To monitor gradual changes in the replication foci distribution during early S phase, different segments of newly synthesized DNA were visualized by immunocytochemical mapping of two consecutively incorporated deoxythymidine analogs in pulse-chase-pulse experiments in HeLa cells. The resulting dual-labeled fluorescence images were evaluated using cross-correlation function (CCF) analysis. General changes of CCF shape due to image deterioration caused by blur, noise, and lateral sampling (pixel size) were also discussed. Using CCF analysis of model images simulating either random initiation of new replication foci, or the firing of new foci in close proximity to completed ones, we were able to ascribe the changes in the early S replication foci distribution to the latter mechanism. In contrast to the data published previously, we monitored the dynamics of all replication foci for up to 3 h. In addition, we showed that the replication foci dynamics is well described by random walk model, so that the average de-localization of individual foci is proportional to square root of the applied chase.

• MeSH
• DNA analysis   MeSH
• Financing, Government MeSH
• Microscopy, Fluorescence MeSH
• Image Interpretation, Computer-Assisted methods   MeSH
• Humans MeSH
• DNA Replication MeSH
• S Phase immunology   MeSH
• Models, Theoretical MeSH
• Check Tag
• Humans MeSH

The intracellular transport of Mason-Pfizer monkey virus (M-PMV) assembled capsids from the pericentriolar region to the plasma membrane (PM) requires trafficking of envelope glycoprotein (Env) to the assembly site via the recycling endosome. However, it is unclear if Env-containing vesicles play a direct role in trafficking capsids to the PM. Using live cell microscopy, we demonstrate, for the first time, anterograde co-transport of Gag and Env. Nocodazole disruption of microtubules had differential effects on Gag and Env trafficking, with pulse-chase assays showing a delayed release of Env-deficient virions. Particle tracking demonstrated an initial loss of linear movement of GFP-tagged capsids and mCherry-tagged Env, followed by renewed movement of Gag but not Env at 4h post-treatment. Thus, while delayed capsid trafficking can occur in the absence of microtubules, efficient anterograde transport of capsids appears to be mediated by microtubule-associated Env-containing vesicles.

• MeSH
• Simian Acquired Immunodeficiency Syndrome metabolism   virology   MeSH
• Cell Membrane virology   MeSH
• Chlorocebus aethiops MeSH
• Gene Products, env genetics   metabolism   MeSH
• Gene Products, gag genetics   metabolism   MeSH
• Macaca mulatta MeSH
• Mason-Pfizer monkey virus genetics   metabolism   MeSH
• Microtubules metabolism   virology   MeSH
• Protein Transport MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Synthesis, import, assembly and turnover of the nuclearly encoded subunits of cytochrome-c oxidase were investigated in cultured human cells depleted of mitochondrial gene products by continuous inhibition of mitochondrial protein synthesis (OP- cells). Immunoprecipitation after pulse labeling demonstrated that the synthesis of the nuclear subunits was not preferentially inhibited, implying that there is no tight regulation in the synthesis of mitochondrial and nuclear subunits of mitochondrial enzyme complexes. Quantitative analysis of the mitochondrial membrane potential in OP- cells indicated that its magnitude was about 30% of that in control cells. This explains the normal import of the nuclearly encoded subunits of cytochrome-c oxidase and other nuclearly encoded mitochondrial proteins into the mitochondria that was found in OP- cells. The turnover rate of nuclear subunits of cytochrome-c oxidase, determined in pulse-chase experiments, showed a specific increase in OP- cells. Moreover, immunoblotting demonstrated that the steady-state levels of nuclear subunits of cytochrome-c oxidase were severely reduced in these cells, in contrast to those of the F1 part of complex V. Native electrophoresis of mitochondrial enzyme complexes showed that assembly of the nuclear subunits of cytochrome-c oxidase did not occur in OP- cells, whereas the (nuclear) subunits of F1 were assembled. The increased turnover of the nuclear subunits of cytochrome-c oxidase in OP- cells is, therefore, most likely due to an increased susceptibility of unassembled subunits to intra-mitochondrial degradation.

