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PubMed2007

The carboxy-terminal domain of glycoprotein N of human cytomegalovirus is required for virion morphogenesis.

Glycoproteins M and N (gM and gN, respectively) are among the few proteins that are conserved across the herpesvirus family. The function of the complex is largely unknown. Whereas deletion from most alphaherpesviruses has marginal effects on the replication of the respective viruses, both proteins are essential for replication of human cytomegalovirus (HCMV). We have constructed a series of mutants in gN to study the function of this protein. gN of HCMV is a type I glycoprotein containing a short carboxy-terminal domain of 14 amino acids, including two cysteine residues directly adjacent to the predicted transmembrane anchor at positions 125 and 126. Deletion of the entire carboxy-terminal domain as well as substitution with the corresponding region from alpha herpesviruses or mutations of both cysteine residues resulted in a replication-incompetent virus. Recombinant viruses containing point mutations of either cysteine residue could be generated. These viruses were profoundly defective for replication. Complex formation of the mutant gNs with gM and transport of the complex to the viral assembly compartment appeared unaltered compared to the wild type. However, in infected cells, large numbers of capsids accumulated in the cytoplasm that failed to acquire an envelope. Transiently expressed gN was shown to be modified by palmitic acid at both cysteine residues. In summary, our data suggest that the carboxy-terminal domain of gN plays a critical role in secondary envelopment of HCMV and that palmitoylation of gN appears to be essential for function in secondary envelopment of HCMV and virus replication.

Amino Acid Sequence↗
PubMed2006

Defining the transcriptome of accelerated and replicatively senescent keratinocytes reveals links to differentiation, interferon signaling, and Notch related pathways.

Epidermal keratinocytes (KCs) undergo highly orchestrated morphological and molecular changes during transition from proliferative compartment into growth arrested early and late differentiation layers, prior to dying in outermost cornified layers of normal skin. Creation of stratum corneum is vital to barrier function protecting against infection. Transcriptional events in KCs regulating complex processes of differentiation and host defense required to maintain constant epidermal thickness and resistance to infection in either young or aged skin are largely unknown. Furthermore, as terminal differentiation is characterized by irreversible loss of replicative potential culminating in dead layers at the skin surface, this process may be viewed as a form of senescence. However, a complete transcriptional profile of senescent (SN) human KCs has not been previously defined to permit delineation of molecular boundaries involving differentiation and senescence. To fill this void, we utilized global transcriptional analysis of KCs maintained in vitro as either cultures of proliferating (PR) cells, early and late confluent (LC) (accelerated senescence) cultures, or KCs undergoing replicative senescence. Global gene expression profiling revealed early confluent (EC) KCs were somewhat similar to PR KCs, while prominent differences were evident when compared to LC KCs; which were also distinct from replicatively SN KCs. While confluent KCs have in common several genes regulating differentiation with replicatively SN KCs, the latter cells expressed elevated levels of genes involved in interferon signaling and inflammatory pathways. These results provide new insights into cell autonomous transcriptional-based programs operative within KCs contributing to replicative senescence, with partial sharing of genes involved in differentiation. In addition, regulation of KC senescence may involve participation of interferon signaling pathways derived from the important role of KCs in protecting skin from infection. Integrating all of the transcriptional data revealed a key role for Notch receptor mediated signaling in the confluency induced differentiation phenotype using this model system.

Apoptosis↗
PubMed1995

Inhibition of the intracellular transport of influenza viral RNA by actinomycin D.

In primary chicken embryo cells infected with fowl plague virus addition of actinomycin D at defined times during the infection cycle has different consequences on viral replication. If actinomycin D is added immediately after infection with a concentration, which inhibits viral RNA synthesis only partially, it interferes with the nucleo-cytoplasmic transport of all viral RNA species (mRNA and vRNA) so far tested. If actinomycin D is present during infection (adsorption, penetration and uncoating) no viral RNA is synthesized, and the nucleocapsid of the infecting virus does not reach the nucleus, as shown by fluorescent antibodies. Therefore the primary effect of actinomycin D on influenza virus replication is on the transport of the incoming vRNPs from the cytoplasm to the cell nucleus, which is the cell compartment where transcription takes place.

Animals↗
PubMed2006

Innate immune recognition of viral infection.

Induction of the antiviral innate immune response depends on recognition of viral components by host pattern-recognition receptors. Members of the Toll-like receptor family have emerged as key sensors that recognize viral components such as nucleic acids. Toll-like receptor signaling results in the production of type I interferon and inflammatory cytokines and leads to dendritic cell maturation and establishment of antiviral immunity. Cells also express cytoplasmic RNA helicases that function as alternative pattern-recognition receptors through recognition of double-stranded RNA produced during virus replication. These two classes of pattern-recognition receptor molecules are expressed in different intracellular compartments and induce type I interferon responses via distinct signaling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1987

Proliferation of human testicular tumours.

