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PubMed1992

Rats are not aversive when exposed to 60-Hz magnetic fields at 3.03 mT.

Thirty-two male rats were tested in two replicates of an experiment to determine whether body currents induced by 60-Hz magnetic fields might lead to avoidance behavior comparable to that which results from exposure to strong 60-Hz electric fields. The test apparatus was a two-compartment Plexiglas shuttlebox enclosed in a sound-attenuating plywood chamber, which in turn was encompassed by two copper bus bars that, when energized, served as a source of 60-Hz magnetic fields. Location of the rat, and traverse activity in the shuttlebox were monitored by nine infra-red photo detectors equally spaced along the length of the apparatus. Rats were divided into 2 groups: 1 group of rats (n = 8 per group per replicate) was sham exposed while rats in the other group (n = 8 per group per replicate) were exposed to a 3.03 mT (30.3 G), 60-Hz magnetic field whenever they traversed to or were located on the side (L or R) predetermined as the exposed side. To control artifact incident to side preference, the side exposed (L or R) was alternated over the exposed rats. Each rat was tested individually in a 1-h session. A 2-factor ANOVA (exposed vs. control, replicate 1 vs. replicate 2) failed to reveal any significant effects due to either factor or to an interaction between factors. These data demonstrate that rats do not avoid exposure to 60-Hz magnetic fields at a flux density of 3.03 mT and further imply that the avoidance by rats of high level 60-Hz electric fields is mediated by something other than the internal body currents induced by the exposure.

Animals↗
PubMed2005

Neuron-to-cell spread of pseudorabies virus in a compartmented neuronal culture system.

Alphaherpesviruses are parasites of the peripheral nervous system in their natural hosts. After the initial infection of peripheral tissues such as mucosal cells, these neurotropic viruses will invade the peripheral nervous system that innervates the site of infection via long-distance axonal transport of the viral genome. In natural hosts, a latent and a nonproductive infection is usually established in the neuronal cell bodies. Upon reactivation, the newly replicated genome will be assembled into capsids and transported back to the site of entry, where a localized infection of the epithelial or mucosal cells will produce infectious virions that can infect naïve hosts. In this paper, we describe an in vitro method for studying neuron-to-cell spread of alphaherpesviruses using a compartmented culture system. Using pseudorabies virus as a model, we infected neuron cell bodies grown in Teflon chambers and observed spread of infection to nonneuronal cells plated in a different compartment. The cells are in contact with the neurons via axons that penetrate the Teflon barrier. We demonstrate that wild-type neuron-to-cell spread requires intact axons and the presence of gE, gI, and Us9 proteins, but does not require gD. We also provide ultrastructural evidence showing that capsids enclosed within vesicles can be found along the entire length of the axon during viral egress.

Animals↗
PubMed2004

Differential outcomes of human cytomegalovirus infection in primitive hematopoietic cell subpopulations.

The cellular reservoir for latent human cytomegalovirus (HCMV) in the hematopoietic compartment, and the mechanisms governing a latent infection and reactivation from latency are unknown. Previous work has demonstrated that HCMV infects CD34+ progenitors and expresses a limited subset of viral genes. The outcome of HCMV infection may depend on the cell subpopulations infected within the heterogeneous CD34+ compartment. We compared HCMV infection in well-defined CD34+ cell subpopulations. HCMV infection inhibited hematopoietic colony formation from CD34+/CD38- but not CD34+/c-kit+ cells. CD34+/CD38- cells transiently expressed a large subset of HCMV genes that were not expressed in CD34+/c-kit+ cells or cells expressing more mature cell surface phenotypes. Although viral genomes were present in infected cells, viral gene expression was undetectable by 10 days after infection. Importantly, viral replication could be reactivated by coculture with permissive fibroblasts only from the CD34+/CD38- population. Strikingly, a subpopulation of CD34+/CD38- cells expressing a stem cell phenotype (lineage-/Thy-1+) supported a productive HCMV infection. These studies demonstrate that the outcome of HCMV infection in the hematopoietic compartment is dependent on the nature of the cell subpopulations infected and that CD34+/CD38- cells support an HCMV infection with the hallmarks of latency.

