Raptor mortality due to West Nile virus in the United States, 2002

West Nile virus (WNV) has affected many thousands of birds since it was first detected in North America in 1999, but the overall impact on wild bird populations is unknown. In mid-August 2002, wildlife rehabilitators and local wildlife officials from multiple states began reporting increasing numbers of sick and dying raptors, mostly red-tailed hawks (Buteo jamaicensis) and great horned owls (Bubo virginianus). Commonly reported clinical signs were nonspecific and included emaciation, lethargy, weakness, inability to perch, fly or stand, and nonresponse to danger. Raptor carcasses from 12 states were received, and diagnostic evaluation of 56 raptors implicated WNV infection in 40 (71%) of these cases. Histologically, nonsuppurative encephalitis and myocarditis were the salient lesions (79% and 61%, respectively). Other causes of death included lead poisoning, trauma, aspergillosis, and Salmonella spp. and Clostridium spp. infections. The reason(s) for the reported increase in raptor mortality due to WNV in 2002 compared with the previous WNV seasons is unclear, and a better understanding of the epizootiology and pathogenesis of the virus in raptor populations is needed.     

Interactive effects of ozone and climate on tree growth and water use in a southern Appalachian forest in the USA

* A lack of data on responses of mature tree growth and water use to ambient ozone (O(3)) concentrations has been a major limitation in efforts to understand and model responses of forests to current and future changes in climate. * Here, hourly to seasonal patterns of stem growth and sap flow velocity were examined in mature trees from a mixed deciduous forest in eastern Tennessee (USA) to evaluate the effects of variations in ambient O(3) exposure and climate on patterns of stem growth and water use. * Ambient O(3) caused a periodic slowdown in seasonal growth patterns that was attributable in part to amplification of diurnal patterns of water loss in tree stems. This response was mediated by statistically significant increases in O(3)-induced daily sap flow and led to seasonal losses in stem growth of 30-50% for most species in a high-O(3) year. * Decreased growth and increased water use of mature forest trees under episodically high ambient O(3) concentrations suggest that O(3) will amplify the adverse effects of increasing temperatures on forest growth and forest hydrology.     

Conformational dynamics of the molecular chaperone Hsp90 in complexes with a co-chaperone and anticancer drugs

The molecular chaperone Hsp90 is essential for the correct folding, maturation and activation of a diverse array of client proteins, including several key constituents of oncogenic processes. Hsp90 has become a focus of cancer research, since it represents a target for direct prophylaxis against multistep malignancy. Hydrogen-exchange mass spectrometry was used to study the structural and conformational changes undergone by full-length human Hsp90beta in solution upon binding of the kinase-specific co-chaperone Cdc37 and two Hsp90 ATPase inhibitors: Radicicol and the first-generation anticancer drug DMAG. Changes in hydrogen exchange pattern in the complexes in regions of Hsp90 remote to the ligand-binding site were observed indicating long-range effects. In particular, the interface between the N-terminal domain and middle domains exhibited significant differences between the apo and complexed forms. For the inhibitors, differences in the interface between the middle domain and the C-terminal domain were also observed. These data provide important insight into the structure of the biologically active form of the protein.     

Mutation in HSF4 associated with early but not late-onset hereditary cataract in the Boston Terrier

Primary hereditary cataract (HC) is one of the most common disorders in purebred dogs and is a leading cause of blindness. Boston Terriers suffer from 2 distinct forms of HC which occur at different ages and which are different in their appearance and progression. Early-onset hereditary cataract (EHC) affects dogs within the first few months of life, is always progressive and bilateral, and results in total blindness, whereas late-onset hereditary cataract (LHC) in general affects dogs over the age of 3 and is more variable in its clinical phenotype, age of onset, progression, and the degree to which vision is impaired. A mutation in HSF4 has recently been reported in a small number of Boston Terriers affected with EHC. In this study, we analyzed 22 additional Boston Terriers affected with early-onset cataract to confirm that the HSF4 mutation is causative for this form of cataract in this breed. In addition, we analyzed 40 Boston Terriers that were either clinically clear or affected with LHC for the presence or absence of the HSF4 mutation. By also sequencing HSF4 in dogs affected with LHC, we conclude that HSF4 is not associated with the development of the late-onset form of cataract and that the 2 forms of cataract in this breed are therefore genetically discrete conditions.     

A NASP (N1/N2)-related protein, Sim3, binds CENP-A and is required for its deposition at fission yeast centromeres

A defining feature of centromeres is the presence of the histone H3 variant CENP-A(Cnp1). It is not known how CENP-A(Cnp1) is specifically delivered to, and assembled into, centromeric chromatin. Through a screen for factors involved in kinetochore integrity in fission yeast, we identified Sim3. Sim3 is homologous to known histone binding proteins NASP(Human) and N1/N2(Xenopus) and aligns with Hif1(S. cerevisiae), defining the SHNi-TPR family. Sim3 is distributed throughout the nucleoplasm, yet it associates with CENP-A(Cnp1) and also binds H3. Cells defective in Sim3 function have reduced levels of CENP-A(Cnp1) at centromeres (and increased H3) and display chromosome segregation defects. Sim3 is required to allow newly synthesized CENP-A(Cnp1) to accumulate at centromeres in S and G2 phase-arrested cells in a replication-independent mechanism. We propose that one function of Sim3 is to act as an escort that hands off CENP-A(Cnp1) to chromatin assembly factors, allowing its incorporation into centromeric chromatin.     

Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1

Mutations in the PTEN‐induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson''s disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser⁶⁵) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1‐dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub‐family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser¹¹¹) in response to PINK1 activation. Using phospho‐specific antibodies raised against Ser¹¹¹ of each of the Rabs, we demonstrate that Rab Ser¹¹¹ phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient‐derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser¹¹¹ phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser¹¹¹ phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser⁶⁵. We further show mechanistically that phosphorylation at Ser¹¹¹ significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser¹¹¹ may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase‐mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson''s disease.     

Nxf3 is expressed in Sertoli cells, but is dispensable for spermatogenesis

In eukaryotes, mRNA is actively exported to the cytoplasm by a family of nuclear RNA export factors (NXF). Four Nxf genes have been identified in the mouse: Nxf1, Nxf2, Nxf3, and Nxf7. Inactivation of Nxf2, a germ cell-specific gene, causes defects in spermatogenesis. Here we report that Nxf3 is expressed exclusively in Sertoli cells of the postnatal testis, in a developmentally regulated manner. Expression of Nxf3 coincides with the cessation of Sertoli cell proliferation and the beginning of their differentiation. Continued expression of Nxf3 in mature Sertoli cells of the adult is spermatogenesis stage-independent. Nxf3 is not essential for spermatogenesis, however, suggesting functional redundancy among Nxf family members. With its unique expression pattern in the testis, the promoter of Nxf3 can be used to drive postnatal Sertoli cell-specific expression of other proteins such as Cre recombinase.