Herpes simplex virus glycoprotein D is sufficient to induce spontaneous pH-independent fusion in a cell line that constitutively expresses the glycoprotein

Spontaneous small polykaryocytes were detected in a cell line designated BJ-o that harbors the BamHI J fragment of herpes simplex virus 1 DNA and expresses constitutively glycoprotein D (gD). The fusion activity of BJ-o cells correlated with gD production and was drastically reduced following exposure of the cells to monoclonal antibody HD1 to gD. Studies on the characteristics and requirements of cell fusion dependent on gD led to the conclusion that the characteristics and requirements for gD-mediated fusion activity of BJ-o cells are similar to those previously reported for cell fusion induced by the virus in that (i) polykaryocytosis was not augmented by exposure to medium of low pH with or without prior exposure to trypsin, (ii) the number of polykaryocytes was reduced following removal of terminal sialic acid residues by neuraminidase, and (iii) the number of polykaryocytes was augmented by masking of high-mannose N-linked oligosaccharides with concanavalin A or with its reduced form, succinyl concanavalin A. This effect was reversed by competition with mannose.     

The synthetic, oxidized C-terminal fragment of the Plasmodium berghei circumsporozoite protein elicits a high protective response

A polypeptide of 69 amino acids (PbCS 242-310) encompassing the C-terminal region of the circumsporozoite protein of Plasmodium berghei (PbCS) was generated using solid-phase peptide synthesis. The immunological and protective properties of peptide PbCS 242-310 were studied in BALB/c mice (H-2d). Two subcutaneous injections, in the presence of IFA at the base of the tail, generated (i) high titers of anti-peptide antibodies which also recognized the native P. berghei CS protein, (ii) cytolytic T cells specific for the Kd-restricted peptide PbCS 245-253 and (iii) partial CD8+-dependent protection against sporozoite-induced malaria. The same frequencies of peptide PbCS 245-253 specific CD8+ T cells were found by IFN-gamma ELISPOT in the draining lymph nodes of animals immunized with the short optimal CTL peptide 245-253 or with the polypeptide 242-310, indicating that the longer polypeptide can be processed and presented in vivo in the context of MHC class I as efficiently as the short CTL peptide. Interestingly, higher levels of IFN-gamma producing CD8 T cells and protection were observed when the four cysteine residues present in the C-terminal peptide were fully oxidized. These findings underline the potential importance of the chemical nature of the C-terminal fragment on the activation of the immune system and concomitant protection.     

Differential protein expression throughout the life cycle of Trypanosoma congolense, a major parasite of cattle in Africa

Trypanosoma congolense is an important pathogen of livestock in Africa. To study protein expression throughout the T. congolense life cycle, we used culture-derived parasites of each of the three main insect stages and bloodstream stage parasites isolated from infected mice, to perform differential protein expression analysis. Three complete biological replicates of all four life cycle stages were produced from T. congolense IL3000, a cloned parasite that is amenable to culture of major life cycle stages in vitro. Cellular proteins from each life cycle stage were trypsin digested and the resulting peptides were labeled with isobaric tags for relative and absolute quantification (iTRAQ). The peptides were then analyzed by tandem mass spectrometry (MS/MS). This method was used to identify and relatively quantify proteins from the different life cycle stages in the same experiment. A search of the Wellcome Trust's Sanger Institute's semi-annotated T. congolense database was performed using the MS/MS fragmentation data to identify the corresponding source proteins. A total of 2088 unique protein sequences were identified, representing 23% of the ～9000 proteins predicted for the T. congolense proteome. The 1291 most confidently identified proteins were prioritized for further study. Of these, 784 yielded annotated hits while 501 were described as "hypothetical proteins". Six proteins showed no significant sequence similarity to any known proteins (from any species) and thus represent new, previously uncharacterized T. congolense proteins. Of particular interest among the remainder are several membrane molecules that showed drastic differential expression, including, not surprisingly, the well-studied variant surface glycoproteins (VSGs), invariant surface glycoproteins (ISGs) 65 and 75, congolense epimastigote specific protein (CESP), the surface protease GP63, an amino acid transporter, a pteridine transporter and a haptoglobin-hemoglobin receptor. Several of these surface disposed proteins are of functional interest as they are necessary for survival of the parasites.     

