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At least 289 records · Page 16Linked to original sources
PubMed2000

Stability of DNA triplexes on shuttle vector plasmids in the replication pool in mammalian cells.

Triple helix-forming oligonucleotides may be useful as gene-targeting reagents in vivo, for applications such as gene knockout. One important property of these complexes is their often remarkable stability, as demonstrated in solution and in cells following transfection. Although encouraging, these measurements do not necessarily report triplex stability in cellular compartments that support DNA functions such as replication and mutagenesis. We have devised a shuttle vector plasmid assay that reports the stability of triplexes on DNA that undergoes replication and mutagenesis. The assay is based on plasmids with novel variant supF tRNA genes containing embedded sequences for triplex formation and psoralen cross-linking. Triple helix-forming oligonucleotides were linked to psoralen and used to form triplexes on the plasmids. At various times after introduction into cells, the psoralen was activated by exposure to long wave ultraviolet light (UVA). After time for replication and mutagenesis, progeny plasmids were recovered and the frequency of plasmids with mutations in the supF gene determined. Site-specific mutagenesis by psoralen cross-links was dependent on precise placement of the psoralen by the triple helix-forming oligonucleotide at the time of UVA treatment. The results indicated that both pyrimidine and purine motif triplexes were much less stable on replicated DNA than on DNA in vitro or in total transfected DNA. Incubation of cells with amidoanthraquinone-based triplex stabilizing compounds enhanced the stability of the pyrimidine triplex.

Animals↗
PubMed1985

Seventh-Day Adventist vegetarians have a quiescent proliferative activity in colonic mucosa.

The proliferation of epithelial cells in colonic mucosa was studied in humans at varying degrees of risk for colon cancer. Seventh-Day Adventist vegetarians, known to have significantly lower mortality from colon cancer than the general U.S. population, had the most quiescent proliferative activity of mucosal epithelial cells. Increased replication and expansion of the proliferative compartment accompanied increased colon cancer risk. The analytical methods of this study may be useful in assessing the influence of dietary components involved in the initiation, promotion or inhibition of colon cancer, and in developing strategies for nutritional intervention.

Adult↗
PubMed2000

Bacterial replication in the host cell cytosol.

Intracellular bacteria in mammalian host cells can either live in a membrane-bound vacuole modified to support bacterial growth, or escape from the primary phagosome into the host cell cytoplasm. Phagosomal escape is best studied in Listeria monocytogenes in which a pore-forming cytolysin and two phospholipases are involved in the lysis of the phagosomal membrane. The mechanisms of and requirements for cytoplasmic growth are less clear but there is growing evidence that proficient replication of bacteria in the cytoplasmic compartment requires specific bacterial and cellular preconditions.

Animals↗
PubMed2001

Transplantation hematopoiesis.

Hematopoietic reconstitution after transplantation relies on a small number of hematopoietic stem cells that selectively migrate from the bloodstream to the hematopoietic microenvironment. In the first phase of engraftment, an extreme proliferative demand causes a limited stem cell fraction and its progeny to divide continuously, showing a significant telomere shortening. When all hematopoietic compartments are fully reconstituted, progenitor cell replication stabilizes and the stem cell pool reverts to a quiescent state. In transplant recipients, the hematopoietic microenvironment and stem cell reservoir remain defective for a prolonged period. However, in most patients donor-derived polyclonal hemopoiesis results, and the system is not exhausted but is capable of sustained normal counts and eventually overcomes stressed conditions.

Animals↗
PubMed1998

Examination of determinants for intranuclear localization and transactivation within the RING finger of herpes simplex virus type 1 IE110k protein.