• MeSH
• Biological Transport MeSH
• Cell Nucleus * enzymology   MeSH
• Cell Compartmentation MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Mitochondria * enzymology   MeSH
• Gene Expression Regulation, Enzymologic MeSH
• Electron Transport Complex IV chemistry   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

Aerobic anoxygenic phototrophs were recently found to constitute a significant portion of the marine microbial community. These bacteria use bacteriochlorophyll-containing reaction centers to perform photoheterotrophic metabolism. A new instrument for routine measurements of both chlorophyll a and bacteriochlorophyll a was used for monitoring anoxygenic phototrophs in the Baltic Sea in late summer 2003. Bacteriochlorophyll a concentration ranged from 8 to 50 ngl(-1), with an average bacteriochlorophyll/chlorophyll ratio of 4.2 x 10(-3). Moreover, diel trends in bacteriochlorophyll a signals were observed, with a distinct decline occurring during daylight hours. Based on laboratory measurements this phenomenon was ascribed to the complete inhibition of bacteriochlorophyll synthesis by light, which, in combination with a concurrent turnover of the cells, resulted in a pigment decline. Following this explanation, we postulate that bacteriochlorophyll a can serve as a natural 'pulse-and-chase' marker, allowing estimation of the mortality rates of anoxygenic phototrophs from the rates of pigment decline. Based on this assumption, we suggest that the Baltic photoheterotrophic community was characterized by high turnover rates, in a range of 0.7-2 d(-1).

• MeSH
• Aerobiosis MeSH
• Bacteriochlorophyll A metabolism   MeSH
• Bacteriological Techniques methods   instrumentation   MeSH
• Time Factors MeSH
• Circadian Rhythm MeSH
• Financing, Organized MeSH
• Fluorometry instrumentation   MeSH
• Photosynthesis MeSH
• Seawater microbiology   MeSH
• Oceans and Seas MeSH
• Plankton growth & development   MeSH
• Sphingomonadaceae growth & development   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Geographicals
• Oceans and Seas MeSH
• Baltic States MeSH

The mammalian D-type cyclins promote progression through a G1 checkpoint by phosphorylating the retinoblastoma protein (pRB), and can contribute to oncogenesis via their deregulated expression achieved through gene amplification, chromosomal rearrangement, or retroviral integration. We now report a novel mechanism of tumour-associated D-cyclin over-abundance, resulting from enhanced protein stability. In two human cell lines established from a single uterine sarcoma biopsy, pRB-positive SK-UT-1B and pRB-deficient SK-UT-1, aberrant accumulation of functional cyclins D1, and D2 and D3 occurred in the absence of gene amplification and/or elevated mRNA expression. The abundance of D-cyclin proteins remained elevated throughout the cell cycle, and pulse-chase experiments revealed six to 10-fold prolongation of their protein half-lives as compared with either diploid fibroblasts or control U-2-OS sarcoma cells. These results point to a critical regulatory role of D-type cyclin turnover, and contribute to refinement of current views of the role played by the cyclin D-CDK-p16-pRB pathway in cell cycle control and tumorigenesis.

• MeSH
• Enzyme Activation MeSH
• Gene Amplification MeSH
• Cyclin D MeSH
• Cyclin-Dependent Kinase 4 MeSH
• Cyclin-Dependent Kinases metabolism   MeSH
• Cyclins genetics   metabolism   MeSH
• Immunoblotting MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Tumor Cells, Cultured MeSH
• Neoplasm Proteins genetics   metabolism   MeSH
• Uterine Neoplasms genetics   metabolism   MeSH
• Proto-Oncogene Proteins * MeSH
• Retinoblastoma Protein genetics   metabolism   MeSH
• Sarcoma metabolism   MeSH
• Drug Stability MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

According to a general paradigm, proper DNA duplication from each replication origin is ensured by two protein complexes termed replisomes. In prokaryotes and in budding yeast Saccharomyces cerevisiae, these two replisomes seem to be associated with one another until DNA replication initiated from the origin has finished. This arrangement results in the formation of the loop of newly synthesized DNA. However, arrangement of replisomes in other eukaryotic organisms including vertebrate cells is largely unknown. Here, we used in vivo labeling of DNA segments in combination with the electron microscopy tomography to describe the organization of replisomes in human HeLa cells. The experiments were devised in order to distinguish between a model of independent replisomes and a model of replisome couples. The comparative analysis of short segments of replicons labeled in pulse-chase experiments of various length shows that replisomes in HeLa cells are organized into the couples during DNA replication. Moreover, our data enabled to suggest a new model of the organization of replicated DNA. According to this model, replisome couples produce loop with the associated arms in the form of four tightly associated 30nm fibers.