ADP-ribosyl Cyclase↗
PubMed1987

Virus vector-mediated gene transfer.

A wide variety of different animal virus groups have been employed to develop virus vector systems for delivery of foreign genes into a host cell or animal. The minimal requirements for a virus vector, replication, packaging and assembly functions, have been defined and both defective and viable virus vectors may be produced. Signals that provide cell or tissue specificity, high rates of transcription, RNA processing, translational controls or specific localization of the foreign gene product into a cellular compartment are all now possible. The combination of safety, specificity and high levels of production make virus vectors a leading choice for the expression of foreign genes in experimental and commercial applications.

Animals↗
PubMed2006

Morphological events during the Trypanosoma cruzi cell cycle.

The replication and segregation of organelles producing two identical daughter cells must be precisely controlled during the cell cycle progression of eukaryotes. In kinetoplastid flagellated protozoa, this includes the duplication of the single mitochondrion containing a network of DNA, known as the kinetoplast, and a flagellum that grows from a cytoplasmic basal body through the flagellar pocket compartment before emerging from the cell. Here, we show the morphological events and the timing of these events during the cell cycle of the epimastigote form of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease. DNA staining, flagellum labeling, bromodeoxyuridine incorporation, and ultra-thin serial sections show that nuclear replication takes 10% of the whole cell cycle time. In the middle of the G2 stage, the new flagellum emerges from the flagellar pocket and grows unattached to the cell body. While the new flagellum is still short, the kinetoplast segregates and mitosis occurs. The new flagellum reaches its final size during cytokinesis when a new cell body is formed. These precisely coordinated cell cycle events conserve the epimastigote morphology with a single nucleus, a single kinetoplast, and a single flagellum status of the interphasic cell.

Animals↗
PubMed2000

Arrest of S-phase progression is impaired in Fanconi anemia cells.

Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability, hypersensitivity to DNA cross-linking agents, and a prolonged G2 phase of the cell cycle. We observed a marked dose-dependent accumulation of FA cells in the G2 compartment after treatment with 4,5',8-trimethylpsoralen (Me(3)Pso) in combination with 365 nm irradiation. Using bivariate DNA distribution methodology, we determined the proportion of replicating and arresting S-phase cells and observed that, whereas normal cells arrested DNA replication in the presence of Me(3)Pso cross-links and monoadducts, FA lymphoblasts failed to arrest DNA synthesis. Taken together, the above data suggest that, in response to damage induced by DNA cross-linking agents, the S-phase checkpoint is inefficient in FA cells. This would lead to accumulation of secondary lesions, such as single- and double-strand breaks and gaps. The prolonged time in G2 phase seen in FA cells therefore exists in order to allow the cells to remove lesions which accumulated during the preceding abnormal S phase.

Cell Line↗
PubMed2006

Population pharmacokinetics of allopurinol in full-term neonates with perinatal asphyxia.

In newborn infants, allopurinol is being tested as a free radical scavenger to prevent brain damage caused by reperfusion and oxygenation after perinatal hypoxia and ischemia (birth asphyxia). To develop rational dosing schemes for future studies, knowledge of the pharmacokinetics in this patient group is essential. In the present study, a population pharmacokinetic model was designed and validated for allopurinol in this specific patient group. One-compartment and 2-compartment models were fitted to plasma concentration time data of 24 newborns entered in 2 clinical trials using nonlinear mixed effects modeling. A bootstrap procedure was performed to check the robustness of the model. The data were best described using a 1-compartment model with linear elimination. Estimated pharmacokinetic parameters were volume of the central compartment (V, 0.79 L/kg) and total body clearance (CL, 0.078 L/h/kg), with 42% and 60% interindividual variability, respectively. The median values for these parameters of 1000 bootstrap replicates were very similar (95% confidence intervals were 0.67 to 0.96 and 0.054 to 0.10 for V and CL, respectively), indicating the robustness of the model. A population pharmacokinetic model has been designed and validated which adequately describes the data of 2 clinical studies in critically ill newborn infants. The model will be used to design dosing strategies for future evaluation of the benefits of allopurinol in these patients.

