The 1:1 N-NS protein complex of vesicular stomatitis virus is essential for efficient genome replication.

We studied the effect pH had on the N-NS protein complex to determine its role in vesicular stomatitis virus (VSV) genome replication, as we had previously shown that VSV genome replication in vitro requires the interaction of the viral N and NS proteins into a 1:1 complex. A previous report showed that the growth of VSV in L cells was sensitive to the pH of the environment (M. Fiszman, J. B. Leaute, C. Chany, and M. Girard, J. Virol. 13:801-808, 1974). We hypothesized that low pH might disrupt the N-NS protein complex, and so we investigated the molecular events leading to inhibition of viral RNA replication in vitro from extracts that were prepared from VSV-infected cells incubated at pH 6.6. We found that viral genome RNA synthesis in vitro was reduced when infected cells were maintained at pH 6.6. Through immunoprecipitation analysis of the viral soluble protein pool, we found that a complex that usually exists between the N and NS proteins at pH 7.4 was altered in extracts from infected cells maintained at pH 6.6, and this was responsible for the observed effects on viral replication. The effect of low pH on the N-NS protein complex could not be abolished by increasing the concentration of the altered complex, indicating that the effects is more than simply a decrease in the level of the protein complex in the cell. Our data provide additional evidence that the 1:1 N-NS protein complex, and not the N protein alone, serves as the substrate for viral RNA replication in vivo.     

Ultrastructural alterations associated with the initiation of follicular atresia

Summary To identify and describe ovarian follicles committed to undergo follicular degeneration (atresia), immature rats were primed with pregnant mare serum gonadotropin (PMSG). After PMSG treatment, preovulatory follicles develop but subsequently degenerate. Prior to the appearance of pyknotic nuclei (Stage I of atresia), degenerative changes were observed in focal areas of the granulosa cell layer. These changes include blebbing of the cytoplasm and alterations in the shape of the granulosa cells. The appearance of these degenerative changes coincides with a decrease in ovarian concentrations of estradiol and testosterone. Since estrogens and androgens maintain the follicle, the decline in estradiol and testosterone could be responsible for the further degenerative alterations that lead to complete deterioration of the preovulatory follicle. In Stage I atretic follicles, lysosome-derived autophagic vacuoles develop and macrophages invade both the thecal and granulosa cell layers. The combined actions of the autophagic vacuoles and macrophages could destroy both the granulosa-cell and thecal layers and thereby transform the preovulatory follicle into an ovarian cyst.     

Fusion properties of cells persistently infected with human parainfluenza virus type 3: participation of hemagglutinin-neuraminidase in membrane fusion.

Cells persistently infected with human parainfluenza virus type 3 (HPF3) exhibit a novel phenotype. They are completely resistant to fusion with each other but readily fuse with uninfected cells. We demonstrate that the inability of these cells to fuse with each other is due to a lack of cell surface neuraminic acid. Neuraminic acid is the receptor for the HPF3 hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. Uninfected CV-1 cells were treated with neuraminidase and then tested for their ability to fuse with the persistently infected (pi) cells. Neuraminidase treatment totally abolished cell fusion. To extend this result, we used a cell line deficient in sialic acid and demonstrated that these cells, like the neuraminidase-treated CV-1 cells, were unable to fuse with pi cells. We then tested whether mimicking the agglutinating function of the HN molecule with lectins would result in cell fusion. We added a panel of five lectins to the neuraminic acid-deficient cells and showed that binding of these cells to the pi cells did not result in fusion; the lectins could not substitute for interaction of neuraminic acid with the HN molecule in promoting membrane fusion. These results provide compelling evidence that the HN molecule of HPF3 and its interaction with neuraminic acid participate in membrane fusion and that cell fusion is mediated by an interaction more complex than mere juxtaposition of the cell membranes.     

An invisible ubiquitin conformation is required for efficient phosphorylation by PINK1

The Ser/Thr protein kinase PINK1 phosphorylates the well‐folded, globular protein ubiquitin (Ub) at a relatively protected site, Ser65. We previously showed that Ser65 phosphorylation results in a conformational change in which Ub adopts a dynamic equilibrium between the known, common Ub conformation and a distinct, second conformation wherein the last β‐strand is retracted to extend the Ser65 loop and shorten the C‐terminal tail. We show using chemical exchange saturation transfer (CEST) nuclear magnetic resonance experiments that a similar, C‐terminally retracted (Ub‐CR) conformation also exists at low population in wild‐type Ub. Point mutations in the moving β5 and neighbouring β‐strands shift the Ub/Ub‐CR equilibrium. This enabled functional studies of the two states, and we show that while the Ub‐CR conformation is defective for conjugation, it demonstrates improved binding to PINK1 through its extended Ser65 loop, and is a superior PINK1 substrate. Together our data suggest that PINK1 utilises a lowly populated yet more suitable Ub‐CR conformation of Ub for efficient phosphorylation. Our findings could be relevant for many kinases that phosphorylate residues in folded protein domains.     

