Conserved features of TBE1 transposons in ciliated protozoa

BACKGROUND: Accessory function (AF) is one way antigen presenting cells generate sufficient secondary signals for optimal T-cell proliferation and IL-2 production. In general, alveolar macrophages (AM) are inferior accessory cells in comparison to monocytes whereas in sarcoidosis AF of AM is increased. METHODS: We compared the accessory index (AI) of AM and peripheral blood monocytes (PBM) of 41 patients with inactive sarcoidosis (SAR I, n = 12); active sarcoidosis with new or progressing symptoms (SAR II, n = 19), active sarcoidosis with spontaneous remission (SAR III, n = 10), tuberculosis (TB, n = 12), hypersensitivity pneumonitis (HP, n = 12), Wegener's disease (WD, n = 2), undefined alveolitis (UA, n = 8) and chronic obstructive pulmonary disease (COPD, n = 6) by employing the histoincompatibility-insensitive Jurkat cells as indicator cells. RESULTS: Compared with the controls (1.08 +/- 0.3) AMs of all groups but SAR I (AI: 0.96 +/- 0.42) exhibited significantly increased AIs (SAR II: 3.6 +/- 3.9; SAR III: 3.2 +/- 2.4; TB: 2.8 +/- 2.2; HP: 3 +/- 2; UA: 2.7 +/- 2.3; COPD: 3.1 +/- 2.2; p < 0.05 for all comparisons). Only in HP, AI of PBM was significantly increased compared with controls (3 +/- 1.5, 1.3 +/- 0.5, respectively; p < 0.001). Alveolar macrophages from patients with arcoidosis, TB, and HB express the costimulatory molecule CD80 on their surface and anti-CD80 antibodies inhibited the IL-2 release of Jurkat cells in this system to 59 +/- 27%. CONCLUSIONS: Our data demonstrate that AM from patients with various diseases have the capability to act as competent accessory cells and that the reported accessory function of these cells is at least in part mediated by the expression of CD80.     

Conserved features in the active site of nonhomologous serine proteases

BACKGROUND: Serine protease activity is critical for many biological processes and has arisen independently in a few different protein families. It is not clear, though, the degree to which these protease families share common biochemical and biophysical properties. We have used a computer program to study the properties that are shared by four serine protease active sites with no overall structural or sequence homology. The program systematically compares the region around the catalytic histidines from the four proteins with a set of noncatalytic histidines, used as controls. It reports the three-dimensional locations and level of statistical significance for those properties that distinguish the catalytic histidines from the noncatalytic ones. The method of analysis is general and can be applied easily to other active sites of interest. RESULTS: As expected, some of the reported properties correspond to previously known features of the serine protease active site, including the catalytic triad and the oxyanion hole. Novel properties are also found, including the spatial distribution of charged, polar, and hydrophobic groups arranged to stabilize the catalytic residues, and a relative abundance of some residues (Val, Tyr, Leu, and Gly) around the active site. CONCLUSIONS: Our findings show that in addition to some properties common to all the proteases examined, there are a set of preferred, but not required, properties that can be reliably observed only by aligning the sites and comparing them with carefully selected statistical controls.     

Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.     

Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs

SNARE [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor] proteins are essential for membrane fusion and are conserved from yeast to humans. Sequence alignments of the most conserved regions were mapped onto the recently solved crystal structure of the heterotrimeric synaptic fusion complex. The association of the four alpha-helices in the synaptic fusion complex structure produces highly conserved layers of interacting amino acid side chains in the center of the four-helix bundle. Mutations in these layers reduce complex stability and cause defects in membrane traffic even in distantly related SNAREs. When syntaxin-4 is modeled into the synaptic fusion complex as a replacement of syntaxin-1A, no major steric clashes arise and the most variable amino acids localize to the outer surface of the complex. We conclude that the main structural features of the neuronal complex are highly conserved during evolution. On the basis of these features we have reclassified SNARE proteins into Q-SNAREs and R-SNAREs, and we propose that fusion-competent SNARE complexes generally consist of four-helix bundles composed of three Q-SNAREs and one R-SNARE.     

