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At least 271 records · Page 15Linked to original sources
PubMed2003

T-cell homeostasis in humans with thymic hypoplasia due to chromosome 22q11.2 deletion syndrome.

Patients with chromosome 22q11.2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome) typically exhibit thymic hypoplasia, conotruncal cardiac defects, and hypoparathyroidism. The immunodeficiency that results from the thymic hypoplasia has been extensively described and consists primarily of T-cell lymphopenia. A curious feature of the T-cell lymphopenia is that the age-related rate of decline of T-cell numbers is slower in patients than controls. This leads to T-cell numbers in adulthood that are minimally decreased compared with controls. This suggests that homeostatic mechanisms might be acting to preserve the peripheral blood T-cell numbers in patients. We characterized changes in CD4/CD45RA and CD4/CD45RO T-cell populations in patients and controls of various ages and determined T-cell recombination excision circles and telomere length within the CD4/CD45RA population. Patients had evidence of accelerated conversion of naive to memory cells and had evidence of more extensive replicative history within the CD4/CD45RA compartment compared with controls. Oligoclonal T-cell receptor (TCR) Vbeta families and missing Vbeta families were seen more often in patients than controls. These data are consistent with homeostatic proliferation of T cells in patients with limited T-cell production due to thymic hypoplasia.

Adolescent↗
PubMed1992

Nuclear distribution of PCNA during embryonic development in Xenopus laevis: a reinvestigation of early cell cycles.

The immunocytological distribution of the proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication, has been examined during the early development of Xenopus laevis. The protein is uniformly detected in nuclei during early stages up to the neurula stage. PCNA is detected by its distinctive cyclical pattern during early development, remaining detectable only during the period of S phase of each cell cycle. Immunological detection of PCNA is therefore a useful and specific non-isotopic marker of S-phase cells in the embryo. PCNA associates with typical karyomeric structures, suggesting that DNA replication starts before the nuclear compartment is entirely formed. At the midblastula transition, a new pattern of PCNA staining becomes apparent. First, a new type of PCNA staining is detected at the nuclear periphery. Second, mitotic clusters with different PCNA distributions suggest that the onset of desynchronization of the cell cycle at this stage is not random.

Animals↗
PubMed2004

Current concepts and controversies in prion immunopathology.

Scrapie in sheep and new variant Creutzfeldt-Jakob disease in humans are typically initiated by extracerebral exposure to prions. Both exhibit early prion accumulation in sites of the peripheral lymphoreticular system, such as splenic or lymph nodal germinal centers. In germinal centers, follicular dendritic cells (FDCs), whose development and maintenance depend on lymphotoxin and tumor necrosis factor signaling, are believed to be the main cell type for efficient prion replication in the periphery. Here, we discuss the molecular requirements for prion replication competence in stromal and lymphoid compartments of lymphoid organs. In addition, we examine the preconditions of transepithelial passage of prions in the mucosal-associated lymphoid system. Our results suggest that under specific conditions, efficient prion replication in mesenteric and inguinal lymph nodes is possible in the absence of mature FDCs. M cells are a plausible candidate for the mucosal portal of prion infection.

Animals↗
PubMed2001

Characterization of a late entry event in the replication cycle of human immunodeficiency virus type 2.

Certain human cell lines and primary macrophage cultures are restricted to infection by some primary isolates of human immunodeficiency virus type 2 (HIV-2), although early steps of the viral life cycle such as fusion at the plasma membrane and reverse transcription are fully supported. The late postintegration events, transcription, translation, assembly, budding, and maturation into infectious virions are functional in restrictive cells. Apart from primary macrophages, the restrictive cell types are actively dividing, and nuclear import of preintegration complexes (PICs) is not required for infection. We therefore postulate that the PICs are trapped in a cellular compartment, preventing subsequent steps in the replication cycle that lead to integration of the provirus. To test this we showed that HIV-2 particles pseudotyped with vesicular stomatitis virus envelope G protein, which delivers HIV into an endocytic compartment, could overcome the block to infection. We suggest that delivery of the viral core into an appropriate cellular compartment is a critical step during the entry process of HIV.