• MeSH
• Bromodeoxyuridine metabolism   MeSH
• Cell Nucleus metabolism   ultrastructure   MeSH
• Chromatin physiology   ultrastructure   MeSH
• Deoxyuracil Nucleotides metabolism   MeSH
• DNA-Directed DNA Polymerase chemistry   metabolism   MeSH
• Financing, Organized MeSH
• HeLa Cells MeSH
• Nucleic Acid Conformation MeSH
• Humans MeSH
• Models, Genetic MeSH
• Multienzyme Complexes chemistry   metabolism   MeSH
• Image Processing, Computer-Assisted MeSH
• DNA Replication physiology   MeSH
• Replicon genetics   MeSH
• Electron Microscope Tomography MeSH
• Check Tag
• Humans MeSH

The cyanobacterium Synechocystis sp. PCC 6803 contains four members of the FtsH protease family. One of these, FtsH (slr0228), has been implicated recently in the repair of photodamaged photosystem II (PSII) complexes. We have demonstrated here, using a combination of blue native PAGE, radiolabeling, and immunoblotting, that FtsH (slr0228) is required for selective replacement of the D1 reaction center subunit in both wild type PSII complexes and in PSII subcomplexes lacking the PSII chlorophyll a-binding subunit CP43. To test whether FtsH (slr0228) has a more general role in protein quality control in vivo, we have studied the synthesis and degradation of PSII subunits in wild type and in defined insertion and missense mutants incapable of proper assembly of the PSII holoenzyme. We discovered that, when the gene encoding FtsH (slr0228) was disrupted in these strains, the overall level of assembly intermediates and unassembled PSII proteins markedly increased. Pulse-chase experiments showed that this was due to reduced rates of degradation in vivo. Importantly, analysis of epitope-tagged and green fluorescent protein-tagged strains revealed that slr0228 was present in the thylakoid and not the cytoplasmic membrane. Overall, our results show that FtsH (slr0228) plays an important role in controlling the removal of PSII subunits from the thylakoid membrane and is not restricted to selective D1 turnover.

• MeSH
• Electrophoresis, Gel, Two-Dimensional MeSH
• Biochemical Phenomena MeSH
• Biochemistry MeSH
• Cell Membrane metabolism   MeSH
• Time Factors MeSH
• Chloroplasts metabolism   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Epitopes chemistry   MeSH
• Financing, Organized MeSH
• Photosystem II Protein Complex metabolism   MeSH
• Genotype MeSH
• Immunoblotting MeSH
• Microscopy, Confocal MeSH
• Metalloendopeptidases physiology   chemistry   MeSH
• Mutation, Missense MeSH
• Mutation MeSH
• Plasmids metabolism   MeSH
• Peptide Hydrolases chemistry   MeSH
• Light MeSH
• Synechocystis metabolism   MeSH
• Thylakoids metabolism   MeSH
• Protein Binding MeSH
• Green Fluorescent Proteins chemistry   metabolism   MeSH

Quantification of carbon (C) fluxes in mycorrhizal plants is one of the important yet little explored tasks of mycorrhizal physiology and ecology. (13)CO2 pulse-chase labelling experiments are increasingly being used to track the fate of C in these plant-microbial symbioses. Nevertheless, continuous monitoring of both the below- and aboveground CO2 emissions remains a challenge, although it is necessary to establish the full C budget of mycorrhizal plants. Here, a novel CO2 collection system is presented which allows assessment of gaseous CO2 emissions (including isotopic composition of their C) from both belowground and shoot compartments. This system then is used to quantify the allocation of recently fixed C in mycorrhizal versus nonmycorrhizal Medicago truncatula plants with comparable biomass and mineral nutrition. Using this system, we confirmed substantially greater belowground C drain in mycorrhizal versus nonmycorrhizal plants, with the belowground CO2 emissions showing large variation because of fluctuating environmental conditions in the glasshouse. Based on the assembled (13)C budget, the C allocation to the mycorrhizal fungus was between 2.3% (increased (13)C allocation to mycorrhizal substrate) and 2.9% (reduction of (13)C allocation to mycorrhizal shoots) of the plant gross photosynthetic production. Although the C allocation to shoot respiration (measured during one night only) did not differ between the mycorrhizal and nonmycorrhizal plants under our experimental conditions, it presented a substantial part (∼10%) of the plant C budget, comparable to the amount of CO2 released belowground. These results advocate quantification of both above- and belowground CO2 emissions in future studies.