Whole tumour perfusion with radioactive DNA precursors reveals that proliferation of human testicular tumours differs widely depending on type and stage of development. In seminomas a homogeneous distribution of proliferating cells at early stages is followed by a shift of the growth fraction to the periphery at advanced stage. Embryonal carcinomas show a chessboard-like pattern of proliferating fractions with high 3H-thymidine labelling indices and short tpot. In mature teratomas proliferation is low. It increases in immature teratocarcinomas. Lack of proliferation is observed in beta-hCG-positive syncytiotrophoblastic cells of choriocarcinomas, the growth fraction being limited to the cytotrophoblastic compartment.

Adult↗
PubMed1986

Renewal and release of hemopoietic stem cells: does clonal succession exist?

The term "clonal succession" was proposed 20 years ago by Kay to characterize the release of stem cells from their primary pool, followed by the expansion of each cell to a clone and the succession of new clones as the mature cells of each clone are lost because of their finite life span. The authors point out that the events described by Kay as clonal succession are necessary features of hemopoietic proliferation. Clonal succession is equivalent to the universally recognized existence of a self-replicating primary stem cell pool that continuously supplies cells for amplification and maturation to subsequent compartments. The authors object to the term "clonal succession" because it has been used by a number of investigators to characterize specific schemes rather than the general aspects of hemopoietic proliferation, thus leading to confusion. They suggest that the descriptive term, "continuous release of stem cells for differentiation," would be preferable to the use or misuse of "clonal succession."

Animals↗
PubMed1994

Intracellular trafficking in Mycobacterium tuberculosis and Mycobacterium avium-infected macrophages.

Despite the potential role of the macrophage in the eradication of invading microbes, Mycobacterium species have evolved mechanisms to ensure their survival and replication inside the macrophage. Particles phagocytosed by macrophages normally will be delivered into acid lysosomal compartments for degradation. Mycobacterium must, in some way, avoid this fate by modulation of their phagosome. Immunoelectron microscopy of macrophages infected with Mycobacterium avium or Mycobacterium tuberculosis indicates that the vacuolar membrane surrounding the bacilli possesses the late endosomal/lysosomal marker, LAMP-1 (lysosomal-associated membrane protein-1), but lacks the vesicular proton-ATPase. Analysis of the intersection of the bacteria-containing vacuoles with the endocytic network of the macrophage supports previous studies indicating that these bacilli restrict the fusion capability of their intracellular compartments. The occurrence of vesicles containing lipoarabinomannan, discrete from those containing Mycobacterium, indicate that material does traffic out from the mycobacterial vacuole. To compensate for this loss of membrane, the vacuole must remain dynamic and fuse with LAMP-1-containing vesicles to maintain the density of this marker.

Animals↗
PubMed1979

Effects of 5-fluorouracil on mouse bone marrow.

5-Fluorouracil (150 mg/kg) was injected intravenously into 11-week-old male mice. Its effects on marrow structure over the following 9 d were assessed using light microscopic examination of semi-thin transverse sections of decalcified humeri. The extravascular compartment became markedly depleted of cells and decreased in size, reaching a minimum on day 5 post-treatment. Proliferating haematopoietic cells disappeared within 2 d. Post-replicative erythrocytic cells underwent extra-vascular phagocytosis by resident macrophages, whilst mature granulocytes continued to pass into the circulation. Repopulation was underway by day 6, and re-expansion of the extravascular compartment by day 7 when megakaryocytes, of larger than normal average size, occupied most of the extravascular compartment. Megakaryocyte dominance gave way over days 8 and 9 as other haematopoietic cells proliferated and the extravascular compartment continued to enlarge. The significance of the results is discussed in relation to marrow micro-circulation, egress of haematopoietic cells into the circulation, and cell proliferation in the marrow. Endotoxin, administered 4 h after 5-fluorouracil, accelerated depletion of the marrow and recovery of haematopoiesis by about 1 d.

Animals↗
PubMed2004

Intracellular trafficking study of a RB51 B. abortus vaccinal strain isolated from cow milk.

