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PubMed1998

Chronic cyclosporine-A injection in rats with damaged blood-brain barrier does not impair retention of passive avoidance.

Recently, we demonstrated that chronic administration of immunosuppressant drug, cyclosporine-A (CsA), does not produce impairment in memory retention of a passive avoidance task in normal adult rats. Since CsA has been used as an adjunctive therapy to avoid xenograft rejection inherent in neural transplantation therapy for neurodegenerative disorders, we replicated our previous study in animals with damaged blood-brain barrier (BBB) simulating that of the neural transplantation protocol. Adult rats with damaged BBB that received either chronic CsA (5, 10, and 20 mg/kg) or vehicle injection did not differ significantly in their memory retention of the passive avoidance task that rewarded 'less mobile activity', in that animals avoided electric shock when they restrained their movements within the safe compartment. General spontaneous locomotor activity also was not altered by CsA, except in animals that received 20 mg/kg, which displayed significant hypoactivity at later post-injection periods of CsA. The absence of potentiation of retention of the passive avoidance task in all CsA-treated animals, including the hypoactive ones, suggests that locomotor activity did not interfere with cognitive behavior. The present results confirm our previous findings that the therapeutic dosage (10 mg/kg) of CsA used for neural transplantation does not produce visible deleterious effects on the performance of memory retention task in immunosuppressed rats with damaged BBB.

Animals↗
PubMed2001

Internally located signal peptides direct hepatitis C virus polyprotein processing in the ER membrane.

An endoplasmic reticulum (ER) signal peptide is an amino acid sequence motif that directs the translocation of nascent polypeptides to the lumen of ER membrane. Most of known ER signal peptides are either N-terminal cleavable or internally uncleavable. In the structural protein region of hepatitis C virus (HCV) polyprotein, however, four internally located cleavable signal peptides are arranged in a tandem array. The published experimental results indicated that the nascent HCV polyprotein is processed in the ER membrane by host signal peptidase(s) to the respective viral proteins. Here we propose that the four ER signal peptides lead the nascent HCV polyprotein to ER membrane, and the four internally located cleavable signal peptides are the sole determinant for the compartment localization of the matured viral proteins. After cleavage at the C-terminus, the signal peptides retain at the C-terminus of mature proteins, and serve as ER membrane anchors. The signal peptide directed polyprotein processing in the ER membrane preludes the virion assembly and budding from the ER membrane. This unique processing may be a general mechanism adopted by many types of virus for virion assembly and replication. The revelation of signal peptidase involved in HCV polyprotein processing presents a novel drug target to suppress HCV viral replication for the much needed HCV therapy.

Amino Acid Sequence↗
PubMed2001

p53-dependent induction of p21(Cip1/WAF1/Sdi1) protects against oxygen-induced toxicity.

The beneficial effects of supplemental oxygen delivered to patients suffering from acute respiratory distress is offset by its reduction to genotoxic reactive oxygen species (ROS) that inhibit proliferation and kill pulmonary cells. Cells respond to oxygen-induced damage by expressing the tumor suppressor p53 and the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1) (p21), which limits proliferation by blocking entry into S phase. Since preventing DNA synthesis during genotoxic stress may enhance survival, the current study examines whether hyperoxia induces p21 through a p53-dependent pathway and whether p21 protects cells from the toxic effects of oxygen. HCT116 colon carcinoma cells and clonal lines lacking p53 or p21were used in this study because they allow direct cytotoxic comparisons between isogenic cells, without complications arising from unknown genetic differences between nonhomologous cell lines. Hyperoxia (95% O2, 5% CO2) increased p53 abundance, phosphorylation of p53 on serine 15, and p21 mRNA and protein in parental HCT116 cells that ceased proliferation. In contrast, p21 was not detected in either p53- or p21-deficient HCT116 cells, which exited the G1 compartment and were arrested in S and G2/M phases during hyperoxia. Trypan blue-dye exclusion revealed that induction of p21 markedly enhanced survival during exposure and colony survival assays showed that p21 enhanced the ability to resume proliferation during recovery in room air. The observation that p53-dependent induction of p21 prevents exit from G1 and promotes survival during hyperoxia is consistent with the importance of limiting DNA replication during genotoxic stress caused by oxygen exposure.

