distinct from the endocytosis signals and promotes LRP sorting to the recycling pathway in the early endosomes.     

Structure of an XPF endonuclease with and without DNA suggests a model for substrate recognition

The XPF/Mus81 structure-specific endonucleases cleave double-stranded DNA (dsDNA) within asymmetric branched DNA substrates and play an essential role in nucleotide excision repair, recombination and genome integrity. We report the structure of an archaeal XPF homodimer alone and bound to dsDNA. Superposition of these structures reveals a large domain movement upon binding DNA, indicating how the (HhH)(2) domain and the nuclease domain are coupled to allow the recognition of double-stranded/single-stranded DNA junctions. We identify two nonequivalent DNA-binding sites and propose a model in which XPF distorts the 3' flap substrate in order to engage both binding sites and promote strand cleavage. The model rationalises published biochemical data and implies a novel role for the ERCC1 subunit of eukaryotic XPF complexes.     

Genetic and Ecological Characterisation of Colour Dimorphism in a Coral Reef Fish

Synopsis Many recognised species of coral reef fishes exhibit two or more colour variants, but it is unknown whether these represent genetically identical phenotypes, genetic polymorphisms or closely related species. We tested between these alternatives for two colour morphs of the coral reef fish, Pseudochromis fuscus, from Lizard Island (Great Barrier Reef). A molecular analysis using mtDNA did not detect any genetic differentiation between co-occurring ‘yellow’ and ‘brown’ colour morphs. A previous study proposed that these two colour morphs are aggressive mimics of yellow and brown damselfishes. Here, a manipulative field experiment was used to evaluate whether the colour dimorphism in P. fuscus is a phenotypic response to the presence of two different model species. Colonies of either yellow or brown damselfish species were established on different patch reefs, and each of the two different P. fuscus morphs was then placed on the different reefs. Contrary to expectations, all yellow individuals that stayed on the reefs changed to brown, regardless of the model species. No brown individuals changed to the yellow colouration. However, P. fuscus were more likely to emigrate from, or suffer higher mortality on, patch reefs where they were not matched with similarly coloured models. Clearly, yellow and brown P. fuscus are members of a single species with sufficient phenotypic plasticity to switch from yellow to brown colouration.     

In vivo up-regulation of brain-derived neurotrophic factor in specific brain areas by chronic exposure to Delta-tetrahydrocannabinol

Cannabinoids are widely abused drugs. Here we show that chronic administration of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the active psychotropic agent in marijuana and hashish, at 1.5 mg per kg per day intraperitoneally for 7 days, increases the expression, at both mRNA and protein levels, of brain-derived neurotrophic factor (BDNF), in specific rat brain areas, notably in those involved in reward and addiction. Real-time PCR revealed a 10-fold up-regulation of BDNF mRNA in the nucleus accumbens (NAc) upon chronic Delta(9)-THC treatment, but there was no change at 3 or 24 h after a single injection. Smaller increases in mRNA levels were found in the ventral tegmental area (VTA), medial prefrontal cortex and paraventricular nucleus (PVN). Immunohistochemistry showed large increases in BDNF-stained cells in the NAc (5.5-fold), posterior VTA (4-fold) and PVN (1.7-fold), but no change was observed in the anterior VTA, hippocampus or dorsal striatum. Altogether, our study indicates that chronic exposure to Delta(9)-THC up-regulates BDNF in specific brain areas involved with reward, and provides evidence for different BDNF expression in the anterior and posterior VTA. Moreover, BDNF is known to modulate synaptic plasticity and adaptive processes underlying learning and memory, leading to long-term functional and structural modification of synaptic connections. We suggest that Delta(9)-THC up-regulation of BDNF expression has an important role in inducing the neuroadaptive processes taking place upon exposure to cannabinoids.     

Induction of long-term protective antiviral endogenous immune response by short neutralizing monoclonal antibody treatment

Long-term immune control of viral replication still remains a major challenge in retroviral diseases. Several monoclonal antibodies (MAbs) have already shown antiviral activities in vivo, including in the clinic but their effects on the immune system of treated individuals are essentially unknown. Using the lethal neurodegeneration induced in mice upon infection of neonates by the FrCas(E) retrovirus as a model, we report here that transient treatment by a neutralizing MAb shortly after infection can, after an immediate antiviral effect, favor the development of a strong protective host immune response containing viral propagation long after the MAb has disappeared. In vitro virus neutralization- and complement-mediated cell lysis assays, as well as in vivo viral challenges and serum transfer experiments, indicate a clear and essential contribution of the humoral response to antiviral protection. Our observation may have important therapeutic consequences as it suggests that short antibody-based therapies early after infection should be considered, at least in the case of maternally infected infants, as adjunctive treatment strategies against human immunodeficiency virus, not only for a direct effect on the viral load but also for favoring the emergence of an endogenous antiviral immune response.     