Molecular cloning,complete sequence,and biological characterization of a xenotropic murine leukemia virus constitutively released from the human B-lymphoblastoid cell line DG-75

A previously undetected retrovirus has been isolated from the human Epstein-Barr virus (EBV)-negative, B-lymphoblastoid DG-75 cell line, widely used for EBV gene transfection studies. The complete 8207-base genome of the DG-75 retrovirus was molecularly cloned from viral mRNA and sequenced (Accession No. AF221065). Northern blot analysis with probes specific for the putative RU-5, gag, pol, and env regions identified a full-length viral RNA and spliced env mRNA. DG-75 viral RNA was isolated from the DG-75 cell sublines UW and KAR, but not from the HAD subline. The DG-75 retrovirus was isolated with primer-binding sites that match tRNA(Thr) and tRNA(Gln2). Homology searches revealed homology to (i) xenotropic NZB-9-1 env mRNA, (ii) Moloney-MLV pol region, and (iii) a truncated Evi-2 endogenous proviral sequence gag and pol region. Viral interference and infectivity assays confirmed the xenotropic nature of the DG-75 retrovirus. The DG-75 retrovirus is the first isolate of an exogenous xenotropic MLV in which the full-length genomic sequence has been characterized.     

von Willebrand Factor A domain-related protein, a novel microneme protein of the malaria ookinete highly conserved throughout Plasmodium parasites.

The mosquito-invasive form of the malarial parasite, the ookinete, develops numerous secretory organelles, called micronemes, in the apical cytoplasm. Micronemal proteins are thought to be secreted during midgut invasion and to play a crucial role in attachment and motility of the ookinete. We found a novel ookinete micronemal protein of rodent malarial parasite Plasmodium berghei, named P. berghei von Willebrand factor A domain-related protein (PbWARP), and report it here as a putative soluble adhesive protein of the ookinete. The PbWARP gene contained a single open reading frame encoding a putative secretory protein of 303 amino acids, with a von Willebrand factor type A module-like domain as a main component. Western blot analysis demonstrated that PbWARP was firstly produced 12 h after fertilization by maturing ookinetes as SDS-resistant complexes. Recombinant PbWARP produced with a baculovirus system also formed SDS-resistant high-order oligomers. Immuno-electron microscopic studies showed that PbWARP was randomly distributed in the micronemes. PbWARP homologues also exist in human malarial parasites, Plasmodium falciparum and Plasmodium vivax. Highly conserved primary structures of PbWARP homologues among these phylogenetically distant Plasmodium species suggest their functional significance and the presence of a common invasion mechanism widely utilized throughout Plasmodium parasites.     

Healthy women and patients with endometriosis show high concentrations of inhibin A, inhibin B, and activin A in peritoneal fluid throughout the menstrual cycle

Inhibin A, inhibin B, and activin A are growth factors which play local autocrine/paracrine roles in reproductive tissues. Since peritoneal fluid hormone content may reflect in part ovarian and endometrial secretory activities, the present study aimed to evaluate: (i) whether inhibin alpha-, activin betaA- and betaB-subunits, and activin receptor type II and type IIB mRNA are expressed in peritoneal tissues; (ii) expression and secretion of inhibin A and B, and activin A in cultured endometriotic cells; and (iii) concentrations of inhibin A and B, and activin A in serum and in peritoneal fluid in healthy women and in patients with endometriosis throughout the menstrual cycle. A group of women (n = 72) was recruited at laparoscopy for infertility investigation and divided into two groups: (i) control healthy women (n = 35), (ii) women with endometriosis (n = 37). Both groups were subdivided according to the follicular and luteal phase of the menstrual cycle. At the time of laparoscopy, specimens of peritoneal tissues were collected from three healthy women, while endometriotic tissue samples were collected and cultured from three women with endometriosis. Peritoneal tissues and cultured endometriotic cells expressed inhibin alpha-, activin betaA-, and betaB-subunits, and activin receptors mRNAs; in addition, inhibin-related proteins were measurable in culture medium. In healthy women, inhibin A and B, and activin A concentrations in peritoneal fluid were significantly higher than in serum (P < 0.001), at both phases of the menstrual cycle. Peritoneal inhibin A and B, and activin A concentrations were not significantly different between healthy women and patients with endometriosis, either when evaluated according to the degree of the disease and/or to the phase of the menstrual cycle. In conclusion, the findings that high concentrations are present in peritoneal fluid and that menstrual cycle-related changes occur suggest that reproductive organs may contribute to inhibin-related proteins in peritoneal fluid.      