Cell-Free System↗
PubMed1986

The stem cell concept revisited: self-renewal capacity is a dynamic property of hemopoietic cells.

A rigid developmental program of stem cell division and progressive maturation into blood cells is challenged. It is proposed that the capacity for self-renewal is not limited to pluripotent stem cells but is shared by committed progenitors and even cells of later compartments. The relative probability of self-replication vs maturation in mitotic cells is controlled by extracellular influences. At the cell population level, the balance between proliferation and maturation and between compartments is regulated by feedback interactions. Inducibility of maturation in response to regulatory signals is smaller at earlier stages; consequently, at steady state primitive cells self-renew while their more differentiated progeny are forced to be transitory. The proposed dynamic linkage between compartments can be destabilized in a number of ways, resulting in defective hemopoiesis or leukemia. At all stages hemopoietic cells are able to change their patterns of gene expression, in an inheritable manner, in response to changes in their microenvironment. In particular, the capacity for self-renewal itself can vary even within a conventionally-defined compartment. On this basis of adaptive differentiation and self-renewal it is possible to account for the progression of chronic myelocytic leukemia and its "blastic conversion"; to analyse the hemopoietic system's response to various physiological and experimental perturbations; and to re-interpret the excessive phenotypic plasticity and apparent "lineage infidelity" manifested by leukemic cells and cell lines.

Animals↗
PubMed1995

Association of Legionella pneumophila with the macrophage endoplasmic reticulum.

Legionella pneumophila replicates within a membrane-bounded compartment that is studded with ribosomes. In this study we investigated whether these ribosomes originate from the cytoplasmic pool or are associated with host endoplasmic reticulum (ER). Immunofluorescence and electron microscopic localization studies of ER proteins in macrophages infected with L. pneumophila indicated that the bacteria reside in a compartment surrounded by ER. An L. pneumophila mutant that grows slowly in macrophages was slow to associate with host ER, providing genetic evidence in support of the hypothesis that this specialized vacuole is required for intracellular bacterial growth. Ultrastructural studies, in which the ER luminal protein BiP was labeled by immunoperoxidase cytochemistry, revealed that L. pneumophila replication vacuoles resemble nascent autophagosomes. Furthermore, short-term amino acid starvation of macrophages, which stimulated host autophagosomes. Furthermore, short-term amino acid starvation of macrophages, which stimulated host autophagy, increased association of the bacteria with the ER and enhanced bacterial growth. These results are compatible with the hypothesis that L. pneumophila exploits the autophagy machinery of macrophages to establish an intracellular niche favorable for replication.

Animals↗
PubMed1998

Clinical implications of HIV dynamics and drug resistance in macrophages.

Macrophages are widely recognized as the second major target of HIV in the body. The cellular characteristics of such resting cells markedly affect the dynamics of virus lifecycle, that is slower but far more prolonged that in lymphocytes. In addition, the limited concentrations of endogenous nucleotide pools in macrophages downregulate the enzymatic activity of reverse transcriptase. As a consequence, both the anti-HIV activity and the development of resistance to antiviral drugs in macrophages are substantially different than those found in activated lymphocytes. These peculiar characteristics of virus replication and efficacy of antiviral drugs in macrophages have a natural in vivo counterpart in extralymphoid tissues, where macrophages account for the majority of cells infected by HIV. Furthermore, the replication of HIV in macrophages of testis and central nervous system is far less affected by antiviral drugs than in lymph nodes, because of the presence of natural barriers that markedly diminish the concentration of such drugs. For all these reasons, HIV infection of macrophages should be taken into account in therapeutic strategies aimed to achieve an optimal therapeutic effect in all tissue compartments where the virus hides and replicates.