From recommendation to action: psychosocial factors influencing physician intention to use Health Technology Assessment (HTA) recommendations

Background

The Evidence-Based Practice (EBP) Project has been investigating the implementation of evidence-based mental health practices (Assertive Community Treatment, Family Psychoeducation, Integrated Dual Diagnosis Treatment, Illness Management and Recovery, and Supported Employment) in state public mental health systems in the United States since 2001. To date, Project findings have yielded valuable insights into implementation strategy characteristics and effectiveness. This paper reports results of an effort to identify and classify state-level implementation activities and strategies employed across the eight states participating in the Project.

Methods

Content analysis and Greenhalgh et al's (2004) definition of innovation were used to identify and classify state-level activities employed during three phases of EBP implementation: Pre-Implementation, Initial Implementation and Sustainability Planning. Activities were coded from site visit reports created from documents and notes from key informant interviews conducted during two periods, Fall 2002 – Spring 2003, and Spring 2004. Frequency counts and rank-order analyses were used to examine patterns of implementation activities and strategies employed across the three phases of implementation.

Results

One hundred and six discreet implementation activities and strategies were identified as innovative and were classified into five categories: 1) state infrastructure building and commitment, 2) stakeholder relationship building and communications, 3) financing, 4) continuous quality management, and 5) service delivery practices and training. Implementation activities from different categories were employed at different phases of implementation.

Conclusion

Insights into effective strategies for implementing EBPs in mental health and other health sectors require qualitative and quantitative research that seeks to: a) empirically test the effects of tools and methods used to implement EBPs, and b) establish a stronger evidence-base from which to plan, implement and sustain such efforts. This paper offers a classification scheme and list of innovative implementation activities and strategies. The classification scheme offers potential value for future studies that seek to assess the effects of various implementation processes, and helps establish widely accepted standards and criteria that can be used to assess the value of innovative activities and strategies.     

Pathogenesis of inclusion bodies in (CAG)n/Qn-expansion diseases with special reference to the role of tissue transglutaminase and to selective vulnerability

At least eight neurodegenerative diseases, including Huntington disease, are caused by expansions in (CAG)n repeats in the affected gene and by an increase in the size of the corresponding polyglutamine domain in the expressed protein. A hallmark of several of these diseases is the presence of aberrant, proteinaceous aggregates in the nuclei and cytosol of affected neurons. Recent studies have shown that expanded polyglutamine (Qn) repeats are excellent glutaminyl-donor substrates of tissue transglutaminase, and that the substrate activity increases with increasing size of the polyglutamine domain. Tissue transglutaminase is present in the cytosol and nuclear fractions of brain tissue. Thus, the nuclear and cytosolic inclusions in Huntington disease may contain tissue transglutaminase-catalyzed covalent aggregates. The (CAG)n/Qn-expansion diseases are classic examples of selective vulnerability in the nervous system, in which certain cells/structures are particularly susceptible to toxic insults. Quantitative differences in the distribution of the brain transglutaminase(s) and its substrates, and in the activation mechanism of the brain transglutaminase(s), may explain in part selective vulnerability in a subset of neurons in (CAG)n-expansion diseases, and possibly in other neurodegenerative disease. If tissue transglutaminase is found to be essential for development of pathogenesis, then inhibitors of this enzyme may be of therapeutic benefit.     

minifly, a Drosophila gene required for ribosome biogenesis

We report here the genetic, molecular, and functional characterization of the Drosophila melanogaster minifly (mfl) gene. Genetic analysis shows that mfl is essential for Drosophila viability and fertility. While P-element induced total loss-of-function mutations cause lethality, mfl partial loss-of-function mutations cause pleiotropic defects, such as extreme reduction of body size, developmental delay, hatched abdominal cuticle, and reduced female fertility. Morphological abnormalities characteristic of apoptosis are found in the ovaries, and a proportion of eggs laid by mfl mutant females degenerates during embryogenesis. We show that mfl encodes an ubiquitous nucleolar protein that plays a central role in ribosomal RNA processing and pseudouridylation, whose known eukaryotic homologues are yeast Cfb5p, rat NAP57 and human dyskerin, encoded by the gene responsible for the X-linked dyskeratosis congenita disease. mfl genetic analysis represents the first in vivo functional characterization of a member of this highly conserved gene family from higher eukaryotes. In addition, we report that mfl hosts an intron encoded box H/ACA snoRNA gene, the first member of this class of snoRNAs identified so far from Drosophila.     