Estimating and validating disability-adjusted life years at the global level: a methodological framework for cancer

ABSTRACT: BACKGROUND: Disability-adjusted life years (DALYs) link data on disease occurrence to health outcomes, and they are a useful aid in establishing country-specific agendas regarding cancer control. The variables required to compute DALYs are however multiple and not readily available in many countries. We propose a methodology that derives global DALYs and validate variables and DALYs based on data from various cancer registries. METHODS: We estimated DALYs for four countries (Norway, Bulgaria, India and Uganda) within each category of the human development index (HDI). The following sources (indicators) were used: Globocan2008 (incidence and mortality), various cancer registries (proportion cured, proportion treated and duration of disease), treatment guidelines (duration of treatment), specific burden of disease studies (sequelae and disability weights), alongside expert opinion. We obtained country-specific population estimates and identified resource levels using the HDI, DALYs are computed as the sum of years of life lost and years lived with disabilities. RESULTS: Using mortality:incidence ratios to estimate country-specific survival, and by applying the human development index we derived country-specific estimates of the proportion cured and the proportion treated. The fit between the estimates and observed data from the cancer registries was relatively good. The final DALY estimates were similar to those computed using observed values in Norway, and in WHOs earlier global burden of disease study. Marked cross-country differences in the patterns of DALYs by cancer sites were observed. In Norway and Bulgaria, breast, colorectal, prostate and lung cancer were the main contributors to DALYs, representing 54 % and 45 %, respectively, of the totals. These cancers contributed only 27 % and 18 %, respectively, of total DALYs in India and Uganda. CONCLUSIONS: Our approach resulted in a series of variables that can be used to estimate country-specific DALYs, enabling global estimates of DALYs and international comparisons that support priorities in cancer control.     

Candida albicans expresses a focal adhesion kinase-like protein that undergoes increased tyrosine phosphorylation upon yeast cell adhesion to vitronectin and the EA.hy 926 human endothelial cell line

The signaling pathways triggered by adherence of Candida albicans to the host cells or extracellular matrix are poorly understood. We provide here evidence in C. albicans yeasts of a p105 focal adhesion kinase (Fak)-like protein (that we termed CaFak), antigenically related to the vertebrate p125Fak, and its involvement in integrin-like-mediated fungus adhesion to vitronectin (VN) and EA.hy 926 human endothelial cell line. Biochemical analysis with different anti-chicken Fak antibodies identified CaFak as a 105-kDa protein and immunofluorescence and cytofluorimetric analysis on permeabilized cells specifically stain C. albicans yeasts; moreover, confocal microscopy evidences CaFak as a cytosolic protein that colocalizes on the membrane with the integrin-like VN receptors upon yeast adhesion to VN. The protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A strongly inhibited C. albicans yeast adhesion to VN and EA.hy 926 endothelial cells. Moreover, engagement of alpha v beta 3 and alpha v beta 5 integrin-like on C. albicans either by specific monoclonal antibodies or upon adhesion to VN or EA.hy 926 endothelial cells stimulates CaFak tyrosine phosphorylation that is blocked by PTK inhibitor. A role for CaFak in C. albicans yeast adhesion was also supported by the failure of VN to stimulate its tyrosine phosphorylation in a C. albicans mutant showing normal levels of CaFak and VNR-like integrins but displaying reduced adhesiveness to VN and EA.hy 926 endothelial cells. Our results suggest that C. albicans Fak-like protein is involved in the control of yeast cell adhesion to VN and endothelial cells.     

Establishment of a MAGI-derived indicator cell line that detects the Nef enhancement of HIV-1 infectivity with high sensitivity

The Nef protein of the simian and human immunodeficiency viruses (SIV and HIV) is regarded as one of the critical determinants of the pathogenicity of HIV-1 in vivo. The positive effect of Nef on viral replication is examined most easily in vitro by the use of indicator cells such as HeLa-CD4-LTR-beta-gal cells (MAGI) or MAGIC5 cells, which are MAGI-derived, CCR5-expressing cells. However, Nef increases the infectivity of many HIV-1 strains no more than 10-fold in these indicator cells. It was noted that MAGI cells expressing a lower level of CD4 enabled us to discriminate more clearly between wild-type and Nef-defective virions. A MAGIC5-derived cell line, MAGNEF, which stably expressed a low level of CD4, was established. The infectivity of the Nef-defective HIV-1 NL4-3 strain was consistently less than one-twentieth of that of the wild type in MAGNEF cells. By using MAGNEF cells, it was shown that Nef enhanced the infectivity of a subtype C HIV-1, Indie-C1 strain, although the effect of Nef on Indie-C1 was significantly less than that on the subtype B strains NL4-3 and SF2. These results validate the versatility of MAGNEF cells for use in the simple and sensitive assay for the level of Nef dependence of various HIV-1 isolates.     