In vitro construction of pseudovirions of human papillomavirus type 16: incorporation of plasmid DNA into reassembled L1/L2 capsids

Lack of permissive and productive cell cultures for the human papillomaviruses (HPVs) has hindered the study of virus-neutralizing antibodies and infection. We developed a cell-free system generating infectious HPV16 pseudovirions. HPV16 L1/L2 capsids, which had been self-assembled in insect cells (Sf9) expressing virion proteins L1 and L2, were disassembled with 2-mercaptoethanol (2-ME), a reducing agent, and reassembled by removal of 2-ME in the presence of a beta-galactosidase expression plasmid. Plasmid DNA purified together with the reassembled capsids was resistant to DNase I digestion. The reassembled pseudovirions mediated DNA transfer to COS-1 cells, as monitored by induced beta-galactosidase activity. Transfer was inhibited by anti-HPV16 L1 antiserum but not by antisera against L1s of HPV6 and HPV18. Construction in vitro of HPV pseudovirions containing marker plasmids would be potentially useful in developing methods to assay virus-neutralizing antibodies and to transfer exogenous genes to HPV-susceptible cells.     

Self-assembly of human papillomavirus type 1 capsids by expression of the L1 protein alone or by coexpression of the L1 and L2 capsid proteins

Vaccinia virus vectors were used to express the major (L1) and minor (L2) capsid proteins of human papillomavirus type 1 (HPV-1) with the vaccinia virus early (p7.5K) or late (pSynth, p11K) promoters. All constructs expressed the appropriate-sized HPV proteins, and both L1 and L2, singly or in combination, localized to the nucleus. Capsids were purified by cesium chloride density gradient centrifugation from nuclei of cells infected with a vaccinia virus-L1 (vac-L1) recombinant or a vac-L1-L2 recombinant but not from vac-L2-infected cells. Electron microscopy showed that the particles were 55 nm in diameter and had icosahedral symmetry. Immunogold-labeled antibodies confirmed the presence of the L1 and L2 proteins in the HPV-1 capsids. Capsids containing L1 alone were fewer and more variable in size and shape than capsids containing the L1 and L2 proteins. The L1-plus-L2 capsids were indistinguishable in appearance from HPV-1 virions obtained from plantar warts. The ability to produce HPV capsids in vitro will be useful in many studies of HPV pathogenicity.     

Detection of BK polyomavirus genotypes in healthy and HIV-positive children

BACKGROUND: Atrial flutter is a common arrhythmia which frequently recurs after cardioversion and is relatively difficult to control with antiarrhythmic agents. AIMS: To evaluate the success rate, recurrence rate and safety of radiofrequency, (RF) ablation for atrial flutter in a consecutive series of patients with drug refractory chronic or paroxysmal forms of the arrhythmia. METHODS: Electrophysiologic evaluation of atrial flutter included activation mapping with a 20 electrode halo catheter placed around the tricuspid annulus and entrainment mapping from within the low right atrial isthmus. After confirmation of the arrhythmia mechanism with these techniques, an anatomic approach was used to create a linear lesion between the inferior tricuspid annulus and the eustachian ridge at the anterior margin of the inferior vena cava. In order to demonstrate successful ablation, mapping techniques were employed to show that bi-directional conduction block was present in the low right atrial isthmus. RESULTS: Successful ablation was achieved in 26/27 patients (96%). In one patient with a grossly enlarged right atrium, isthmus block could not be achieved. Of the 26 patients with successful ablation, there has been one recurrence of typical flutter (4%) during a mean follow-up period of 5.5 +/- 2.7 months. This patient underwent a successful repeat ablation procedure. Of eight patients with documented clinical atrial fibrillation (in addition to atrial flutter) prior to the procedure, five continued to have atrial fibrillation following the ablation. There were no procedural complications and all patients had normal AV conduction at the completion of the ablation. CONCLUSIONS: RF ablation is a highly effective and safe procedure for cure of atrial flutter. In patients with chronic or recurrent forms of atrial flutter RF ablation should be considered as a first line therapeutic option.     

Radiation-resistant and radiation-sensitive forms of host resistance to polyomavirus

Newborn mice of several inbred strains develop few or no tumors following inoculation with highly tumorigenic strains of polyomavirus. Here we show that such resistant strains can be divided into two groups based on the responses of adult mice to radiation followed by virus inoculation. Most strains show a radiation-sensitive form of resistance (Rr-s) and develop tumors following radiation and virus challenge. This type of resistance has previously been recognized as immunological, based on T-cell responses against virus-encoded neoantigen(s) expressed in tumor cells. Other strains exhibit a radiation-resistant form of resistance (Rr-r) and fail to develop tumors when treated in the same manner. Three additional properties of Rr-r mice distinguish them from Rr-s mice: (i) survival of newborns following inoculation with a highly virulent and usually lethal strain of virus, (ii) resistance to virus spread in newborns inoculated with either tumorigenic or virulent virus strains, and (iii) dominant or semidominant transmission of resistance in crosses with a highly susceptible strain. The Rr-r phenotype reflects a constitutive nonimmunological type of resistance that is targeted to the virus and blocks its dissemination.     

An inactivated avian polyomavirus vaccine is safe and immunogenic in various Psittaciformes

The safety and immunogenicity of adjuvanted and nonadjuvanted inactivated avian polyomavirus vaccines, administered either intramuscularly or subcutaneously (s.c.), were evaluated in a group of mixed species Psittaciformes. In 233 vaccinates representing species of macaws, cockatoos, conures, and parrots, gross reactions were limited to small scab formation at the s.c. injection site in three African grey parrots. Both vaccines stimulated a virus neutralizing (VN) antibody response, particularly in birds that were seronegative prior to vaccination. Ninety-three percent of the birds that were seronegative at the beginning of the study seroconverted (greater than fourfold increase in VN antibody titer) by 2 weeks after the second vaccination. Seventy-six percent of all the vaccinates had at least a fourfold increase in VN antibody titer at this time. There was no significant difference in seroconversion between the birds vaccinated with adjuvanted or nonadjuvanted vaccines. This study indicates that an inactivated avian polyomavirus vaccine can be used to safely immunize various species of psittacine birds in a field setting.     

Amplification mediated by polyomavirus large T antigen defective in replication

The polyomavirus large T antigen promotes homologous recombination at high rates when expressed in rat cells carrying the viral replication origin and two repeats of viral DNA sequences stably integrated into the cellular genome. Recombination consists of both reciprocal and nonreciprocal events and is promoted by mutants defective in the initiation of viral DNA synthesis (L. St-Onge, L. Bouchard, and M. Bastin, J. Virol. 67:1788-1795, 1993). We have extended our studies to a rat cell line undergoing amplification of the viral insert. We show that large T antigen promotes amplification independently of its replicative function but that its origin-specific DNA binding activity is not sufficient to promote homologous recombination.     

Detection of human papillomavirus DNA and clinicopathological study of vulvar human papillomavirus infection

We studied 217 vulvar HPV infection patients by clinically, pathologically, and virologically. From 90.6% of the cauliflower-like vulvar lesions and 29.7% of the papillomatous and finger-like lesions, we detected HPV 6/11 DNA by dot blotting hybridization. The patients in 90.0% of the cauliflower-like group and 9.8% of the papillomatous and finger-like group had a high risk factor to intercourse with different sex partners (P < 0.0001). The pathological characteristics, nature history, and response to treatment were different. According to clinical, pathological, and virological findings divided three types: vulvar HPV infection type 1 (or condylomata acuminata), vulvar HPV infection type 2, and vulvar HPV infection type 3.     

Anogenital papillomavirus infection and neoplasia in immunodeficient women

This article reviews the impact of infection with human immunodeficiency virus (HIV) on HPV infections and HPV-associated lesions of the female anogenital tract. Studies investigating HPV infections in HIV-seropositive women are presented as well as the possibility that HIV can influence HPV expression directly through molecular interactions between viral genes and indirectly through immunosuppression. Studies linking HIV infection to invasive cervical cancer and cervical intraepithelial neoplasia are reviewed; recommended protocols for cervical cancer screening in HIV-seropositive women for cervical disease also are presented.     

Conserved domains in DNA repair proteins and evolution of repair systems

A detailed analysis of protein domains involved in DNA repair was performed by comparing the sequences of the repair proteins from two well-studied model organisms, the bacterium Escherichia coli and yeast Saccharomyces cerevisiae, to the entire sets of protein sequences encoded in completely sequenced genomes of bacteria, archaea and eukaryotes. Previously uncharacterized conserved domains involved in repair were identified, namely four families of nucleases and a family of eukaryotic repair proteins related to the proliferating cell nuclear antigen. In addition, a number of previously undetected occurrences of known conserved domains were detected; for example, a modified helix-hairpin-helix nucleic acid-binding domain in archaeal and eukaryotic RecA homologs. There is a limited repertoire of conserved domains, primarily ATPases and nucleases, nucleic acid-binding domains and adaptor (protein-protein interaction) domains that comprise the repair machinery in all cells, but very few of the repair proteins are represented by orthologs with conserved domain architecture across the three superkingdoms of life. Both the external environment of an organism and the internal environment of the cell, such as the chromatin superstructure in eukaryotes, seem to have a profound effect on the layout of the repair systems. Another factor that apparently has made a major contribution to the composition of the repair machinery is horizontal gene transfer, particularly the invasion of eukaryotic genomes by organellar genes, but also a number of likely transfer events between bacteria and archaea. Several additional general trends in the evolution of repair proteins were noticed; in particular, multiple, independent fusions of helicase and nuclease domains, and independent inactivation of enzymatic domains that apparently retain adaptor or regulatory functions.     

Conserved neuron promoting activity in Drosophila and vertebrate laminin alpha1

Drosophila S2 cells were transfected with constructs that code for two portions of the Drosophila laminin alpha chain. Construct recalphaL coded for domains III, I/II, and G of laminin alpha. Construct recalphaS coded for only the COOH-most 12% of the I/II domain and the G domain. The corresponding polypeptides were isolated and characterized from the culture media. The recalphaL chain partly formed disulfide-linked heterotrimers with the endogenously produced beta and gamma laminin chains. Like normal Drosophila laminin, a substrate coating of either recalphaL or recalphaS supported neuron differentiation and neurite extension of primary Drosophila embryo cell cultures. However, at the same low concentrations, only Drosophila laminin-1, but neither recalphaL nor recalphaS supported myogenesis in these cultures. Previously, an overlapping set of dodecapeptides that covered a region of the murine laminin alpha1 chain similar to recalphaS had been synthesized and tested for cell culture support properties (Nomizu, M., Kim, W. H., Yamamura, K., Utani, A., Otaka, A., Roller, P. P., Kleinman, H. K., and Yamada, Y. (1995) J. Biol. Chem. 270, 20583-20590). The Drosophila laminin alpha homologues of the six most active vertebrate dodecapeptides were now synthesized and tested as substrates for differentiation of primary Drosophila embryo cells. Peptides that contained either the Drosophila sequence SIKVGV or the murine homologue, SIKVAV, provided support for neurite extension.     

Persistent papillomavirus infection in a cat

The purpose of this study was to further define the cellular response to titanium and polymethylmethacrylate (PMMA) particles in aseptic loosening, and to determine if the use of pamidronate may be effective in inhibiting bone resorption associated with this response. Macrophages and osteoblasts were cocultured to simulate the environment around an aseptically loose prosthesis. Macrophages were plated on the bottom of six well plates and osteoblasts were plated on culture dish inserts, and placed into the wells with the macrophages. Incubation of macrophages with PMMA in this system led to release of prostaglandin E (PGE2), granulocyte macrophage-colony stimulating factor (GM-CSF), and interleukin-6 (IL-6). Incubation with titanium led to release of tumor necrosis factor (TNF) and IL-6. Exposure of calvaria to media from cells exposed to either PMMA or titanium led to release of calcium 45. Incubation of calvaria with pamidronate was able to inhibit release of calcium 45 associated with exposure to the macrophage/osteoblast/particle conditioned medium. Bone resorption at the interface between implant and bone is a consistent feature leading to loosening of orthopedic implants. By inhibiting bone resorption associated with the inflammatory response to implant particulates, pamidronate or other bisphosphonates may have clinical utility in the treatment or prevention or aseptic loosening.