Blotting, Western↗
PubMed2006

Intracellular survival of Burkholderia cenocepacia in macrophages is associated with a delay in the maturation of bacteria-containing vacuoles.

Strains of the Burkholderia cepacia complex (Bcc) are opportunistic bacteria that can cause life-threatening infections in patients with cystic fibrosis and chronic granulomatous disease. Previous work has shown that Bcc isolates can persist in membrane-bound vacuoles within amoeba and macrophages without bacterial replication, but the detailed mechanism of bacterial persistence is unknown. In this study, we have investigated the survival of the Burkholderia cenocepacia strain J2315 within RAW264.7 murine macrophages. Strain J2315 is a prototypic isolate of the widespread and transmissible ET12 clone. Unlike heat-inactivated bacteria, which reach lysosomes shortly after internalization, vacuoles containing live B. cenocepacia J2315 accumulate the late endosome/lysosome marker LAMP-1 and start fusing with lysosomal compartments only after 6 h post internalization. Using fluorescent fluid-phase probes, we also demonstrated that B. cenocepacia-containing vacuoles continued to interact with newly formed endosomes, and maintained a luminal pH of 6.4 +/- 0.12. In contrast, vacuoles containing heat-inactivated bacteria had an average pH of 4.8 +/- 0.03 and rapidly merged with lysosomes. Additional experiments using concanamycin A, a specific inhibitor of the vacuolar H+-ATPase, revealed that vacuoles containing live bacteria did not exclude the H+-ATPase. This mode of bacterial survival did not require type III secretion, as no differences were found between wild type and a type III secretion mutant strain. Collectively, our results suggest that intracellular B. cenocepacia cause a delay in the maturation of the phagosome, which may contribute to facilitate bacterial escape from the microbicidal activities of the host cell.

Animals↗
PubMed1986

Response of the microflora in outdoor experimental streams to pentachlorophenol: environmental factors.

The 2nd year of a 2-year study of the fate of pentachlorophenol in outdoor artificial streams focused on details of microbial degradation by a combination of in situ and laboratory measurements. Replicate streams were dosed continuously at pentachlorophenol concentrations of 0, 48, and 144 micrograms/L, respectively, for an 88-d period during the summer of 1983. Pentachlorophenol was degraded both aerobically and anaerobically. Aerobic degradation was more rapid than anaerobic degradation. Mineralization of pentachlorophenol was concommitant with pentachlorophenol disappearance under aerobic conditions, but lagged behind loss of the parent molecule under anaerobic conditions. Biodegradation in the streams, or in specific stream compartments such as the sediment or water column, was characterized by an adaptation period (3-5 weeks for the stream as a whole, and reproducible from the previous year), which was inversely dependent on the concentration of pentachlorophenol and microbial biomass. The adaptation in the streams could be attributed to the time necessary for selective enrichment of an initially low population of pentachlorophenol degraders on surface compartments. The extent of biodegradation in the streams (percent loss of initial concentration of pentachlorophenol) increased with increasing pentachlorophenol input, which was explicable by an increase in the pentachlorophenol degrader population with increasing pentachlorophenol concentration. The sediment zone most significant to overall pentachlorophenol biodegradation was the top 0.5- to 1-cm layer as shown by pentachlorophenol migration rates and depth profiles of degrader density within the sediment. Pentachlorophenol profiles in sediment cores taken during and after the adaptation period for degradation showed that diffusion of pentachlorophenol into the sediment was rate limiting to degradation in this compartment.(ABSTRACT TRUNCATED AT 250 WORDS)

Aerobiosis↗
PubMed1987

The distribution of a 'mitosis-specific' antigen during Drosophila development.

We have used MPM-2, a monoclonal antibody raised against mitotic HeLa cells, to stain a Drosophila cell line, whole mounts of Drosophila embryos, and sectioned tissue from embryonic and larval stages of development. MPM-2 recognizes a major phosphoprotein of approximately 125 X 10(3) Mr in Drosophila tissue culture cells that, like the mammalian MPM-2 antigen, appears to be recognized only in mitotic cells. During early embryogenesis, when the embryonic nuclei divide as a syncytium with a very short nuclear division time, MPM-2 antigen is observed within the spindle compartment at all stages of the nuclear division cycle. Upon cellularization of the embryo and lengthening of the duration of the cycle, the antigen is predominantly seen in mitotic cells. Drosophila larvae contain both diploid and polytene tissues: in diploid tissue MPM-2 staining is specifically observed over mitotic cells, as expected from its distribution in cellularized embryos. Surprisingly, antigen is also detected in the nuclei of polytene cells that replicate their DNA but do not undergo mitosis.