Anti-HIV Agents↗
PubMed2001

Microinjection and growth of bacteria in the cytosol of mammalian host cells.

Most facultative intracellular bacteria replicate in specialized phagosomes after being taken up by mammalian cells. Relatively few intracellular bacteria escape the phagosomal compartment with the help of cytolytic (pore-forming) proteins and replicate in the host cell cytosol. Without such toxins, intracellular bacteria cannot reach this cellular compartment. To circumvent the requirement of an "escape" step, we developed a procedure allowing the efficient direct injection of bacteria into the cytosol of mammalian cells. With this technique, we show that most bacteria, including extracellular bacteria and intracellular pathogens that normally reside in a vacuole, are unable to replicate in the cytosol of the mammalian cells. In contrast, microorganisms that replicate in the cytosol, such as Listeria monocytogenes, Shigella flexneri, and, to some extent, enteroinvasive Escherichia coli, are able to multiply in this cellular compartment after microinjection. Further L. monocytogenes with deletion in its PrfA-regulated hpt gene was found to be impaired in replication when injected into the cytosol. Complementation of the hpt mutation with a plasmid carrying the wild-type hpt gene restored the replication ability in the cytosol. These data indicate that cytosolic intracellular pathogens have evolved specific mechanisms to grow in this compartment of mammalian cells.

Animals↗
PubMed2004

Alternate, virus-induced membrane rearrangements support positive-strand RNA virus genome replication.

All positive-strand RNA [(+)RNA] viruses replicate their RNA on intracellular membranes, often in association with spherular invaginations of the target membrane. For brome mosaic virus, we previously showed that such spherules serve as compartments or mini-organelles for RNA replication and that their assembly, structure, and function have similarities to the replicative cores of retrovirus and double-stranded RNA virus virions. Some other (+)RNA viruses conduct RNA replication in association with individual or clustered double-membrane vesicles, appressed double membranes, or other structures whose possible relationships to the spherular invaginations are unclear. Here we show that modulating the relative levels and interactions of brome mosaic virus replication factors 1a and 2a polymerase (2apol) shifted the membrane rearrangements associated with RNA replication from small invaginated spherules to large, karmellae-like, multilayer stacks of appressed double membranes that supported RNA replication as efficiently as spherules. Spherules were induced by expressing 1a, which has functional similarities to retrovirus virion protein Gag, or 1a plus low levels of 2apol. Double-membrane layers were induced by 1a plus higher levels of 2apol and were suppressed by deleting the major 1a-interacting domain from 2apol. The stacked, double-membrane layers alternated with spaces that, like spherule interiors, were 50-60 nm wide, connected to the cytoplasm, and contained 1a and 2apol. These and other results suggest that seemingly diverse membrane rearrangements associated with RNA replication by varied (+)RNA viruses may represent topologically and functionally related structures formed by similar protein-protein and protein-membrane interactions and interconverted by altering the balances among those interactions.

Bromovirus↗
PubMed1991

Altered regulation of growth and expression of differentiation-associated keratins in benign mouse skin tumors.

Alterations in the pattern of epidermal cell differentiation and proliferation in mouse skin and benign skin tumors were studied by two-color immunofluorescence using monospecific antibodies. Replicating cells were identified by 5-bromo-deoxyuridine (BrdU) pulse-labeling and differentiating cells by keratins K1 and K10. In normal mouse skin, pulse-chase experiments for 120 h revealed that replication was restricted to a single layer of basal cells. Replicating cells did not express K1 or K10, but these keratins were sequentially expressed in post-mitotic basal cells 18 and 24 h following DNA synthesis respectively, and cells expressing these keratins migrated into the suprabasal layers. In phorbol-ester- or cantharidin-stimulated hyperplastic skin, replicating cells were also confined to the basal cell compartment and suprabasal cells expressed keratins 1 and 10. In papillomas induced by initiation with 7,12-dimethylbenz[a]anthracene and promotion with 12-O-tetradecanoylphorbol-13-acetate, replication occurred predominantly in cells in an expanded basal cell compartment (two to four layers above the basement membrane). Cells in these basal layers did not express K1 or K10, but more superficial cells did. After a 1 h pulse of BrdU, replication was also identified in suprabasal cells expressing the differentiation-associated keratins. These and other results suggest that benign tumor cells escape the obligatory growth arrest associated with differentiation. Replication of K1- and K10-expressing suprabasal cells may represent an early alteration during mouse skin carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1982

Frog virus 3 DNA replication occurs in two stages.