Receptor-induced conformational changes in the SU subunit of the avian sarcoma/leukosis virus A envelope protein: implications for fusion activation

The avian sarcoma/leukosis virus (ASLV) is activated for fusion by a two-step mechanism. For ASLV subgroup A (ASLV-A), association with its receptor (Tva) at neutral pH converts virions to a form that can bind target membranes and, in some assays, induce the lipid-mixing stage of fusion. Low pH is necessary to complete the fusion reaction. ASLV-A env (EnvA) exists on the viral surface as a trimer of heterodimers consisting of receptor binding (SU-A) and fusion-mediating (TM-A) subunits. As the receptor binding and fusion-mediating functions reside in separate subunits, we hypothesize that SU-A and TM-A are conformationally coupled. To begin to understand the effect of the binding of a soluble 47-residue domain of the receptor (sTva) on this coupling and the subsequent function of low pH, we prepared recombinant proteins representing full-length SU-A and a nested set of deletion mutant proteins. Full-length SU-A binds sTva with high affinity, but even small deletions at either the N or the C terminus severely impair sTva binding. We have purified the full-length SU-A subunit and characterized its interactions with sTva and the subsequent effect of low pH on the complex. sTva binds SU-A with an apparent KD of 3 pM. Complex formation occludes hydrophobic surfaces and tryptophan residues and leads to a partial loss of alpha-helical structure in SU-A. Low pH does not alter the off rate for the complex, further alter the secondary structure of SU-A, or induce measurable changes in tryptophan environment. The implications of these findings for fusion are discussed.     

Localization of myosin II to chromosome arms and spindle fibers in PtK1 cells: a possible role for an actomyosin system in mitosis

Summary. The enzymes of importance in moving chromosomes are called motor proteins and include dynein, kinesin, and possibly myosin II. These three molecules are all included in the category of ATPases, in that they have the ability to convert chemical energy into mechanical energy. Both dynein and kinesin have been documented as molecules that “walk” along microtubules in the mitotic spindle, carrying cargo such as chromosomes. Myosin II, analogous to the muscle contraction system, transiently interacts along actin filaments and associates with kinetochore microtubules. In this paper we present evidence that a third ATPase, myosin II, may act as a “thruster” to propel chromosomes during the mitotic process. Double-label immunocytochemistry to actin and myosin II shows that myosin II is localized on chromosome arms at the beginning of mitosis and remains localized to the chromosomes throughout mitosis. Specific staining of myosin II is relegated to the outside of chromosomes with the highest density of staining occurring between the spindle poles and the chromosomes. This specific localization could account for the movement of chromosomes during mitosis, since they segregate towards the spindle poles, along kinetochore microtubules containing actin filaments, after aligning at the equatorial region of the cell at metaphase. We conclude from this study that there is an actomyosin system present in the mitotic spindle and that myosin is attached to chromosome arms and may act as a thruster in moving chromosomes during the mitotic process. Correspondence and reprints: Department of Biological Sciences, University of Denver, 2190 E Iliff Avenue, Denver, CO 80208, U.S.A.     

Diseases and mortality in free-ranging brown bear (Ursus arctos), gray wolf (Canis lupus), and wolverine (Gulo gulo) in Sweden

Ninety-eight brown bears (Ursus arctos), 20 gray wolves (Canis lupus), and 27 wolverines (Gulo gulo), all free-ranging, were submitted to the National Veterinary Institute, Uppsala, Sweden, during 1987-2001 for investigation of diseases and causes of mortality. The most common cause of natural death in brown bears was infanticide. Infanticide also was observed in wolverines but not in wolves. Traumatic injuries, originating from road or railway accidents, were the most common cause of death in wolves and occurred occasionally in brown bears. Most wolverines were submitted as forensic cases in which illegal hunting/poaching was suspected. Sarcoptic mange was observed in several wolves but not in brown bears or wolverines. Sarcoptic mange most likely was acquired from infected red foxes (Vulpes vulpes) that were killed by wolves. Other parasites and infectious diseases were only found sporadically.