Brucella is responsible for one of the major worldwide zoonoses. Over the last century, several vaccines have been used against brucellosis. Among these, the rough vaccine Brucella abortus RB51 was introduced with the idea that it would not interfere with the diagnosis of brucellosis. Recently, RB51 has been isolated from milk and vaginal exudates from vaccinated cows, thus raising the possibility of extensive bacterial replication in these animals. We hypothesized that shedding of RB51 might be related to a change in its intracellular cell cycle. Therefore, we have compared the intracellular trafficking in CHO cells of the virulent B. abortus 2308 and two RB51 strains, the vaccinal strain and the one isolated from cow milk. Both RB51 strains were transiently observed in phagosomes characterized by the presence of the early endosomal marker EEA1 and then were found in cathepsin D-enriched lysosomal compartments, in which they eventually underwent degradation at later post-infection times. In contrast, the virulent 2308 strain replicated within the endoplasmic reticulum. These results suggest that a change in intracellular trafficking cannot account for Brucella shedding in adult vaccinated cows.

Animals↗
PubMed2001

The conserved serine 177 in the delta antigen of hepatitis delta virus is one putative phosphorylation site and is required for efficient viral RNA replication.

Hepatitis delta virus (HDV) small delta antigen (S-HDAg) plays a critical role in virus replication. We previously demonstrated that the S-HDAg phosphorylation occurs on both serine and threonine residues. However, their biological significance and the exact phosphorylation sites of S-HDAg are still unknown. In this study, phosphorylated S-HDAg was detected only in the intracellular compartment, not in viral particles. In addition, the number of phosphorylated isoforms of S-HDAg significantly increased with the extent of viral replication in transfection system. Site-directed mutagenesis showed that alanine replacement of serine 177, which is conserved among all the known HDV strains, resulted in reduced phosphorylation of S-HDAg, while the mutation of the other two conserved serine residues (2 and 123) had little effect. The S177A mutant dramatically decreased its capability in assisting HDV RNA replication, with a preferential and profound impairment of the antigenomic RNA replication. Furthermore, the viral RNA editing, a step relying upon antigenomic RNA replication, was also abolished by this mutation. These results suggested that phosphorylation of S-HDAg, with serine 177 as a presumable site, plays a critical role in viral RNA replication, especially in augmenting the replication of antigenomic RNA.

Amino Acid Sequence↗
PubMed1988

Replication program of active and inactive multigene families in mammalian cells.

In a comprehensive study, the temporal replication of tissue-specific genes and flanking sequences was compared in nine cell lines exhibiting different tissue-specific functions. Some of the rules we have determined for the replication of these tissue specific genes include the following. (i) Actively transcribed genes usually replicate during the first quarter of the S phase. (ii) Some immunoglobulin genes replicate during the first half of S phase even when no transcriptional activity is detected but appear to replicate even earlier in cell lines where they are transcribed. (iii) Nontranscribed genes can replicate during any interval of S phase. (iv) Multigene families arranged in clusters of 250 kilobases or less define a temporal compartment comprising approximately one-quarter of S phase. While these rules, and others that are discussed, apply to the tissue-specific genes studied here, all tissue-specific genes may not follow this pattern. In addition, housekeeping genes did not follow some of these rules. These results provide the first molecular evidence that the coordinate timing of replication of contiguous sequences within a multigene family is a general property of the mammalian genome. The relationship between replication very early during S phase and the transcriptional activity within a chromosomal domain is discussed.

Animals↗
PubMed1981

Origin of life between Scylla and Charybdis.

The "package model" discussed here is concerned with the preservation of genetic information by primordial compartments. Each viable "package" encloses a complete set of unlinked genes in varying numbers of copies. Due to stochastic distribution and error-prone replication two potent perils endanger the informational integrity of packages: fluctuation and mutation. A computer simulation was used to quantify the effects of fluctuation, mutation, and package death by accident. Assuming reasonable rates for these parameters it is suggested that life started out with compartments containing not more than 3 different genes.

Biological Evolution↗
PubMed2000

Chromosome territories, interchromatin domain compartment, and nuclear matrix: an integrated view of the functional nuclear architecture.