Allopurinol↗
PubMed2006

Intracellular Salmonella enterica redirect exocytic transport processes in a Salmonella pathogenicity island 2-dependent manner.

During intracellular life, Salmonella enterica proliferate within a specialized membrane compartment, the Salmonella-containing vacuole (SCV), and interfere with the microtubule cytoskeleton and cellular transport. To characterize the interaction of intracellular Salmonella with host cell transport processes, we utilized various model systems to follow microtubule-dependent transport. The vesicular stomatitis virus glycoprotein (VSVG) is a commonly used marker to follow protein transport from the Golgi to the plasma membrane. Using a VSVG-GFP fusion protein, we observed that virulent intracellular Salmonella alter exocytotic transport and recruit exocytotic transport vesicles to the SCV. This virulence function was dependent on the function of the type III secretion system encoded by Salmonella Pathogenicity Island 2 (SPI2) and more specifically on a subset of SPI2 effector proteins. Furthermore, the Golgi to plasma membrane traffic of the shingolipid C(5)-ceramide was redirected to the SCV by virulent Salmonella. We propose that Salmonella modulates the biogenesis of the SCV by deviating this compartment from the default endocytic pathway to an organelle that interacts with the exocytic pathway. This observation might reveal a novel element of the intracellular survival and replication strategy of Salmonella.

Animals↗
PubMed2004

The induction of acute ileitis by a single microbial antigen of Toxoplasma gondii.

The role of specific microbial Ags in the induction of experimental inflammatory bowel disease is poorly understood. Oral infection of susceptible C57BL/6 mice with Toxoplasma gondii results in a lethal ileitis within 7-9 days postinfection. An immunodominant Ag of T. gondii (surface Ag 1 (SAG1)) that induces a robust B and T cell-specific response has been identified and a SAG1-deficient parasite (Deltasag1) engineered. We investigated the ability of Deltasag1 parasite to induce a lethal intestinal inflammatory response in susceptible mice. C57BL/6 mice orally infected with Deltasag1 parasites failed to develop ileitis. In vitro, the mutant parasites replicate in both enterocytes and dendritic cells. In vivo, infection with the mutant parasites was associated with a decrease in the chemokine and cytokine production within several compartments of the gut-associated cell population. RAG-deficient (RAG1(-/-)) mice are resistant to the development of the ileitis after T. gondii infection. Adoptive transfer of Ag-specific CD4(+) effector T lymphocytes isolated from C57BL/6-infected mice into RAG(-/-) mice conferred susceptibility to the development of the intestinal disease. In contrast, CD4(+) effector T lymphocytes from mice infected with the mutant Deltasag1 strain failed to transfer the pathology. In addition, resistant mice (BALB/c) that fail to develop ileitis following oral infection with T. gondii were rendered susceptible following intranasal presensitization with the SAG1 protein. This process was associated with a shift toward a Th1 response. These findings demonstrate that a single Ag (SAG1) of T. gondii can elicit a lethal inflammatory process in this experimental model of pathogen-driven ileitis.

Acute Disease↗
PubMed1996

Relationship between intracellular free calcium concentrations and the intracellular development of Toxoplasma gondii.

We measured intracellular free calcium concentrations ([Ca++]i) in the subcellular compartments of Toxoplasma gondii infected living cells using microspectrofluorometry and Indo-1 staining. [Ca++]i mapping was defined in infected and uninfected cells and in the neoformed parasitophorous vacuole (PV) 24 and 48 hr after parasite inoculation. At 24 hr after infection, a [Ca++]i gradient (PV/cytoplasm) was observed in favor of the PV in 72% of infected cells (p<0.001). Inside of the PV (lumen and parasites), [Ca++]i values appeared to be homogeneously distributed. At 48 hr after infection, the parasites had replicated and formed typical rosettes of more than 16 parasites. At this step, a positive [Ca++]i gradient (PV/cytoplasm) was detected in all analyzed cells (p<0.001). This result suggests that the PV (lumen and parasites) represents an individual subcellular compartment within the host cell that includes an independent [Ca++]i. Moreover, after 48 hr the cytoplasmic [Ca++]i decreased significantly (39 nM) compared with that measured from uninfected cells (53 nM) (p <0.05). Furthermore, the exit of Toxoplasma mediated by the calcium ionophore 4BrA23187 was preceded by a rise of [Ca++]i to 1 mM in the PV. The [Ca++]i rise and the liberation of parasites from their host appear to be correlated. On the basis of these observations, we suggest that the increase of [Ca++]i in the vacuole may act as a signal that triggers the egress of T. gondii.