The herpesvirus regulatory protein IE110k possesses a cysteine-rich, RING finger motif required for its role in transactivation and virus replication. IE110k also localizes to subnuclear compartments termed PODs (PML oncogenic domains). Localization to PODs induces redistribution of the proteins associated with this nuclear compartment, including the cellular RING finger protein, PML. Here we construct a series of deletions, RING domain swaps and point mutations to analyse specific requirements within the IE110k RING finger for subnuclear localization, redistribution of PML and transactivation and we examine the relationship between these activities. We find that IE110k localizes to distinct nuclear subdomains that are more numerous than the cellular PODs and that mutation of two residues within a predicted loop of the RING finger, or replacing the IE110k RING finger with a RING finger from a cellular gene abrogates the ability of IE110k to localize to these extra compartments and traps IE110k in the original PODs. We further demonstrate that RING fingers from the cellular genes mdm-2 and Bmi I, when placed within IE110k, alter the nuclear distribution of IE110k, do not transactivate, and do not redistribute PML. We also demonstrate that the majority of wild-type IE110k, like PML, is associated with the nuclear matrix. Although substitutions and deletions within the RING finger abolish transactivation, these mutant proteins remain tightly associated with the matrix. These results further dissect the determinants involved in different aspects of nuclear compartmentalization of IE110k and are discussed in relation to PML, PODs and the IE110k RING finger.

Amino Acid Sequence↗
PubMed2005

Balamuthia mandrillaris, free-living ameba and opportunistic agent of encephalitis, is a potential host for Legionella pneumophila bacteria.

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of granulomatous encephalitis in humans and other mammalian species. Other free-living amebas, such as Acanthamoeba and Hartmannella, can provide a niche for intracellular survival of bacteria, including the causative agent of Legionnaires' disease, Legionella pneumophila. Infection of amebas by L. pneumophila enhances the bacterial infectivity for mammalian cells and lung tissues. Likewise, the pathogenicity of amebas may be enhanced when they host bacteria. So far, the colonization of B. mandrillaris by bacteria has not been convincingly shown. In this study, we investigated whether this ameba could host L. pneumophila bacteria. Our experiments showed that L. pneumophila could initiate uptake by B. mandrillaris and could replicate within the ameba about 4 to 5 log cycles from 24 to 72 h after infection. On the other hand, a dotA mutant, known to be unable to propagate in Acanthamoeba castellanii, also did not replicate within B. mandrillaris. Approaching completion of the intracellular cycle, L. pneumophila wild-type bacteria were able to destroy their ameboid hosts. Observations by light microscopy paralleled our quantitative data and revealed the rounding, collapse, clumping, and complete destruction of the infected amebas. Electron microscopic studies unveiled the replication of the bacteria in a compartment surrounded by a structure resembling rough endoplasmic reticulum. The course of intracellular infection, the degree of bacterial multiplication, and the ultrastructural features of a L. pneumophila-infected B. mandrillaris ameba resembled those described for other amebas hosting Legionella bacteria. We hence speculate that B. mandrillaris might serve as a host for bacteria in its natural environment.

Amoeba↗
PubMed1996

Ability of Escherichia coli isolates that cause meningitis in newborns to invade epithelial and endothelial cells.

Escherichia coli isolates that cause meningitis in newborns are able to invade the circulation and subsequently cross the blood-brain barrier. One mechanism for traversing the blood-brain barrier might involve transcytosis through the endothelial cells. The ability of the meningitis isolate E. coli IHE3034, of serotype 018:K1:H7, to invade epithelial (T24) and endothelial (EA-hy926) cells was investigated by the standard gentamicin survival assay and by electron microscopy. Human bladder epithelial and endothelial cells were efficiently invaded by strain IHE3034, whereas epithelial human colon Caco-2 cells, canine kidney MDCK cells, and the opossum [correction of opposum] epithelial kidney cell line OK were not invaded. The ability to invade human epithelial cells of the bladder could also be demonstrated for several other newborn meningitis E. coli strains and one septicemic E. coli strain. Studies utilizing inhibitors which act on eukaryotic cells revealed a dependence on microfilaments as well as on microtubules in the process of E. coli IHE3034 entry into T24 and EA-hy926 cells. These results indicated that cell cytoskeletal rearrangements are involved in bacterial uptake and suggest that there are either two pathways (microtubule dependent and microfilament dependent) or one complex pathway involving both microtubules and microfilaments. The intracellular IHE3034 organisms were contained in a host-membrane-confined compartment mainly as single microorganisms. Intracellular replication of 1HE3034 was not detected, nor did the number of intracellular bacteria decrease significantly during a 48-h period. The ability of E. coli O18:K1 to invade and survive within certain eukaryotic cells may be another virulence factor of meningitis-associated E. coli.

Actin Cytoskeleton↗
PubMed2001

Brucella suis-impaired specific recognition of phagosomes by lysosomes due to phagosomal membrane modifications.

Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells. We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell. By in vitro reconstitution experiments, we observed a specific association between killed B. suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments.

Animals↗
PubMed2002

Role of cholesterol and the ganglioside GM(1) in entry and short-term survival of Brucella suis in murine macrophages.

Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. We have previously shown that the maturation inhibition of the Brucella-containing phagosome appears to be restricted at the phagosomal membrane, but the precise molecular mechanisms and factors involved in this inhibition have yet to be identified. Interestingly, recent studies have revealed that caveolae or lipid rafts are implicated in the entry of some microorganisms into host cells and mediate an endocytic pathway avoiding fusion with lysosomes. In this study, we investigated the role of cholesterol and the ganglioside GM(1), two components of lipid rafts, in entry and short-term survival of Brucella suis in murine macrophages, by using cholesterol-sequestering (filipin and beta-methyl cyclodextrin) and GM(1)-binding (cholera toxin B) molecules. Our results suggest that lipid rafts may provide a portal for entry of Brucella into murine macrophages under nonopsonic conditions, thus allowing phagosome-lysosome fusion inhibition, and provide further evidence to support the idea that the phagosome maturation inhibition is restricted at the phagosomal membrane.

Animals↗
PubMed2003

Role of the Brucella suis lipopolysaccharide O antigen in phagosomal genesis and in inhibition of phagosome-lysosome fusion in murine macrophages.

Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both cell types. However, the molecular mechanisms and the microbial factors involved are poorly understood. Smooth lipopolysaccharide (LPS) of Brucella has been reported to be an important virulence factor, although its precise role in pathogenesis is not yet clear. In this study, we show that the LPS O side chain is involved in inhibition of the early fusion between Brucella suis-containing phagosomes and lysosomes in murine macrophages. In contrast, the phagosomes containing rough mutants, which fail to express the O antigen, rapidly fuse with lysosomes. In addition, we show that rough mutants do not enter host cells by using lipid rafts, contrary to smooth strains. Thus, we propose that the LPS O chain might be a major factor that governs the early behavior of bacteria inside macrophages.

Animals↗
PubMed2004

Interaction of Chlamydia trachomatis serovar L2 with the host autophagic pathway.

Chlamydiae are obligate intracellular pathogens that replicate within a membrane-bound compartment (the inclusion) and are associated with important human diseases, such as trachoma, pneumonia, and atherosclerosis. We have examined the interaction of the host autophagic pathway with Chlamydia trachomatis serovar L2 by using the specific autophagosomal stain monodansylcadaverine, antibodies to autophagosome-associated markers, and traditionally used autophagic inhibitors, particularly 3-methyladenine and amino acids. Chlamydial inclusions did not sequester monodansylcadaverine, suggesting absence of fusion with autophagosomes. Interestingly, exposure of cultures infected for 19 h to 3-methyladenine or single amino acids until the end of infection (44 h) caused various degrees of abnormalities in the inclusion maturation and in the progeny infectivity. Incubation of host cells with chemicals throughout the entire period of infection modulated the growth of Chlamydia even more dramatically. Remarkably, autophagosomal markers MAP-LC3 and calreticulin were redistributed to the inclusion of Chlamydia, a process that appears to be sensitive to 3-methyladenine and some amino acids. The present data indicate the lack of autophagosomal fusion with the inclusion because it was devoid of monodansylcadaverine and no distinct rim of autophagosomal protein-specific staining around the inclusion could be observed. However, high sensitivity of Chlamydia to conditions that could inhibit host autophagic pathway and the close association of MAP-LC3 and calreticulin with the inclusion membrane still suggest a potential role of host autophagy in the pathogenesis of Chlamydia.

Adenine↗
PubMed2002

Measles-related appendicitis.

Owing to the characteristic Warthin-Finkeldey giant cells found in hyperplastic mucosa-associated lymphoid tissue, it has been emphasized that pathologists can make a diagnosis of measles from appendectomy specimens even in the prodromal stage before diagnostic rashes develop. However, to date, those reported cases of measles-related appendicitis have dealt with the histologic features of the prodromal stage and we found no reports in the English literature describing the histopathologic findings of appendicitis during the full-blown stage of measles. Here, we describe 2 cases of measles-related appendicitis that show contrasting histologic features according to stage, one discovered during the prodromal stage and the other occurring during the full-blown stage. This report describes heretofore unreported histopathologic findings of measles-related appendicitis observed during the full-blown stage of the infection and highlights histopathologic changes caused by replication of the virus in different compartments of the same organ during the course of infection.