Viral DNA synthesis in frog virus 3 (FV3)-infected cells occurs both in the nucleus and in the cytoplasm (Goorha et al., Virology 84:32-51, 1978). Relationships between viral DNA molecules synthesized in these two compartments and their role in the virus replication were examined. The data presented here suggest that (i) FV3 DNA replicated in two stages and (ii) nucleus and cytoplasm were the sites of stages 1 and 2 of DNA replication, respectively. Stages 1 and 2 were further distinguished by their temporal appearance during infection and by the sizes of the replicating DNA as determined by sedimentation in neutral sucrose gradients. In stage 1, replicating molecules, between the size of unit and twice the unit length, were produced early in infection (2 h postinfection). In contrast, stage 2 of DNA replication occurred only after 3 h postinfection, and replicating molecules were large concatemers. Results of pulse-chase experiments showed that the concatemeric DNA served as the precursor for the production of mature FV3 DNA. Denaturation of concatemeric DNA with alkali or digestion with S1 nuclease reduced it to less than genome size molecules, indicating the presence of extensive single-stranded regions. Analysis of replicating DNA by equilibrium centrifugation in CsCl gradients after a pulse-chase suggested that these single-stranded regions were subsequently repaired. Based on these and previous data, a scheme of FV3 replication is presented. According to this scheme, FV3 utilizes the nucleus for early transcription and stage 1 of DNA replication. The viral DNA is then transported to the cytoplasm, where it participates in stage 2 DNA replication to form a concatemeric replication complex. The processing of concatemers to produce mature viral DNA and virus assembly also occurs in the cytoplasm. This mode of replication is strikingly different from any other known DNA virus.

Animals↗
PubMed2002

Disruption of the Salmonella-containing vacuole leads to increased replication of Salmonella enterica serovar typhimurium in the cytosol of epithelial cells.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that inhabits a vacuolar compartment, called the Salmonella-containing vacuole (SCV), in infected host cells. Maintenance of the SCV is accomplished by SifA, and mutants of this Salmonella pathogenicity island 2 type III effector replicate more efficiently in epithelial cells. Here we demonstrate that enhanced replication of sifA mutants occurs in the cytosol of these cells. Increased replication of wild-type bacteria was also observed in cells treated with wortmannin or expressing Rab5 Q79L or Rab7 N125I, all of which caused a loss of SCV integrity. Our findings demonstrate the requirement of the host cell endosomal system for maintenance of the SCV and that loss of this compartment allows increased replication of serovar Typhimurium in the cytosol of epithelial cells.

Antigens, CD↗
PubMed1997

Inhibition of replication of HIV in primary monocyte/macrophages by different antiviral drugs and comparative efficacy in lymphocytes.