• MeSH
• Photosynthesis physiology   MeSH
• Glomeromycota physiology   MeSH
• Plant Roots metabolism   MeSH
• Medicago truncatula metabolism   microbiology   MeSH
• Mycorrhizae metabolism   MeSH
• Carbon Dioxide chemistry   metabolism   MeSH
• Carbon metabolism   MeSH
• Plant Shoots metabolism   MeSH
• Publication type
• Journal Article MeSH

Immature capsids of the Betaretrovirus, Mason-Pfizer Monkey virus (M-PMV), are assembled in the pericentriolar region of the cell, and are then transported to the plasma membrane for budding. Although several studies, utilizing mutagenesis, biochemistry, and immunofluorescence, have defined the role of some viral and host cells factors involved in these processes, they have the disadvantage of population analysis, rather than analyzing individual capsid movement in real time. In this study, we created an M-PMV vector in which the enhanced green fluorescent protein, eGFP, was fused to the carboxyl-terminus of the M-PMV Gag polyprotein, to create a Gag-GFP fusion that could be visualized in live cells. In order to express this fusion protein in the context of an M-PMV proviral backbone, it was necessary to codon-optimize gag, optimize the Kozak sequence preceding the initiating methionine, and mutate an internal methionine codon to one for alanine (M100A) to prevent internal initiation of translation. Co-expression of this pSARM-Gag-GFP-M100A vector with a WT M-PMV provirus resulted in efficient assembly and release of capsids. Results from fixed-cell immunofluorescence and pulse-chase analyses of wild type and mutant Gag-GFP constructs demonstrated comparable intracellular localization and release of capsids to untagged counterparts. Real-time, live-cell visualization and analysis of the GFP-tagged capsids provided strong evidence for a role for microtubules in the intracellular transport of M-PMV capsids. Thus, this M-PMV Gag-GFP vector is a useful tool for identifying novel virus-cell interactions involved in intracellular M-PMV capsid transport in a dynamic, real-time system.

• MeSH
• Biological Transport MeSH
• Cell Membrane metabolism   MeSH
• Fluorescent Dyes metabolism   MeSH
• Genetic Vectors genetics   MeSH
• Gene Products, gag genetics   metabolism   MeSH
• HEK293 Cells MeSH
• Capsid metabolism   MeSH
• Kinetics MeSH
• Humans MeSH
• Mason-Pfizer monkey virus genetics   metabolism   physiology   MeSH
• Microtubules metabolism   virology   MeSH
• Molecular Imaging MeSH
• Movement MeSH
• Proviruses genetics   metabolism   physiology   MeSH
• Recombinant Fusion Proteins genetics   metabolism   MeSH
• Virus Assembly MeSH
• Protein Transport MeSH
• Cell Survival MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Thylakoid biogenesis is an intricate process requiring accurate and timely assembly of proteins, pigments and other cofactors into functional, photosynthetically competent membranes. PSII assembly is studied in particular as its core protein, D1, is very susceptible to photodamage and has a high turnover rate, particularly in high light. PSII assembly is a modular process, with assembly steps proceeding in a specific order. Using aqueous two-phase partitioning to separate plasma membranes (PM) and thylakoid membranes (TM), we studied the subcellular localization of the early assembly steps for PSII biogenesis in a Synechocystis sp. PCC6803 cyanobacterium strain lacking the CP47 antenna. This strain accumulates the early D1-D2 assembly complex which was localized in TM along with associated PSII assembly factors. We also followed insertion and processing of the D1 precursor (pD1) by radioactive pulse-chase labeling. D1 is inserted into the membrane with a C-terminal extension which requires cleavage by a specific protease, the C-terminal processing protease (CtpA), to allow subsequent assembly of the oxygen-evolving complex. pD1 insertion as well as its conversion to mature D1 under various light conditions was seen only in the TM. Epitope-tagged CtpA was also localized in the same membrane, providing further support for the thylakoid location of pD1 processing. However, Vipp1 and PratA, two proteins suggested to be part of the so-called 'thylakoid centers', were found to associate with the PM. Together, these results suggest that early PSII assembly steps occur in TM or specific areas derived from them, with interaction with PM needed for efficient PSII and thylakoid biogenesis.

• MeSH
• Bacterial Proteins metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Photosynthesis radiation effects   MeSH
• Photosystem II Protein Complex metabolism   MeSH
• Light MeSH
• Synechocystis metabolism   radiation effects   MeSH
• Thylakoids metabolism   radiation effects   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH