Postmortem Degradation Alters Fluorographic Labeling Patterns and Affinities of Benzodiazepine Binding Proteins

To investigate the effect of endogenous proteolysis on the molecular weights of the benzodiazepine binding proteins, brains of trout, chicken, and rat were removed immediately after death and stored at room temperature for various periods of time before they were frozen. Photoaffinity labeling of membranes with [3H]flunitrazepam, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, revealed proteolytic fragments of 47K in trout, chicken, and rat. The proteolysis set in rapidly after death. Seemingly in parallel with the degradation observed fluorographically, the affinity for [3H]flunitrazepam increased without systematic changes in receptor density. The degradation pattern was not identical to that of the photolabeled trypsinized benzodiazepine binding proteins. The endogenous proteolytic fragments were deglycosylated in two steps. In conclusion, proteolytic effects must be taken into account when interpreting labeling patterns and binding parameters.     

A demonstration of pattern formation without positional information in Dictyostelium

Although positional information, conveyed by morphogen gradients, is a widely accepted way of forming patterns during development, an alternative method is conceivable, based on the intermingled differentiation of cells with different fates, followed by their sorting into discrete pattern elements. It has been proposed that Dictyostelium prestalk and prespore cells behave in this way at the mound stage of development. However, it has been difficult to conclusively demonstrate that they initially differentiate intermingled, because rapid cell movement within the mound makes it impossible to be sure where prestalk and prespore cells originate. We have taken a novel approach to address this problem by blocking cell movement at different stages in development, using the actin-depolymerizing drug, latrunculin-A. Prestalk and prespore cells differentiate with essentially normal efficiency and timing in such paralyzed structures. When movement is blocked sufficiently early, the major cell types all subsequently differentiate at scattered positions throughout the aggregate, and even in the streams leading into it. Our work strongly supports the idea that the prestalk/prespore pattern in Dictyostelium forms without positional information and demonstrate that latrunculin-A may provide a useful tool for the investigation of patterning in other organisms.     

Anti-Glycoprotein H Antibody Impairs the Pathogenicity of Varicella-Zoster Virus in Skin Xenografts in the SCID Mouse Model