Advances in the specific fluorescent labeling of chromatin in fixed and living human cells in combination with three-dimensional (3D) and 4D (space plus time) fluorescence microscopy and image analysis have opened the way for detailed studies of the dynamic, higher-order architecture of chromatin in the human cell nucleus and its potential role in gene regulation. Several features of this architecture are now well established: 1. Chromosomes occupy distinct territories in the cell nucleus with preferred nuclear locations, although there is no evidence of a rigid suprachromosomal order. 2. Chromosome territories (CTs) in turn contain distinct chromosome arm domains and smaller chromatin foci or domains with diameters of some 300 to 800 nm and a DNA content in the order of 1 Mbp. 3. Gene-dense, early-replicating and gene-poor, middle-to-late-replicating chromatin domains exhibit different higher-order nuclear patterns that persist through all stages of interphase. In mitotic chromosomes early replicating chromatin domains give rise to Giemsa light bands, whereas middle-to-late-replicating domains form Giemsa dark bands and C-bands. In an attempt to integrate these experimental data into a unified view of the functional nuclear architecture, we present a model of a modular and dynamic chromosome territory (CT) organization. We propose that basically three nuclear compartments exist, an "open" higher-order chromatin compartment with chromatin domains containing active genes, a "closed" chromatin compartment comprising inactive genes, and an interchromatin domain (ICD) compartment (Cremer et al., 1993; Zirbel et al., 1993) that contains macromolecular complexes for transcription, splicing, DNA replication, and repair. Genes in "open," but not in "closed" higher-order chromatin compartments have access to transcription and splicing complexes located in the ICD compartment. Chromatin domains that build the "open" chromatin compartment are organized in a way that allows the direct contact of genes and nascent RNA to transcription and splicing complexes, respectively, preformed in the ICD compartment. In contrast, chromatin domains that belong to the "closed" compartment are topologically arranged and compacted in a way that precludes the accessibility of genes to transcription complexes. We argue that the content of the ICD compartment is highly enriched in DNA depleted biochemical matrix preparations. The ICD compartment may be considered as the structural and functional equivalent of the in vivo nuclear matrix. A matrix in this functional sense is compatible with but does not necessitate the concept of a 3D nuclear skeleton existing of long, extensively arborized filaments. In the absence of unequivocal evidence for such a structural matrix in the nucleus of living cells we keep an agnostic attitude about its existence and possible properties in maintaining the higher-order nuclear architecture. Quantitative modeling of the 3D and 4D human genome architecture in situ shows that such an assumption is not necessary to explain presently known aspects of the higher-order nuclear architecture. We expect that the interplay of quantitative modeling and experimental tests will result in a better understanding of the compartmentalized nuclear architecture and its functional consequences.

Animals↗
PubMed2000

Coinfection of fibroblasts with Coxiella burnetti and Toxoplasma gondii: to each their own.

Intracellular pathogens have evolved distinct strategies to subvert host cell defenses. At diametrically opposed ends of the spectrum with regard to the host endosomal/lysosomal defenses are the obligate intracellular protozoan Toxoplasma gondii and the bacterium Coxiella burnetti. While the intracellular replication of T. gondii requires complete avoidance of the host endocytic cascade, C. burnetti actively subverts it. This results in these organisms establishing and growing in very different vacuolar compartments. In this study we examined the potential interaction between these distinct compartments following coinfection of mammalian fibroblasts. When present within the same cell, these organisms exhibit minimal interaction with each other. Colocalization of T. gondii and C. burnetti within the same vacuole occurs at a low frequency in doubly infected cells. In such instances only one of the organisms appears to be replication competent, emphasizing the different requirements for survival and/or intracellular growth. The potential basis for both the lack of interaction between these distinct pathogen-containing compartments, and the mechanisms to address their low frequency of colocalization are discussed in the context of our understanding of the biology of the organisms and membrane traffic in eukaryotic cells.

Animals↗
PubMed2001

Changes in soil solution Zn and pH and uptake of Zn by arbuscular mycorrhizal red clover in Zn-contaminated soil.

Red clover plants inoculated with Glomus mosseae were grown in a sterile pasture soil containing 50 mg Zn kg(-1) in 'Plexiglas' (acrylic) containers with nylon net partitions (30 microm mesh) designed to separate the soil into a central root zone and two outer zones for hyphal growth with no root penetration. Two porous plastic soil moisture samplers were installed in each pot, one in the root compartment and the other in one of the hyphal compartments. The soil in the outer compartments was amended with one of the four application rates of Zn (as ZnSO4) ranging from 0 to 1000 mg kg(-1). Non-mycorrhizal controls were included, and there were five replicates of each treatment in a randomised block in a glasshouse. Uninoculated plants received supplementary P to avoid yield limitation due to low soil P status. Plants grew in the central compartment for nine weeks. Soil moisture samples were collected 4, 24 and 62 days after sowing to monitor changes in the Zn concentration and pH of the soil solution. At harvest, the mean mycorrhizal infection rate of inoculated plants ranged from 29% to 34% of total root length and was little affected by Zn application. Root and shoot yields were not affected by mycorrhizal infection. Plant Zn concentration and uptake were lower in mycorrhizal plants than non-mycorrhizal controls, and this effect was more pronounced with increasing Zn application rate to the soil. Soil solution Zn concentrations were lower and pH values were higher in mycorrhizal treatments than non-mycorrhizal controls and the mycorrhiza effect was more pronounced at higher Zn application rates. The protective effect of mycorrhiza against plant Zn uptake may have been associated with changes in Zn solubility mediated by changes in the soil solution pH, or by immobilisation of Zn in the extraradical mycelium.