Several anti-HIV drugs acting on different steps of virus replication were tested in our experimental model of primary monocyte/macrophages; the results were compared with the activity found in lymphocytes. Nucleoside analogues (AZT, ddI, ddC, d4T, PMEA, 3TC etc.) show greater activity in macrophages (M/M) than in lymphocytes. In particular, the EC50 of AZT, ddC, and ddI in M/M is 2- to 100-fold lower than that found in lymphocytes. This greater efficacy of nucleoside analogues in M/M depends on the enhancement of their chain-terminating activity by the low levels of endogenous deoxynucleoside-triphosphates (dNTP) usually found in resting cells such as M/M. Non-nucleoside reverse transcriptase inhibitors (NNRTI) do not act as chain terminators (thus their antiviral effect is not related to the intracellular concentrations of dNTP); as a consequence the activity of TSAO, HEPT, TIBO, and other NNRTI tested in M/M is similar to that found in lymphocytes. Regarding inhibitors of binding and fusion of HIV, we found that their anti-HIV activity is markedly decreased (or even nullified) when M/M are treated with cytokine activators of M/M function and enhancers of HIV replication. More relevant from a clinical standpoint, protease inhibitors are able to inhibit HIV replication in chronically infected macrophages (i.e., cells carrying the proviral genome already integrated in the host genome). All other inhibitors of late stage of virus life cycle tested (antisense-rev, anti-tat, interferon-alpha and -gamma, phosphorothioate analogues, GLQ-223, etc.) were totally inactive in chronically infected macrophages. The different effects of various classes of HIV inhibitors in lymphocytes and macrophages suggests that AIDS therapy should consider all aspects of the pathogenesis of HIV infection and must be restricted to drugs, or combinations of drugs, active against both lymphocytes and M/M in all body compartments where the virus hides and replicates.

Anti-HIV Agents↗
PubMed2003

The escape of the mink embryo from obligate diapause.

The obligate embryonic diapause that characterizes gestation in mink engenders a developmental arrest at the blastocyst stage. The characteristics of escape from obligate diapause were investigated in embryos reactivated by treatment of the dams with exogenous prolactin. Protein and DNA synthesis showed marked increases within 72 h after the reinitiation of development, and embryo diameter increased thereafter. Trophoblast cells from embryos at Day 5 after activation proliferated more readily in vitro than trophoblasts from diapause or from Day 9 after activation, while in Day 9 embryos, cells from the inner cell mass (ICM) replicated comparatively more readily in vitro. There was evidence of expression of fibroblast growth factor-4 (FGF4) in both diapause and activated embryos and in ICM, but not the trophoblast. FGF receptor-2 was present in embryos from Day 5 after reactivation in both trophoblast and ICM cell lines. Trophoblast cell lines established from mink embryos proliferated in culture in the presence of FGF4 with a doubling time of 1.4 days, while in its absence, the doubling time was 4.0 days. We conclude that, during reinitiation of embryogenesis in the mink after diapause, embryo growth is characterized by gradual increases in protein synthesis, accompanied by mitosis of the trophoblast and ICM. There appears to be a pattern of differential proliferation between cells derived from these embryonic compartments, with the trophoblast phase of replication occurring mainly in the early reactivation phase, while the ICM proliferates more rapidly nearer to the time of implantation.

Animals↗
PubMed2005

LidA, a translocated substrate of the Legionella pneumophila type IV secretion system, interferes with the early secretory pathway.

Legionella pneumophila uses a type IV secretion system to deliver effector molecules into the host cell and establish its replication vacuole. In this study, we investigated the role of LidA, a translocated substrate associated with the surface of the L. pneumophila-containing vacuole. LidA is secreted into the host cell throughout the replication cycle of the bacteria and associates with compartments of the early secretory pathway. When overexpressed in mammalian cells or yeast, LidA interferes with the early secretory pathway, probably via a domain predicted to be rich in coiled-coil structure. Finally, during intracellular replication, the replication vacuoles are in close contact with the endoplasmic reticulum-Golgi intermediate compartment and the Golgi apparatus, suggesting a positive correlation between intracellular growth and association of the vacuole with compartments of the early secretory pathway. We propose that LidA is involved in the recruitment of early secretory vesicles to the L. pneumophila-containing vacuole and that the vacuole associates with the secretory pathway to facilitate this process.

Animals↗
PubMed2003

Origins of mitochondrial thymidine triphosphate: dynamic relations to cytosolic pools.