Animals↗
PubMed2001

Biogenesis of the Semliki Forest virus RNA replication complex.

The nonstructural (ns) proteins nsP1 to -4, the components of Semliki Forest virus (SFV) RNA polymerase, were localized in infected cells by confocal microscopy using double labeling with specific antisera against the individual ns proteins. All ns proteins were associated with large cytoplasmic vacuoles (CPV), the inner surfaces of which were covered by small invaginations, or spherules, typical of alphavirus infection. All ns proteins were localized by immuno-electron microscopy (EM) to the limiting membranes of CPV and to the spherules, together with newly labeled viral RNA. Along with earlier observations by EM-autoradiography (P. M. Grimley, I. K. Berezesky, and R. M. Friedman, J. Virol. 2:326-338, 1968), these results suggest that individual spherules represent template-associated RNA polymerase complexes. Immunoprecipitation of radiolabeled ns proteins showed that each antiserum precipitated the other three ns proteins, implying that they functioned as a complex. Double labeling with organelle-specific and anti-ns-protein antisera showed that CPV were derivatives of late endosomes and lysosomes. Indeed, CPV frequently contained endocytosed bovine serum albumin-coated gold particles, introduced into the medium at different times after infection. With time, increasing numbers of spherules were also observed on the cell surfaces; they were occasionally released into the medium, probably by secretory lysosomes. We suggest that the spherules arise by primary assembly of the RNA replication complexes at the plasma membrane, guided there by nsP1, which has affinity to lipids specific for the cytoplasmic leaflet of the plasma membrane. Endosomal recycling and fusion of CPV with the plasma membrane can circulate spherules between the plasma membrane and the endosomal-lysosomal compartment.

Animals↗
PubMed2006

Cytoskeletal components of an invasion machine--the apical complex of Toxoplasma gondii.

The apical complex of Toxoplasma gondii is widely believed to serve essential functions in both invasion of its host cells (including human cells), and in replication of the parasite. The understanding of apical complex function, the basis for its novel structure, and the mechanism for its motility are greatly impeded by lack of knowledge of its molecular composition. We have partially purified the conoid/apical complex, identified approximately 200 proteins that represent 70% of its cytoskeletal protein components, characterized seven novel proteins, and determined the sequence of recruitment of five of these proteins into the cytoskeleton during cell division. Our results provide new markers for the different subcompartments within the apical complex, and revealed previously unknown cellular compartments, which facilitate our understanding of how the invasion machinery is built. Surprisingly, the extreme apical and extreme basal structures of this highly polarized cell originate in the same location and at the same time very early during parasite replication.

Animals↗
PubMed2000

Mechanisms for adaptation of simian immunodeficiency virus to replication in alveolar macrophages.