Animals↗
PubMed2001

Retention time of macerated alfalfa hay and silage in sheep.

Fresh alfalfa was mowed and conditioned mechanically at four levels: a control (rubber rolls), macerated once (a single passage through three finely corrugated rolls set at 1-mm clearance), macerated twice (two passages), and macerated thrice (three passages). Alfalfa was then field-wilted either for 45 h and conserved as chopped silage at 30% dry matter (DM) or for 94 h and stored as baled hay at 85% DM. The eight forage treatments (four mechanical conditioning levels x two conservation systems) were fed to 24 sheep (three replications per treatment) during 5 wk. At the beginning of wk 5, a 15-g sample of chromium-mordanted forage (3.5% Cr) was fed to each sheep, and feces samples were collected at 30 different times over 7 d, between 10 h and 168 h after Cr ingestion. Four models were used to estimate the passage rates, the time delay, and the mean retention time (MRT). A two-compartment time-dependent model and a multicompartment model produced the best fit (average r2 of 0.96) to represent the Cr concentration in the feces over time. When compared with alfalfa hay, alfalfa silage had a higher (P < 0.01) time-dependent turnover rate (0.0949 vs 0.0733/h), a lower (P = 0.03) time delay (9.1 vs 11 h), and a lower (P = 0.04) MRT (57.8 vs 64.4 h). Maceration did not affect significantly (P > 0.10) the time delay or the MRT. However, the MRT of macerated alfalfa hay tended to be higher than the MRT of control hay. Experimental data based on marker concentration in the feces can be used satisfactorily to assess differences in MRT between treatments, but they should be used with caution to estimate the partition of retention time within the gastrointestinal tract.

Animals↗
PubMed1980

Regulation of red blood cell production by erythropoietin: normal mouse marrow in vitro.

The response of immediate erythroid precursors to timed exposures of erythropoietin (Ep) were investigated by examining the formation of murine erythroid colonies in vitro. At various times after the initiation of normal marrow cultures with Ep, enough anti-erythropoietin serum was added to neutralize the actions of the hormone for the rest of the 48 h culture period in plasma clots. Results show that a significant number of erythroid precursors develop to a mature colony after very short Ep exposures (as little as 18 min), but they account for only about 13% of the total colonies generated when Ep is active for 48 h. Only if Ep is active for more than 6 h do additional colonies form, in direct proportion to the length of the Ep exposure. Maximum erythroid colony production requires prolonged exposure to Ep, and the longer Ep is active, the larger the percentage of erythroid colonies that reach 17 cells or more. The mechanism(s) behind these two effects appear intimately connected. These results suggest the existence of two populations of Ep-responsive cells in the immediate erythroid precursor compartment of mice: (1) A CFUE pool, which is on the verge of commitment into the recognizable erythron, and requires very short Ep exposures for this entry; and (2) a "pre-CFUE" stage which feeds into the CFUE pool in the presence of Ep, and which produces colonies only after longer Ep exposures. It is proposed that the pre-CFUE response to Ep involves 1-2 self-replications before entering the CFUE population.

Animals↗
PubMed1993

Folimycin (concanamycin A), an inhibitor of V-type H(+)-ATPase, blocks cell-surface expression of virus-envelope glycoproteins.

Folimycin (concanamycin A) inhibited syncytium formation without affecting glycoprotein synthesis. Excretion of the glycoprotein (G protein) of vesicular stomatitis virus into the medium was blocked. Inaccessibility of the cell-associated viral G protein to protease added extracellularly indicates that cell-surface expression of the viral G protein is blocked by folimycin. The viral G protein accumulated intracellularly in the presence of folimycin electrophoresed a little faster than the control mature one excreted to the medium. Glycopeptides derived from the viral G protein bound to concanavalin A-agarose and were endo H-sensitive. Taken together these results indicate that processing of N-glycosidic oligosaccharide is incomplete in the presence of folimycin and suggest that intracellular trafficking is arrested before reaching at the latest to the trans Golgi compartments.