Appendicitis↗
PubMed2003

The human cytomegalovirus UL82 gene product (pp71) accelerates progression through the G1 phase of the cell cycle.

As viruses are reliant upon their host cell to serve as proper environments for their replication, many have evolved mechanisms to alter intracellular conditions to suit their own needs. For example, human cytomegalovirus induces quiescent cells to enter the cell cycle and then arrests them in late G(1), before they enter the S phase, a cell cycle compartment that is presumably favorable for viral replication. Here we show that the protein product of the human cytomegalovirus UL82 gene, pp71, can accelerate the movement of cells through the G(1) phase of the cell cycle. This activity would help infected cells reach the late G(1) arrest point sooner and thus may stimulate the infectious cycle. pp71 also induces DNA synthesis in quiescent cells, but a pp71 mutant protein that is unable to induce quiescent cells to enter the cell cycle still retains the ability to accelerate the G(1) phase. Thus, the mechanism through which pp71 accelerates G(1) cell cycle progression appears to be distinct from the one that it employs to induce quiescent cells to exit G(0) and subsequently enter the S phase.

Animals↗
PubMed1989

Replication timing of 10 developmentally regulated genes in Physarum polycephalum.

We have tested the hypothesis which stipulates that only early-replicating genes are capable of expression. Within one cell type of Physarum - the plasmodium - we defined the temporal order of replication of 10 genes which were known to be variably expressed in 4 different developmental stages of the Physarum life cycle. Southern analysis of density-labeled, bromodesoxyuridine-substituted DNA reveals that 4 genes presumably inactive within the plasmodium, were not restricted to any temporal compartment of S-phase: 1 is replicated in early S-phase, 2 in mid S-phase and 1 in late S-phase. On the other hand, 4 out of 6 active genes analysed are duplicated early, with the first 30% of the genome. Surprisingly, the two others active genes are replicated late in S-phase. By gene-dosage analysis, based on quantitation of hybridization signals from early and late replicating genes throughout S-phase, we could pinpoint the replication of one of these two genes at a stage where 80-85% of the genome has duplicated. Our results demonstrate that late replication during S-phase does not preclude gene activity.

Blotting, Southern↗
PubMed2006

Lack of decay of HIV-1 in gut-associated lymphoid tissue reservoirs in maximally suppressed individuals.

Although peripheral blood mononuclear cells (PBMCs) and lymph nodes represent a principal reservoir, the contribution of gut-associated lymphoid tissue (GALT) has not been evaluated. In 15 HIV-1-infected subjects with maximal suppression of HIV replication by highly active antiretroviral therapy, we quantified HIV-1 DNA and RNA in mucosal biopsy specimens, PBMCs, and plasma with ultrasensitive assays. We also calculated compartmental burdens of HIV-1 DNA-positive cells and characterized the temporal decay of these reservoirs in a period of 1 year (with projections to >50 years). HIV-1 RNA was detected in 20% of the subjects' mucosal biopsy specimens and in 80% of the PBMC samples. Mucosal HIV-1 DNA was detected in 80% of the subjects and in 100% of the PBMC samples. Calculated numbers of lymphoid cells containing "potentially replication-competent" HIV-1 DNA showed that the PBMC compartment contained approximately 70,000 such cells, and GALT contained approximately 160,000 cells. Rates of decay slopes for all 15 subjects in both compartments were not statistically significantly different when compared with each other or with zero slope. Our data indicate that GALT is a quantitatively important reservoir of potentially replicative cells containing HIV-1 DNA, harboring at least as many or more of such cells as the PBMC compartment. In well-suppressed patients on highly active antiretroviral therapy, the GALT compartment showed no clear pattern of HIV-1 decay, similar to that in the PBMCs.

DNA, Viral↗
PubMed1998

Stem cell features of benign and malignant prostate epithelial cells.