Restriction of vesicular stomatitis virus in a nonpermissive rabbit cell line is at the level of protein synthesis

Replication of vesicular stomatitis virus (VSV) is restricted in a line of rabbit cornea (RC-60) cells; less than one infectious particle is produced per infected cell. We show that VSV is blocked at the level of viral-specific protein synthesis. VSV proteins are synthesized early in infection but the rate of VSV protein synthesis declines rapidly as infection proceeds. At times when synthesis of VSV proteins is barely detectable, VSV mRNA is produced and polyribosome structures are present. The VSV mRNA recovered from the polysome region directs the synthesis of VSV proteins in an in vitro reticulocyte translation system. This suggests that protein synthesis is blocked at some step beyond the level of initiation possibly at the level of elongation. Coinfection with vaccinia virus converts RC-60 cells to a permissive host. In contrast to the abortive infection with VSV alone, VSV proteins are synthesized throughout the replication cycle in doubly infected cells. Vaccinia supplies a product essential for sustained protein synthesis in the abortive system. We have confirmed that the replication of genome length 42 S RNA does not occur at late times in the abortive infection. This lack of 42 S RNA replication is explained by the shut-off of VSV protein synthesis, since continuous protein synthesis is required for the replication of VSV 42 S RNA.     

Quantitative evidence that the genetic basis of human mortality operates at the translation level: a preliminary assessment.

The dying off of human population cohorts due to different age related diseases rigorously follows stochastically derived rules. The existence of such rules suggests a common, fundamental mechanism for human mortality. We show here, by a simple quantitative analysis, that the progressive loss with age of genetic triplet code information (anticodons) in body cells, as described by the statistics of extremes in order theory, serves as such a mechanism.     

A vaccinia virus MVA-T7-mediated recovery of infectious hepatitis A virus from full-size cDNA or from two cDNAs,both by themselves unable to complete the virus life cycle

The replication-deficient vaccinia virus (VV) MVA-T7 produces large amounts of T7 RNA polymerase and permits efficient protein expression from cDNA of T7-promoted genes. Yet, unlike recombinant VV vTF7-3, (VV) MVA-T7 produces no cytopathic effect in primate cells, thus allowing the study of processes with slow kinetics. We have applied MVA-T7 to aid genome expression of HAV, a representative of the Picornaviridae family that is well known for its inefficient replication in mammalian cell cultures. After cDNA transfection and MVA-T7 infection, empty capsids and mature HAV particles were formed with different kinetics and were characterized by their morphology, protein content, and infectivity. The data suggests that HAV genome replication is initiated from RNA, which was transcribed in vivo by the MVA-T7-encoded T7 RNA polymerase. HAV genome replication was also demonstrated in a recombination assay. After co-expression of two subgenomic HAV cDNAs, both by themselves unable to complete the viral life cycle, infectious HAV was rescued, indicating that replication-dependent genetic recombination has occurred. We propose that the high-level genome expression mediated in vivo by the VV-encoded T7 RNA polymerase augments the amount of viral RNA, such that replication of viruses poorly replicating in cell cytoplasm is detectable.     

Evidence for a 6.5-day minimum exoerythrocytic cycle for Plasmodium falciparum in humans and confirmation that immunization with a synthetic peptide representative of a region of the circumsporozoite protein retards infection.

Immunization with a synthetic peptide which is representative of part of the repeating region of Plasmodium falciparum circumsporozoite protein resulted in an immunity which allowed vaccinees to retard the development of patent malaria as compared to nonimmunized controls. Analysis of infection dynamics showed that immunity could be attributed to either neutralization of about 92% of inoculated sporozoites, delayed development of the majority of parasites, or a combination of neutralization and delayed development. In spite of this impressive antiplasmodial capacity, all volunteers after being bitten by infected mosquitoes developed malaria, and seven of eight developed parasitemia between 6.5 and 7.0 days after infective mosquito bites.