In contrast to the simian immunodeficiency virus SIVmac239, which replicates poorly in rhesus monkey alveolar macrophages, a variant with nine amino acid changes in envelope (SIVmac239/316E) replicates efficiently and to high titer in these same cells. We examined levels of viral DNA, RNA, antigen, and infectious virus to identify the nature of the block to SIVmac239 replication in these cells. Low levels of viral antigen (0.1 to 1.0 ng of p27 per ml) and infectious virus (100 to 1,000 infectious units per ml) were produced in the supernatant 1 to 4 days after SIVmac239 infection, but these levels did not increase subsequently. SIVmac239 DNA was synthesized in these macrophage cultures during the initial 24 h after infection, but the levels did not increase subsequently. Quantitation of the numbers of infectious cells in cultures over time and the results of experiments in which cells were reexposed to SIVmac239 after the initial exposure indicated that only a small proportion of cells were susceptible to SIVmac239 infection in these alveolar macrophage cultures and that the vast majority (>95%) of cells were refractory to SIVmac239 infection. In contrast to the results with SIVmac239, the levels of viral antigen, infectious virus, and viral DNA increased exponentially 2 to 7 days after infection by SIVmac239/316E, reaching levels greater than 100 ng of p27 per ml and 100,000 infectious units per ml. Since SIVmac239/316E has previously been described as a virus capable of infecting cells in a relatively CD4-independent fashion, we examined the levels of CD4 expression on the surface of fresh and cultured alveolar macrophages from rhesus monkeys. The levels of CD4 expression were extremely low, below the limit of detection by flow cytometry, on greater than 99% of the macrophages. CCR5(+) cells were profoundly depleted only from alveolar macrophage cultures infected with SIVmac239/316E. High concentrations of an antibody to CD4 delayed but did not block replication of SIVmac239/316E. The results suggest that the adaptation of SIVmac316 to efficient replication in alveolar macrophages results from its ability to infect these cells in a CD4-independent fashion or in a CD4-dependent fashion even at extremely low levels of surface CD4 expression. Since resident macrophages in brains and lungs of humans also express little or no CD4, our findings predict the presence of human immunodeficiency virus type 1 that is relatively CD4 independent in the lung and brain compartments of infected people.

Adaptation, Physiological↗
PubMed2004

The chromoplasts of Or mutants of cauliflower (Brassica oleracea L. var. botrytis).

The Or mutation in cauliflower (Brassica oleracea L. var. botrytis) leads to abnormal accumulations of beta-carotene in orange chromoplasts, in tissues in which leucoplasts are characteristic of wild-type plants. Or chromoplasts were investigated by light microscopy of fresh materials and electron microscopy of glutaraldehyde- and potassium permanganate-fixed materials. Carotenoid inclusions in Or chromoplasts resemble those found in carrot root chromoplasts in their optical activity and angular shape. Electron microscopy revealed that the inclusions are made up of parallel, membrane-bound compartments. These stacks of membranes are variously rolled and folded into three-dimensional objects. We classify Or chromoplasts as "membranous" chromoplasts. The Or mutation also limits plastid replication so that a single chromoplast constitutes the plastidome in most of the affected cells. There are one to two chromoplasts in each cell of a shoot apex. The ability of differentiated chromoplasts to divide in the apical meristems of Or mutant plants resembles the ability of proplastids to maintain plastid continuity from cell to cell in meristems of Arabidopsis thaliana mutants in which plastid replication is drastically limited. The findings are used to discuss the number of levels of regulation involved in plastid replication.

Brassica↗
PubMed1999

Localization of isoprenylated antigen of hepatitis delta virus by anti-farnesyl antibodies.

Hepatitis delta virus (HDV) is a subviral pathogen that requires pre-existing or concurrent infection with hepatitis B virus (HBV). HDV expresses two forms of a single protein, the delta antigen (HDAg), which are identical except for an additional 19 residues at the C terminus of the large form. Within this C-terminal extension a cysteine residue is isoprenylated; this isoprenylation is critical for interaction with HBV envelope proteins to enable virus assembly and release into the medium. Therefore, large HDAg must be recruited to an extracellular compartment. However, immuno-staining with HDAg-specific antibodies has localized the large antigen mainly to the nucleus and supports the notion that large HDAg suppresses virus replication in the nucleus. Since isoprenylation would increase the hydrophobicity of the protein and may favour transport towards specific membranes, the question remains whether the large HDAg detected in the nucleus carries an isoprenyl group. To address this issue, antibodies against the farnesyl modification were generated to allow direct visualization of the antigen by immunofluorescence microscopy. The anti-farnesyl antibodies specifically stained large HDAg expressed in Huh-7 cells, and the signal was largely restricted to the nucleus; the staining pattern could be superimposed on those of cells stained for large HDAg. The large HDAg translocated into the nucleus was therefore isoprenylated. In addition, antibodies specific for the farnesyl modification should be applicable to the study of other similarly isoprenylated proteins.

Amino Acid Sequence↗
PubMed2000

Cryptococcus neoformans is a facultative intracellular pathogen in murine pulmonary infection.