Animals↗
PubMed2006

Long-range directional movement of an interphase chromosome site.

Increasing evidence suggests functional compartmentalization of interphase nuclei. This includes preferential interior localization of gene-rich and early replicating chromosome regions versus peripheral localization of gene-poor and late replicating chromosome regions , association of some active genes with nuclear speckles or transcription "factories", and association of transcriptionally repressed genes with heterochromatic regions. Dynamic changes in chromosome compartmentalization imply mechanisms for long-range interphase chromatin movements. However, live cell imaging in mammalian cells has revealed limited chromatin mobility, described as "constrained diffusion". None of these studies, though, have examined a chromosome locus undergoing an inducible repositioning between two different nuclear compartments. Here we demonstrate migration of an interphase chromosome site from the nuclear periphery to the interior 1-2 hr after targeting a transcriptional activator to this site. Spot redistribution is perturbed by specific actin or nuclear myosin I mutants. Extended periods of chromosome immobility are interspersed with several minute periods in which chromosomes move unidirectionally along curvilinear paths oriented roughly perpendicular to the nuclear envelope at velocities of 0.1-0.9 microm/min over distances of 1-5 microm. Our results suggest an active mechanism for fast and directed long-range interphase chromosome movements dependent directly or indirectly on actin/myosin.

Actins↗
PubMed2000

Infection by porcine endogenous retrovirus after islet xenotransplantation in SCID mice.

Animal donors such as pigs could provide an alternative source of organs for transplantation. However, the promise of xenotransplantation is offset by the possible public health risk of a cross-species infection. All pigs contain several copies of porcine endogenous retroviruses (PERV), and at least three variants of PERV can infect human cell lines in vitro in co-culture, infectivity and pseudotyping experiments. Thus, if xenotransplantation of pig tissues results in PERV viral replication, there is a risk of spreading and adaptation of this retrovirus to the human host. C-type retroviruses related to PERV are associated with malignancies of haematopoietic lineage cells in their natural hosts. Here we show that pig pancreatic islets produce PERV and can infect human cells in culture. After transplantation into NOD/SCID (non-obese diabetic, severe combined immunodeficiency) mice, we detect ongoing viral expression and several tissue compartments become infected. This is the first evidence that PERV is transcriptionally active and infectious cross-species in vivo after transplantation of pig tissues. These results show that a concern for PERV infection risk associated with pig islet xenotransplantation in immunosuppressed human patients may be justified.

Animals↗
PubMed2025

MIA-Jet: Multi-scale Identification Algorithm of Chromatin Jets.

The mammalian genome is organized into large-scale chromosome territories, compartments, domains, and at the smallest scale, chromatin loops and stripes. The newest element is a chromatin jet, a diffused line perpendicular to the main diagonal in the Hi-C contact map, which was reported in quiescent mammalian lymphocytes supporting a two-sided symmetric cohesin loop extrusion model. A similar structure is observed in Repli-HiC data, where relatively thin and straight chromatin fountains indicate coupling of DNA replication forks. However, the precise biological implications of these jet-like structures are unknown due to the limitations in computational methods. We developed MIA-Jet, a multi-scale ridge detection algorithm that can accurately detect jets of variable lengths, widths, and angles. When tested on Hi-C, Repli-HiC, ChIA-PET, ChIA-Drop, and Micro-C data in mouse, human, roundworm, and zebrafish cells, MIA-Jet outperformed existing methods. In human cells, jets were enriched in cohesin loading sites and early replication initiation zones. Applying MIA-Jet to Hi-C data generated from protein-degraded cells revealed that jets are dependent on cohesin but not YY1, and jet signals are strengthened after depleting WAPL. We envision MIA-Jet to be broadly applicable to any 3D genome mapping data, thereby providing new insights into the functional roles of chromatin jets.

3D genome mapping↗
PubMed2006

Inhibition of protein trafficking by coxsackievirus b3: multiple viral proteins target a single organelle.