Nuclear and mitochondrial (mt) DNA replication occur within two physically separated compartments and on different time scales. Both require a balanced supply of dNTPs. During S phase, dNTPs for nuclear DNA are synthesized de novo from ribonucleotides and by salvage of thymidine in the cytosol. Mitochondria contain specific kinases for salvage of deoxyribonucleosides that may provide a compartmentalized synthesis of dNTPs. Here we investigate the source of intra-mt thymidine phosphates and their relationship to cytosolic pools by isotope-flow experiments with [3H]thymidine in cultured human and mouse cells by using a rapid method for the clean separation of mt and cytosolic dNTPs. In the absence of the cytosolic thymidine kinase, the cells (i) phosphorylate labeled thymidine exclusively by the intra-mt kinase, (ii) export thymidine phosphates rapidly to the cytosol, and (iii) use the labeled dTTP for nuclear DNA synthesis. The specific radioactivity of dTTP is highly diluted, suggesting that cytosolic de novo synthesis is the major source of mt dTTP. In the presence of cytosolic thymidine kinase dilution is 100-fold less, and mitochondria contain dTTP with high specific radioactivity. The rapid mixing of the cytosolic and mt pools was not expected from earlier data. We propose that in proliferating cells dNTPs for mtDNA come largely from import of cytosolic nucleotides, whereas intra-mt salvage of deoxyribonucleosides provides dNTPs in resting cells. Our results are relevant for an understanding of certain genetic mitochondrial diseases.

Animals↗
PubMed1979

An automaton analogue of unicellularity.

There is presented in outline form an abstract model of a cell in an evolutionary context. The design is based on an elaboration of John Holland's one-dimensional, abstract universe recently posed for the study of the emergence of self-replicating systems. Eight new ingredients constitute the elaboration. They suggest how compartmentation of a set of "molecules" in a one-dimensional universe can be achieved and how a suitable, compartmentalized set of molecules can replicate in a manner analogous to real cell replication, i.e., there is segregation and semi-conservative replication of the genetic material, and there is division of the compartment through the construction of an "inner membrane". The approach to self-replication of a spatially delimited entity exemplified by this design differs from the abstract models of replication of the von Neumann or Laing type. In these the replicating entity constructs a copy external to itself and does not undergo any essential replacement of any of its parts. Also, while in these models the concern is primarily with questions of the "logical" type, our design has in mind features identifiable with both energy and information considerations. Thus, the rules which define the underlying "physics and chemistry" imply that the self-replicating entity is a dissipative structure. A constant flux of energy is required to maintain the system far from thermodynamic equilibrium in order to account for multiple steady states, and hence dynamic structure.

Cell Compartmentation↗
PubMed2004

Recovery of Campylobacter from broiler feces during extended storage of transport cages.

Feces deposited in transport cages by a Campylobacter-positive flock can cause the spread of Campylobacter to subsequent flocks placed in the same cages. This experiment was designed to determine the effect of extended cage storage on the viability of Campylobacter in feces deposited on the cage floor during commercial transport and holding. After 4 h of feed (but not water) withdrawal, Campylobacter-positive broilers were caught by commercial catching crews, placed into 3 new commercial cages and transported with the rest of the flock to the holding area at a commercial processing facility. Broilers were allowed to remain in the cages for 8 h before being unloaded by facility personnel. After removal of the broilers, empty cages were held under a shed and sampled at 7 intervals for the presence of viable Campylobacter. Cages were sampled by removing all the feces out of a different randomly assigned compartment in each cage at 0.5, 2, 4, 6, 8, 24, and 48 h after unloading. No decrease in Campylobacter numbers was noted through 8 h of storage. After 24 h in both replications, Campylobacter was detected in 2 of 3 compartments by direct plating and detected in the third by enrichment only. After 48 h, Campylobacter was detected in one replication by enrichment only, and was not detected in the second replication at all. Storing soiled transport cages for 48 h between uses results in lower numbers of Campylobacter in feces, but may not eliminate Campylobacter entirely. Due to cage cost and space requirements, routine cage storage between uses would not be practical.

Animals↗