Cyclic expression of endothelin-converting enzyme-1 mediates the functional regulation of seminiferous tubule contraction.

The potent smooth muscle agonist endothelin-1 (ET-1) is involved in the local control of seminiferous tubule contractility, which results in the forward propulsion of tubular fluid and spermatozoa, through its action on peritubular myoid cells. ET-1, known to be produced in the seminiferous epithelium by Sertoli cells, is derived from the inactive intermediate big endothelin-1 (big ET-1) through a specific cleavage operated by the endothelin-converting enzyme (ECE), a membrane-bound metalloprotease with ectoenzymatic activity. The data presented suggest that the timing of seminiferous tubule contractility is controlled locally by the cyclic interplay between different cell types. We have studied the expression of ECE by Sertoli cells and used myoid cell cultures and seminiferous tubule explants to monitor the biological activity of the enzymatic reaction product. Northern blot analysis showed that ECE-1 (and not ECE-2) is specifically expressed in Sertoli cells; competitive enzyme immunoassay of ET production showed that Sertoli cell monolayers are capable of cleaving big ET-1, an activity inhibited by the ECE inhibitor phosphoramidon. Microfluorimetric analysis of intracellular calcium mobilization in single cells showed that myoid cells do not respond to big endothelin, nor to Sertoli cell plain medium, but to the medium conditioned by Sertoli cells in the presence of big ET-1, resulting in cell contraction and desensitization to further ET-1 stimulation; in situ hybridization analysis shows regional differences in ECE expression, suggesting that pulsatile production of endothelin by Sertoli cells (at specific "stages" of the seminiferous epithelium) may regulate the cyclicity of tubular contraction; when viewed in a scanning electron microscope, segments of seminiferous tubules containing the specific stages characterized by high expression of ECE were observed to contract in response to big ET-1, whereas stages with low ECE expression remained virtually unaffected. These data indicate that endothelin-mediated spatiotemporal control of rhythmic tubular contractility might be operated by Sertoli cells through the cyclic expression of ECE-1, which is, in turn, dependent upon the timing of spermatogenesis.     

Progesterone regulates granulosa cell viability through a protein kinase G-dependent mechanism that may involve 14-3-3sigma

Progesterone (P4) inhibits granulosa cell and spontaneously immortalized granulosa cell (SIGC) apoptosis by regulating membrane-initiated events. However, the nature of the signal transduction pathway that is induced by these membrane-initiated events has not been defined. To gain insights into the P4-regulated signal transduction pathway, mouse granulosa cells and SIGCs were cultured with 8-br-cGMP and P4. In culture, 8-br-cGMP mimicked P4's antiapoptotic actions. Because cGMP activates protein kinase G (PKG), the effect of PKG antagonists on P4-regulated SIGC viability was assessed. P4's antiapoptotic action was attenuated by the PKG inhibitors, Rp-8-pCPT-cGMP, KT5823, the PKG-1alpha-specific inhibitor, DT-3, and a dominant negative PKG-1alpha. Further, the type I isoform of PKG was shown to be expressed by SIGCs and activated by P4. P4's antiapoptotic action was not affected by the PKA inhibitor, KT5720. Collectively, these findings indicate that P4 maintains SIGC viability by activating PKG-1alpha. PKG-1alpha-GFP was shown to localize predominantly to the cytoplasm of SIGCs. To identify potential cytoplasmic targets of PKG-1alpha, SIGCs were cultured for 5 h with P4 in the presence or absence of DT-3. Cell lysates were prepared and subjected to two-dimensional electrophoresis. The resulting gels were sequentially stained with ProQ-Diamond Gel Stain and Coomassie Blue to reveal phosphorylated proteins. The two-dimensional gels revealed one major protein, the phosphorylation status of which was abrogated by DT-3. Mass spectrometric analysis identified this protein as 14-3-3sigma, with 14-3-3sigma being phosphorylated on tyrosine 19, serine 28, serine 69, serine 74, threonine 90, threonine 98, and serine 116. Finally, difopein, a specific 14-3-3 inhibitor, was shown to induce apoptosis even in the presence of serum. These data suggest that 1) P4 regulates the phosphorylation status of 14-3-3sigma through a PKG-dependent pathway and 2) 14-3-3sigma plays a central and essential role in maintaining the viability of SIGCs.