Despite replicating to very high titers, coxsackieviruses do not elicit strong CD8 T-cell responses, perhaps because antigen presentation is inhibited by virus-induced disruption of host protein trafficking. Herein, we evaluated the effects of three viral nonstructural proteins (2B, 2BC, and 3A) on intracellular trafficking. All three of these proteins inhibited secretion, to various degrees, and directly associated with the Golgi complex, causing trafficking proteins to accumulate in this compartment. The 3A protein almost completely ablated trafficking and secretion, by moving rapidly to the Golgi, and causing its disruption. Using an alanine-scanning 3A mutant, we show that Golgi targeting and disruption can be uncoupled. Thus, coxsackieviruses rely on the combined effects of several gene products that target a single cellular organelle to successfully block protein secretion during an infection. These findings have implications for viral pathogenesis.

Amino Acid Substitution↗
PubMed2000

Cellular receptor traffic is essential for productive duck hepatitis B virus infection.

We have investigated the mechanism of duck hepatitis B virus (DHBV) entry into susceptible primary duck hepatocytes (PDHs), using mutants of carboxypeptidase D (gp180), a transmembrane protein shown to act as the primary cellular receptor for avian hepatitis B virus uptake. The variant proteins were abundantly produced from recombinant adenoviruses and tested for the potential to functionally outcompete the endogenous wild-type receptor. Overexpression of wild-type gp180 significantly enhanced the efficiency of DHBV infection in PDHs but did not affect ongoing DHBV replication, an observation further supporting gp180 receptor function. A gp180 mutant deficient for endocytosis abolished DHBV infection, indicating endocytosis to be the route of hepadnaviral entry. With further gp180 variants, carrying mutations in the cytoplasmic domain and characterized by an accelerated turnover, the ability of gp180 to function as a DHBV receptor was found to depend on a wild-type-like sorting phenotype which largely avoids transport toward the endolysosomal compartment. Based on these data, we propose a model in which a distinct intracellular DHBV traffic to the endosome, but not beyond, is a prerequisite for completion of viral entry, i.e., for fusion and capsid release. Furthermore, the deletion of the two enzymatically active carboxypeptidase domains of gp180 did not lead to a loss of receptor function.

Adenoviridae↗
PubMed2005

Dynamic cell type specificity of SRC-1 coactivator in modulating uterine progesterone receptor function in mice.

Regulation of gene transcription by the progesterone receptor (PR) in cooperation with coactivator/corepressor complexes coordinates crucial processes in female reproduction. To investigate functional relationships between PR and steroid receptor coactivators (SRCs) in distinct cell types of uterine tissue during gene transcription, we generated a new transgenic mouse model utilizing a Progesterone Receptor Activity Indicator (PRAI) system that could monitor PR activity in vivo. The PRAI system consists of a modified PR bacterial artificial chromosome (BAC) clone in which the DNA binding domain of the PR was replaced with the yeast Gal4 DNA binding domain. A humanized green fluorescent protein (hrGFP) reporter controlled by the Upstream Activating Sequences for the Gal4 gene (UAS(G)) was inserted in tandem with the modified PR gene. Expression of hrGFP in the uterus demonstrated that the PRAI animal model faithfully replicated PR signaling under various endocrine states. Bigenic PRAI-SRC-1(-/-) mice revealed that SRC-1 modulates PR activity in the uterus in a cell-specific fashion and is involved in PR gene activation in stroma and myometrium of the uterus in response to estrogen and progesterone. In contrast, SRC-1 was involved in the down-regulation of PR target gene expression in the luminal and glandular epithelial compartments of the uterus after chronic progesterone treatment. Finally, we dissected the means by which SRC-1 dynamically regulates PR activity in each uterine cell compartment and demonstrated that it involves the differential ability of SRC-1 to modulate expression levels of distinct coactivators, corepressors, and PR in a cell-specific fashion.

Animals↗
PubMed2004

Ex vivo homeostatic proliferation of CD4+ T cells in rheumatoid arthritis is dysregulated and driven by membrane-anchored TNFalpha.