To produce chronic infection, microbial pathogens must escape host immune defenses. Infection with the human pathogenic fungus Cryptococcus neoformans is typically chronic. To understand the mechanism by which C. neoformans survives in tissue after the infection of immunocompetent hosts, we systematically studied the course of pulmonary infection in mice by electron microscopy. The macrophage was the primary phagocytic cell at all times of infection, but neutrophils also ingested yeast. Alveolar macrophages rapidly internalized yeast cells after intratracheal infection, and intracellular yeast cells were noted at all times of infection from 2 h through 28 days. However, the proportion of yeast cells in the intracellular and extracellular spaces varied with the time of infection. Early in infection, yeast cells were found predominantly in the intracellular compartment. A shift toward extracellular predominance occurred by 24 h that was accompanied by macrophage cytotoxicity and disruption. Later in infection, intracellular persistence in vivo was associated with replication, residence in a membrane-bound phagosome, polysaccharide accumulation inside cells, and cytotoxicity to macrophages, despite phagolysosomal fusion. Many phagocytic vacuoles with intracellular yeast had discontinuous membranes. Macrophage infection resulted in cells with a distinctive appearance characterized by large numbers of vacuoles filled with polysaccharide antigen. Similar results were observed in vitro using a macrophage-like cell line. Our results show that C. neoformans is a facultative intracellular pathogen in vivo. Furthermore, our observations suggest that C. neoformans occupies a unique niche among the intracellular pathogens whereby survival in phagocytic cells is accompanied by intracellular polysaccharide production.

Animals↗
PubMed2005

Decreased T-cell receptor excision circles in cutaneous T-cell lymphoma.

PURPOSE: The T cell repertoire in patients with advanced cutaneous T cell lymphoma (CTCL) is significantly contracted despite the presence of relatively normal absolute numbers of T cells. We propose that many normal T cells were being lost in patients with CTCL, with the remaining normal T cells expanding clonally to fill the T cell compartment. T-cell receptor excision circles (TREC) form as a result of the initial gene rearrangement in naïve T cells. Although they are stable, they do not replicate and are subsequently diluted with the expansion of a population of T cells. Their concentration is therefore a measure of unexpanded naïve T cells relative to T cells that have undergone expansion. EXPERIMENTAL DESIGN: We analyzed TRECs from unfractionated peripheral blood T cells from 108 CTCL patients by quantitative PCR. In patients with obvious peripheral blood involvement, we also analyzed TRECs from clonal and nonclonal T cells. RESULTS: We found a decrease in the number of TRECs in peripheral blood of patients with CTCL at all stages of disease, and this decrease was proportional to the loss of complexity of the T cell repertoire as measured by complementarity-determining region 3 spectratyping. In patients with leukemic CTCL and a numerically expanded clone, we also found a significantly lower-than-expected number of TRECs in the nonclonal normal T cells. CONCLUSIONS: We hypothesize that the nonmalignant T cells have proliferated to fill the empty T cell repertoire space left by the loss of other T cells, leading to diminished TRECs and loss of T-cell receptor diversity.

Adult↗
PubMed1998

Mechanism of Borna disease virus entry into cells.

We have investigated the entry pathway of Borna disease virus (BDV). Virus entry was assessed by detecting early viral replication and transcription. Lysosomotropic agents (ammonium chloride, chloroquine, and amantadine), as well as energy depletion, prevented BDV infection, indicating that BDV enters host cells by endocytosis and requires an acidic intracellular compartment to allow membrane fusion and initiate infection. Consistent with this hypothesis, we observed that BDV-infected cells form extensive syncytia upon low-pH treatment. Entry of enveloped viruses into animal cells usually requires the membrane-fusing activity of viral surface glycoproteins (GPs). BDV GP is expressed as two products of 84 and 43 kDa (GP-84 and GP-43, respectively). We show here that only GP-43 is present at the surface of BDV-infected cells and therefore is likely the viral polypeptide responsible for triggering fusion events. We also present evidence that GP-43, which corresponds to the C terminus of GP-84, is generated by cleavage of GP-84 by the cellular protease furin. Hence, we propose that BDV GP-84 is involved in attachment to the cell surface receptor whereas its furin-cleaved product, GP-43, is involved in pH-dependent fusion after internalization of the virion by endocytosis.

Amantadine↗