Varicella-zoster virus (VZV) infection is usually mild in healthy individuals but can cause severe disease in immunocompromised patients. Prophylaxis with varicella-zoster immunoglobulin can reduce the severity of VZV if given shortly after exposure. Glycoprotein H (gH) is a highly conserved herpesvirus protein with functions in virus entry and cell-cell spread and is a target of neutralizing antibodies. The anti-gH monoclonal antibody (MAb) 206 neutralizes VZV in vitro. To determine the requirement for gH in VZV pathogenesis in vivo, MAb 206 was administered to SCID mice with human skin xenografts inoculated with VZV. Anti-gH antibody given at 6 h postinfection significantly reduced the frequency of skin xenograft infection by 42%. Virus titers, genome copies, and lesion size were decreased in xenografts that became infected. In contrast, administering anti-gH antibody at 4 days postinfection suppressed VZV replication but did not reduce the frequency of infection. The neutralizing anti-gH MAb 206 blocked virus entry, cell fusion, or both in skin in vivo. In vitro, MAb 206 bound to plasma membranes and to surface virus particles. Antibody was internalized into vacuoles within infected cells, associated with intracellular virus particles, and colocalized with markers for early endosomes and multivesicular bodies but not the trans-Golgi network. MAb 206 blocked spread, altered intracellular trafficking of gH, and bound to surface VZV particles, which might facilitate their uptake and targeting for degradation. As a consequence, antibody interference with gH function would likely prevent or significantly reduce VZV replication in skin during primary or recurrent infection.Varicella-zoster virus (VZV) causes chicken pox (varicella) upon primary infection. Lifelong latency is established in neurons of the sensory ganglia, and reactivation leads to shingles (herpes zoster) (1). Disease is usually inconsequential in immunocompetent people but can be severe in immunocompromised patients. The current prophylaxis for these high-risk individuals exposed to VZV is high-titer immunoglobulin to VZV administered within 96 h of exposure. This prophylaxis does not always prevent disease, but the severity of symptoms and mortality rates are usually reduced (32).Glycoprotein H (gH) is a type 1 transmembrane protein that is required for virus-cell and cell-cell spread in all herpesviruses studied (12, 15, 24, 26). gH is an important target of the host immune system. Individuals who have had primary infection with VZV or herpes simplex virus (HSV), the most closely related human alphaherpesvirus, have humoral and cellular immunity against gH (1, 56). Immunization of mice with a recombinant vaccinia virus expressing VZV gH and its chaperone, glycoprotein L (gL), induced specific antibodies capable of neutralizing VZV in vitro (28, 37). Immunization of mice with purified HSV gH/gL protein resulted in the production of neutralizing antibodies and protected mice from HSV challenge (5, 44), and administration of an anti-HSV gH monoclonal antibody (MAb) protected mice from HSV challenge (16). Antibodies to HSV and Epstein-Barr virus gH effectively neutralize during virus penetration but not during adsorption in vitro, indicating an essential role for gH in the fusion of viral and cellular membranes but not in initial attachment of the virus to the cell (18, 33).Anti-gH MAb 206, an immunoglobulin G1 (IgG1) antibody which recognizes a conformation-dependent epitope on the mature glycosylated form of gH, neutralizes VZV infection in vitro in the absence of complement (35). MAb 206 inhibits cell-cell fusion in vitro, based on reductions in the number of infected cells and the number of infected nuclei within syncytia, and appears to inhibit the ability of virus particles to pass from the surface of an infected epithelial cell to a neighboring cell via cell extensions (8, 35, 43). When infected cells were treated with MAb 206 for 48 h postinfection (hpi), virus egress and syncytium formation were not apparent, but they were evident within 48 h after removal of the antibody, suggesting that the effect of the antibody was reversible and that there was a requirement for new gH synthesis and trafficking to produce cell-cell fusion. Conversely, nonneutralizing antibodies to glycoproteins E (gE) and I (gI), as well as an antibody to immediate-early protein 62 (IE62), had no effect on VZV spread (46).Like that of other herpesviruses, VZV entry into cells is presumed to require fusion of the virion envelope with the cell membrane or endocytosis followed by fusion. One of the hallmarks of VZV infection is cell fusion and formation of syncytia (8). Cell fusion can be detected as early as 9 hpi in vitro, although VZV spread from infected to uninfected cells is evident within 60 min (45). In vivo, VZV forms syncytia through its capacity to cause fusion of epidermal cells. Syncytia are evident in biopsies of varicella and herpes zoster skin lesions during natural infection and in SCIDhu skin xenografts (34). VZV gH is produced, processed in the Golgi apparatus, and trafficked to the cell membrane, where it might be involved in cell-cell fusion (11, 29, 35). gH then undergoes endocytosis and is trafficked back to the trans-Golgi network (TGN) for incorporation into the virion envelope (20, 31, 42). Since VZV is highly cell associated in vitro, little is known about the glycoproteins required for entry, but VZV gH is present in abundance in the skin vesicles during human chickenpox and zoster (55).Investigating the functions of gH in the pathogenesis of VZV infection in vivo is challenging because it is an essential protein and VZV is species specific for the human host. The objective of this study was to investigate the role of gH in VZV pathogenesis by establishing whether antibody-mediated interference with gH function could prevent or modulate VZV infection of differentiated human tissue in vivo, using the SCIDhu mouse model. The effects of antibody administration at early and later times after infection were determined by comparing infectious virus titers, VZV genome copies, and lesion formation in anti-gH antibody-treated xenografts. In vitro experiments were performed to determine the potential mechanism(s) of MAb 206 interference with gH during VZV replication, virion assembly, and cell-cell spread. The present study has implications for understanding the contributions of gH to VZV replication in vitro and in vivo, the mechanisms by which production of antibodies to gH by the host might restrict VZV infection, and the use of passive antibody prophylaxis in patients at high risk of serious illness caused by VZV.     

Species loss of stoneflies (Plecoptera) in the Czech Republic during the 20th century

1. Rapid expansion and intensification of anthropogenic activities in the 20th century has caused profound changes in freshwater assemblages. Unfortunately, knowledge of the extent and causes of species loss (SL) is limited due to the lack of reliable historical data. An unusual data set allows us to compare changes in the most sensitive of aquatic insect orders, the Plecoptera, at some 170 locations in the Czech Republic between two time periods, 1955–1960 and 2006–2010. Historical data (1890–1911) on assemblages of six lowland rivers allow us to infer even earlier changes. 2. Regional stonefly diversity decreased in the first half of the 20th century. Streams at lower altitudes lost a substantial number of species, which were never recovered. In the second half of the century, large‐scale anthropogenic pressure caused SL in all habitats, leading to a dissimilarity of contemporary and previous assemblages. The greatest changes were found at sites affected by organic pollution and a mixture of organic pollution and channelisation or impoundment. Colonisation of new habitats was observed in only three of the 80 species evaluated. 3. Species of moderate habitat specialisation and tolerance to organic pollution were most likely to be lost. Those with narrow specialisations in protected habitats were present in both historical and contemporary collections. 4. Contemporary assemblages are the consequence of more than a 100 years of anthropogenic impacts. In particular, streams at lower altitude and draining intensively exploited landscapes host a mere fragment of the original species complement. Most stonefly species are less frequently present than before, although their assemblages remain almost intact in near‐natural mountain streams. Our analyses demonstrate dramatic restriction of species ranges and, in some cases, apparent changes in altitudinal preference throughout the area.     