The systemic CD4(+) T cell compartment in patients with rheumatoid arthritis (RA) is characterized by TCR repertoire contraction, shortened telomere lengths, and decreased numbers of recent thymic emigrants, suggesting a disturbed CD4(+) T cell homeostasis. In mice, homeostatic proliferation of peripheral CD4(+) T cells is regulated by TCR interaction with self peptide-MHC complexes (pMHC) and can be reproduced in vitro. We have established an ex vivo model of homeostatic proliferation, in which self-replication of human CD4(+) T cells is induced by cell-cell contact with autologous monocytes. In healthy individuals, blockade of TCR-pMHC class II contact resulted in decreased CD4(+) T cell division. In contrast, homeostatic proliferation in RA patients was not inhibited by pMHC blockade, but increased during the initial culture period. The anti-TNF-alpha Ab cA2 inhibited homeostasis-driven ex vivo proliferation in healthy controls and in RA patients. In addition, treatment of RA patients with infliximab decreased the ex vivo rate of homeostatic proliferation of CD4(+) T cells. Our results suggest a disturbed regulation of CD4(+) T cell homeostasis leading to the repertoire aberrations reported in RA. Membrane-anchored TNF-alpha appears to be a cell-cell contact-dependent stimulus of homeostatic proliferation of CD4(+) T cells, possibly favoring self-replication of autoreactive CD4(+) T cells in patients with RA.

Adult↗
PubMed2006

Resveratrol down-regulates the growth and telomerase activity of breast cancer cells in vitro.

A number of previous studies investigated the in vitro effects of resveratrol on malignant human breast epithelial cell replication. The aim of the present study was to evaluate the activity of resveratrol on human metastatic breast cancer cells. The study was performed on the MCF-7 tumor cell line. Cell growth, cell cycle perturbation and apoptosis were evaluated by trypan blue dye exclusion assay, flow cytometric analysis and confocal fluorescence microscopy. TRAP assay and Western blot analysis respectively detected levels of telomerase activity and levels of hTERT in intracellular compartments of MCF-7 cells treated with resveratrol. Resveratrol has a direct inhibitory effect on cell proliferation. The results demonstrate that the drug induces apoptosis in MCF-7 cells, in a time- and concentration-related manner. Our results also show that the growth-inhibitory effect of resveratrol on malignant cells is mainly due to its ability to induce S-phase arrest and apoptosis in association with reduced levels of telomerase activity. In particular, TRAP assay and Western blot analysis respectively showed that resveratrol treatment down-regulates the telomerase activity of target cells and the nuclear levels of hTERT, the reverse transcriptase subunit of the telomerase complex. In our experimental model of breast cancer, resveratrol shows direct antiproliferative and pro-apoptotic effects. Studies on telomerase function and intracellular hTERT distribution point out that this agent is endowed with additional suppressive functions on critical tumor biological properties. These results speak in favor of a potential role of resveratrol in chemoprevention/chemotherapy of breast cancer.

Antineoplastic Agents, Phytogenic↗
PubMed2002

Toward the engineering of minimal living cells.

The article focuses on the notion of a synthetic or semi-synthetic minimal cell, defined as a system that has the minimal and sufficient structural conditions for cellular life. It is emphasized that two complementary approaches are in principle possible, defined as "bottom-up" and "top-down" approaches. The first one aims at the construction of a minimal cell starting from scratch, and it is argued that a very serious bottle-neck to this pathway lies in the origination of specific macro-molecular sequences, as in nature those were constructed most likely by a particular contingent set of conditions. The top-down approaches utilize extant genes and enzymes, and the work in this case is based on the incorporation of the minimal and sufficient amount of such macromolecules into liposomes, as models for the shell of biological cells. The first phase of this ambitious project foresees the study of conditions under which complex molecular biology reactions takes place in the compartments of liposomes. Examples of these reactions are provided, for example, the production of RNA throughout Q-beta replicase in a self-reproducing vesicle system; or PC Reaction in phospholipid vesicles; or even the incorporation of ribosomes in liposomes, with the production of polypeptide chains. The use of giant vesicles is also illustrated. These systems, due to their large size, offer the advantage that by way of special micro-injection techniques, all sort of biochemical agents can be directly introduced in the compartment; and that the reaction can be followed by optical microscopy. In the final part of the article, the outlook of increasing the complexity of these liposome systems so as to arrive at first semi-synthetic cells is discussed.

Biopolymers↗