PURPOSE: We present a new hypothesis suggesting that the different malignant potential of benign prostatic hyperplasia (BPH) and high grade prostatic intraepithelial neoplasia may be explained by distinct alterations in stem cell-like properties. MATERIALS AND METHODS: We used our results and the recent literature to develop this hypothesis in the context of an updated prostate stem cell model. RESULTS: While high grade prostatic intraepithelial neoplasia is a likely precursor lesion to many prostatic adenocarcinomas, BPH rarely if ever progresses directly to carcinoma. Prostate epithelium contains basal and secretory compartments. Secretory cells appear to differentiate from basal cells. Thus, prostatic stem cells most likely reside in the basal compartment. In BPH there is a slight increase in epithelial proliferation, yet most replicating epithelial cells within BPH maintain their normal restriction to the basal compartment. In high grade prostatic intraepithelial neoplasia there is a marked increase in cell proliferation. In contrast to BPH, the majority of proliferating cells in high grade prostatic intraepithelial neoplasia reside in the secretory compartment. The biological significance of this topographic infidelity of proliferation in high grade prostatic intraepithelial neoplasia remains unclear but may relate mechanistically to down regulation of the cyclin dependent kinase inhibitor, p27kip1. Normal basal cells express GSTP1, an enzyme that inactivates reactive electrophiles and organic hydroperoxides, and that may protect cells from deoxyribonucleic acid damaging agents. In contrast, normal secretory cells and high grade prostatic intraepithelial neoplasia cells do not express this enzyme. CONCLUSIONS: We propose that topographic infidelity of proliferation produces a population of secretory cells replicating in the absence of key genome protective mechanisms, thus setting the stage for an accumulation of genomic alterations and instability in high grade prostatic intraepithelial neoplasia. This action occurs along with activation of telomerase, resulting in an immortal clone capable of developing into invasive carcinoma. The model predicts that genome protection remains intact in BPH, minimizing its malignant potential.

Animals↗
PubMed2005

Increased human immunodeficiency virus type 1 (HIV-1) env compartmentalization in the presence of HIV-1-associated dementia.

The human immunodeficiency virus type 1 (HIV-1) surface Env protein has been implicated in the development of HIV-1-associated dementia (HAD). HIV-1 env diversity was analyzed by heteroduplex tracking assay in 27 infected subjects with various neurological statuses. env compartmentalization between the blood and cerebral spinal fluid (CSF) was apparent with all neurological categories. However, in subjects with HAD, significantly more CSF virus was represented by CNS-unique env variants. Variants specialized for replication in the CNS may play a larger role in the development of HAD. Alternatively, HAD may be associated with a more pronounced state of immunosuppression that permits more extensive replication and independent evolution within the CNS compartment.

AIDS Dementia Complex↗
PubMed2005

Intracellular trafficking of Parachlamydia acanthamoebae.

Parachlamydia acanthamoebae is an obligate intracellular bacterium that naturally infects free-living amoebae. It is a potential agent of pneumonia that resists destruction by human macrophages. However, the strategy used by this Chlamydia-like organism in order to resist to macrophage destruction is unknown. We analysed the intracellular trafficking of P. acanthamoebae within monocyte-derived macrophages. Infected cells were immunolabelled for the bacteria and for various intracellular compartments by using specific antibodies. We analysed the bacteria colocalization with the different subcellular compartments by using epifluorescence and confocal microscopy. Bacterial replication took place 4-6 h post infection within acidic vacuoles. At that time, P. acanthamoebae colocalized with Lamp-1, a membrane marker of late endosomal and lysosomal compartments. A transient accumulation of EEA1 15 min post infection, and of rab7 and the mannose 6-phosphate receptor 30 min post infection confirmed that P. acanthamoebae traffic through the endocytic pathway. The acquisition of Lamp-1 was not different after infection with living and heat-inactivated bacteria. However, 24.5% and 79.5% of living and heat-inactivated P. acanthamoebae, respectively, colocalized with the vacuolar proton ATPase. Moreover, P. acanthamoebae did not colocalized with cathepsin D, a lysosomal hydrolase, suggesting that P. acanthamoebae interferes with maturation of its vacuole. Thus, P. acanthamoebae survives to destruction by human macrophages probably by controlling the vacuole biogenesis.

Acanthamoeba↗