The equine herpesvirus 1 EICP27 protein enhances gene expression via an interaction with TATA box-binding protein

The mechanism(s) by which the early EICP27 gene product cooperates with other equine herpesvirus 1 (EHV-1) regulatory proteins to achieve maximal promoter activity remains unknown. Transient transfection assays revealed that deletion of residues 93-140 of the 470-aa EICP27 protein substantially diminished its activation of the immediate-early (IE) promoter, whereas deletion of residues 140-470 that contain a zinc-finger motif abolished this activity. Fluorescence microscopy of cells expressing the full-length EICP27 protein or portions of this protein revealed that an arginine-rich sequence spanning residues 178-185 mediates nuclear entry. Experiments employing the mammalian Gal4 two-plasmid system revealed that the EICP27 protein does not possess an independent trans-activation domain (TAD). Protein-protein interaction assays using purified proteins revealed that residues 124-220 of the EICP27 protein mediate its direct interaction with TATA box-binding protein (TBP). Partial deletion of this TBP-binding domain attenuated the ability of the EICP27 protein to stimulate the IE and early EICP0 promoters by 68% and 71%, respectively, indicating the importance of this protein-protein interaction.     

Direct mapping of the human TATA box-binding protein (TBP) gene to 6q27 by fluorescencein situ hybridization

Summary TBP (TATA box-binding protein) participates in the expression of eukaryotic genes transcribed by RNA polymerases I, II, and III. Molecular cloning of human TBP revealed that the N-terminal region contains a polymorphic (CAG)_n repeat. We report here the direct localization of human TBP gene to chromosome 6q2705qter region by fluorescencein situ hybridization, using the cDNA clone with or without the (CAG)_n repeat as a probe.     

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.     

Objective semen analysis: has the target been reached?

Objectivity in assessing critical sperm characteristics such as concentration, motility and morphology, has been the target of many systems and devices. Methods based on physical principles such as turbidimetry, spectrophotometry, laser Doppler technology, are too imprecise or technically too complicated to be applied routinely in the laboratory. Microscopic images providing photographical, cinematographical or video data acquisition followed by manual or computer-assisted image analysis (CASA) suffer from practical and/or device deficiencies, making them tedious and time-consuming, or unreliable because of confounding factors and uncontrolled artefacts. A single-step computer system using visual evaluation of the microscopic field and manual tracking of sperm movement yields more reliable results, but technician expertise remains essential for correct performance. The scientific basis of multiple errors in semen aspiration, chamber quality, data acquisition and analysis is given. Biased interpretations of results or incorrect handling of statistical data are criticised.     

The conserved 18,000-molecular-weight outer membrane protein of Haemophilus ducreyi has homology to PAL.

Haemophilus ducreyi expresses an 18,000-molecular-weight outer membrane protein that contains a conserved surface-exposed epitope recognized by monoclonal antibody 3B9. Monoclonal antibody 3B9 cross-reacts with proteins of similar molecular weight found in many Haemophilus sp. strains, including P6, a candidate vaccine for Haemophilus influenzae. The gene encoding the 18,000-molecular-weight outer membrane protein was identified by screening a lambdagt11 genomic library with 3B9. The coding sequence of the gene was localized to a 471-bp open reading frame, designated pal (peptidoglycan-associated lipoprotein). Translation of pal predicted a mature polypeptide with a molecular weight of 15,000 that had extensive homology with P6 and Escherichia coli PAL. The predicted signal peptide had features characteristic of a prokaryotic lipoprotein, and processing of PAL was sensitive to globomycin in H. ducreyi. The sequences encoding mature H. ducreyi PAL were subcloned into the vector pRSET B and expressed as a polyhistidine-containing fusion protein that bound 3B9. In Western blot (immunoblot) analysis, serum samples obtained from healthy subjects and patients with chancroid or other genital ulcer diseases contained antibodies to purified PAL. Antibodies that bound to PAL were removed by absorption with a lysate of Haemophilus sp. antigens, suggesting that patients with chancroid do not develop an H. ducreyi-specific antibody response to PAL.     

Expression and immune recognition of the conserved MSP4 outer membrane protein of Anaplasma marginale.

Defining conserved, protective epitopes is essential to the design of an effective vaccine against bovine anaplasmosis. MSP4, one of six initial body proteins recognized by a neutralizing serum, is conserved among Anaplasma marginale isolates at both the protein and the DNA levels. Sera from cattle immunized with an outer membrane fraction of A. marginale and protected from a virulent challenge bind MSP4. The gene for MSP4 has been cloned, and the recombinant protein has been expressed, isolated, and demonstrated to share epitopes with the native protein expressed on initial bodies. MSP4 may have a greater potential to protect cattle from a challenge by heterologous isolates than other A. marginale surface proteins, which vary widely in size and structure.     

Vaccine effectiveness in older individuals: what has been learned from the influenza-vaccine experience

Vaccination policies in most high-income countries attempt to reduce the adverse impact of influenza targeting people aged at least 60 years. However, while it is widely believed that the current immunization strategy saves many lives, influenza infection still remains a severe burden in aged individuals leading to a wide debate on the exact magnitude of the benefit of vaccination in this population. The first aim of the present review is to examine how effective current influenza-vaccine strategies are in aged adults, by analysing which are the most important factors modulating the interpretation of study results in this population. Furthermore, consideration will be given to how immune factors influence the measurement of vaccine efficacy/effectiveness, where advancing age leads to deleterious changes in the adaptive immune system, resulting in less than optimal responses to infectious agents and vaccination. Finally this review concludes with possible strategies to improve the ability of the senescent immune system to respond to vaccination.     

Plasmodium falciparum rhoptry neck protein 4 has conserved regions mediating interactions with receptors on human erythrocytes and hepatocyte membrane

Plasmodium falciparum-related malaria represents a serious worldwide public health problem due to its high mortality rates. P. falciparum expresses rhoptry neck protein 4 (PfRON4) in merozoite and sporozoite rhoptries, it participates in tight junction-TJ formation via the AMA-1/RON complex and is refractory to complete genetic deletion. Despite this, which PfRON4 key regions interact with host cells remain unknown; such information would be useful for combating falciparum malaria. Thirty-two RON4 conserved region-derived peptides were chemically synthesised for determining and characterising PfRON4 regions having high host cell binding affinity (high activity binding peptides or HABPs). Receptor-ligand interaction/binding assays determined their specific binding capability, the nature of their receptors and their ability to inhibit in vitro parasite invasion. Peptides 42477, 42479, 42480, 42505 and 42513 had greater than 2% erythrocyte binding activity, whilst peptides 42477 and 42480 specifically bound to HepG2 membrane, both of them having micromolar and submicromolar range dissociation constants (Kd). Cell-peptide interaction was sensitive to treating erythrocytes with trypsin and/or chymotrypsin and HepG2 with heparinase I and chondroitinase ABC, suggesting protein-type (erythrocyte) and heparin and/or chondroitin sulphate proteoglycan receptors (HepG2) for PfRON4. Erythrocyte invasion inhibition assays confirmed HABPs’ importance during merozoite invasion. PfRON4 800–819 (42477) and 860–879 (42480) regions specifically interacted with host cells, thereby supporting their inclusion in a subunit-based, multi-antigen, multistage anti-malarial vaccine.