Fabaceae↗
PubMed2001

Population pharmacokinetic modeling and model validation of a spicamycin derivative, KRN5500, in phase 1 study.

PURPOSE: KRN5500, a novel spicamycin derivative, shows the greatest activity against a human tumor xenograft model and the highest therapeutic index among spicamycin derivatives. KRN5500 is currently under clinical development in Japan and the United States. The objective of this study was to develop a population pharmacokinetic model that describes the KRN5500 plasma concentration versus time data. METHODS: Data were collected from 18 patients entered in a phase 1 study. These patients received KRN5500 3-21 mg/m2 as a 2-h infusion. A total of 219 concentration measurements were available. The data were analyzed using the nonlinear mixed effect model (NONMEM) program. In addition, the basic and final population pharmacokinetic models were evaluated using bootstrapping resampling. RESULTS: The basic model selected was a two-compartment model with a combination of additive and constant coefficient of variation error models. The basic model fitted well not only the original data, but also 100 bootstrap replicates generated from the original data set. With regard to the effect of covariates selected by generalized additive modeling analysis, gender (SEX) and performance status were found to be possible determinants of the volume of central compartment by NONMEM analysis. The final regression model for V1 was V1 = theta V1 (1--SEX x theta SEX), where V1 is the typical population value of the volume of central compartment, and SEX = 0 if the patient is male, otherwise SEX = 1. The final model was fitted to the 200 bootstrapped samples. The mean parameter estimates were within 15% of those obtained with the original data set. CONCLUSIONS: The KRN5500 plasma concentration versus time data obtained from the phase 1 study were well described by the population pharmacokinetic model. Further evaluation by bootstrapping showed that the population pharmacokinetic model was stable.

Adult↗
PubMed1990

Improved method for estimating Ca uptake in vascular smooth muscle using compartmental analysis.

A comparative study on the modeling aspects of Ca uptake in vascular smooth muscle is presented with particular emphasis on determination of the influx rate and its standard error for one- and two-compartment models. Experimental data from our laboratory of 45Ca uptake by dog carotid arteries were optimally fitted to a one-compartment model and were used to compare different estimation methods and experiment designs. Reparameterization of the model equation yielded an expression that allows direct estimation of the influx rate and its standard error. Experiment design with replicated sampling at three to four times were found to provide the highest estimation precision and successful comparisons of influx rates under treatment and control conditions. Two-compartment model data reported in the literature for Ca uptake by cells were reprocessed, yielding standard errors for the rate constant of the fast component an order of magnitude larger than the mean estimate. For this case, a three-parameter variant of the one-compartment model was developed that described the data with acceptable standard errors. Overall we found that the choice of the model that fitted Ca uptake data best required consideration of parameter estimate precision comparisons in addition to F tests of significance between alternate models.

Animals↗
PubMed2002

Virion disassembly is required for apoptosis induced by reovirus.

Reovirus infection leads to apoptosis in cultured cells and in vivo. Binding of viral attachment protein final sigma 1 to both sialic acid and junction adhesion molecule is required for induction of apoptosis. However, it is not known whether viral engagement of receptors is sufficient to elicit this cellular response. To determine whether steps in reovirus replication subsequent to viral attachment are required for reovirus-induced apoptosis, we used inhibitors of viral disassembly and RNA synthesis, viral disassembly intermediates, temperature-sensitive (ts) reovirus mutants, and reovirus particles deficient in genomic double-stranded RNA (dsRNA). We found that reovirus-induced apoptosis is abolished in the presence of the viral disassembly inhibitors ammonium chloride and E64. Infectious subvirion particles (ISVPs), which are intermediates in reovirus disassembly that can be generated in vitro by protease treatment, are capable of inducing apoptosis in the presence or absence of these inhibitors. Treatment of cells with the viral RNA synthesis inhibitor ribavirin does not diminish the capacity of reovirus to induce apoptosis, and reovirus ts mutants arrested at defined steps in viral replication produce apoptosis with efficiency similar to that of wild-type virus. Furthermore, reovirus particles lacking dsRNA are capable of inducing apoptosis. Finally, we found that viral attachment and disassembly must occur within the same cellular compartment for reovirus to elicit an apoptotic response. These results demonstrate that disassembly of reovirus virions to form ISVPs, but not viral transcription or subsequent steps in viral replication, is required for reovirus to induce apoptosis.

Ammonium Chloride↗