Involvement of CXCR4 Chemokine Receptor in Metastastic HER2-Positive Esophageal Cancer

A functional linkage of the structurally unrelated receptors HER2 and CXCR4 has been suggested for breast cancer but has not been evaluated for esophageal carcinoma. The inhibition of HER2 leads to a reduction of primary tumor growth and metastases in an orthotopic model of esophageal carcinoma. The chemokine receptor CXCR4 has been implicated in metastatic dissemination of various tumors and correlates with poor survival in esophageal carcinoma. The aim of this study was to investigate a correlation between the expression levels of HER2 and CXCR4 and to evaluate the involvemnent of CXCR4-expression in HER2-positive esophageal carcinoma. The effects of HER2-inhibition with trastuzumab and of CXCR4-inhibition with AMD3100 on primary tumor growth, metastatic homing, and receptor expression were evaluated in vitro and in an orthotopic model of metastatic esophageal carcinoma using MRI for imaging. The clinical relevance of HER2- and CXCR4-expression was examined in esophageal carcinoma patients. A significant correlation of HER2- and CXCR4-expression in primary tumor and metastases exists in the orthotopic model. Trastuzumab and AMD3100 treatment led to a significant reduction of primary tumor growth, metastases and micrometastases. HER2-expression was significantly elevated under AMD3100 treatment in the primary tumor and particularly in the metastases. The positive correlation between HER2- and CXCR4-expression was validated in esophageal cancer patients. The correlation of CXCR4- and HER2-expression and the elevation of HER2-expression and reduction of metastases through CXCR4-inhibition suggest a possible functional linkage and a role in tumor dissemination in HER2-positive esophageal carcinoma.     

Functions of the ORF9-to-ORF12 gene cluster in varicella-zoster virus replication and in the pathogenesis of skin infection

The gene cluster composed of varicella-zoster virus (VZV) open reading frame 9 (ORF9) to ORF12 encodes four putative tegument proteins and is highly conserved in most alphaherpesviruses. In these experiments, the genes within this cluster were deleted from the VZV parent Oka (POKA) individually or in combination, and the consequences for VZV replication were evaluated with cultured cells in vitro and with human skin xenografts in SCID mice in vivo. As has been reported for ORF10, ORF11 and ORF12 were dispensable for VZV replication in melanoma and human embryonic fibroblast cells. In contrast, deletion of ORF9 was incompatible with the recovery of infectious virus. ORF9 localized to the virion tegument and formed complexes with glycoprotein E, which is an essential protein, in VZV-infected cells. Recombinants lacking ORF10 and ORF11 (POKADelta10/11), ORF11 and ORF12 (POKADelta11/12), or ORF10, ORF11 and ORF12 (POKADelta10/11/12) were viable in cultured cells. Their growth kinetics did not differ from those of POKA, and nucleocapsid formation and virion assembly were not disrupted. In addition, these deletion mutants showed no differences compared to POKA in infectivity levels for primary human tonsil T cells. Deletion of ORF12 had no effect on skin infection, whereas replication of POKADelta11, POKADelta10/11, and POKADelta11/12 was severely reduced, and no virus was recovered from skin xenografts inoculated with POKADelta10/11/12. These results indicate that with the exception of ORF9, the individual genes within the ORF9-to-ORF12 gene cluster are dispensable and can be deleted simultaneously without any apparent effect on VZV replication in vitro but that the ORF10-to-ORF12 cluster is essential for VZV virulence in skin in vivo.     

Co-transfer of two plasmids determining bacteriocin production and sucrose utilization in Streptococcus faecium

Abstract Streptococcus faecium strain 25 produced a bacteriocin (enterococcin Sf25), metabolized sucrose and contained three plasmids of 2.4, 4.7 and 13.0 MDa. Plasmids Sfp2.4 and Sfp4.7 were cotransferred in a filter mating procedure to sucrose negative and bacteriocin negative S. faecium strain M16. Strain M16 harboured a nonselftransferable plasmid Sfp 19.1 MDa, which was responsible for erythromycin resistance. Transcipient cells of S. faecium M16 contained the 19.1-MDa plasmid and plasmids Sfp2.4 and Sfp4.7, produced the enterococcin Sf25 and gained the ability to degrade sucrose.