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PubMed2001

Mobile factories: Golgi dynamics in plant cells.

The plant Golgi apparatus plays a central role in the synthesis of cell wall material and the modification and sorting of proteins destined for the cell surface and vacuoles. Earlier perceptions of this organelle were shaped by static transmission electron micrographs and by its biosynthetic functions. However, it has become increasingly clear that many Golgi activities can only be understood in the context of its dynamic organization. Significant new insights have been gained recently into the molecules that mediate this dynamic behavior, and how this machinery differs between plants and animals or yeast. Most notable is the discovery that plant Golgi stacks can actively move through the cytoplasm along actin filaments, an observation that has major implications for trafficking to, through and from this organelle.

Biological Transport↗

PubMed2000

[Magnetic resonance tomography of the brain in workers with chronic occupational manganese dioxide exposure].

AIM: Changes within the brain detected by MRI after chronic manganese poisoning raised the question whether morphological changes of the basal ganglia, particularly of the globus pallidus, could be detected after chronic occupational exposure to manganese dioxide. METHOD: In a cross-sectional study, healthy workers (48 male and 27 female) at a dry cell battery factory were examined. Actual internal exposure was quantified by the analysis of manganese in the blood using atomic absorption spectrometry. Chronic exposure was defined as a cumulative index (CBI) including duration of exposure, individual workplace factors, and previously measured concentrations of MnO2 in dust samples. A Philips Gyroscan T5-II (0.5 T) was used for the MRI of the brain. The following indicators were taken to ascertain possible manganese-induced changes; Pallidum-Index (PI), width of 3rd ventricle and cella media index in addition to clinical examinations. RESULTS: No cases of parkinsonism were detected in clinical examinations or by other means. The mean manganese concentration in blood was 12 micrograms/l (range: 3.9-23.3 micrograms/l). In comparison to the upper reference value of 10 micrograms/l, 42 workers (56%) had a higher body burden. A significant positive correlation between manganese levels in blood and the PI (indicated by T1-shortening) was observed as well as between the CBI and workplace-specific exposure. Brain atrophy was not detected in any of the observed cases. CONCLUSIONS: Long-term exposure to manganese dioxide dust correlates with the Pallidum-Index in MRI scans. Although the MRI findings have no current clinical relevance for individuals, further studies are necessary to evaluate specificity and potential prognostic value.

Adult↗

PubMed1997

Genetic immunization: a new era in vaccines and immune therapeutics.

DNA inoculation represents a novel approach to vaccine and immune therapeutic development. The direct injection of gene expression cassettes into a living host transforms a number of cells into factories for production of the introduced gene products. Expression of these delivered genes has important immunological consequences and may result in the specific immune activation of the host against the novel expressed antigens. The recent demonstration by laboratories that these immune responses are protective in some infectious disease experimental models as well as cancers is viewed with cautious optimism. Further, the relatively short development times, ease of large-scale production, low development, manufacturing, and distribution costs all combine with immunological effectiveness to suggest that this technology will dramatically influence the production of a new generation of experimental vaccines and immune therapies. It is hoped that DNA inoculation will ultimately lead to new vaccines that are immunologically effective and economically accessible to all nations.

Animals↗

PubMed1989

Host-vector systems.

In 1980 it was only possible to express foreign genes in bacteria and a few easily cultured animal cells. During the subsequent eight years specialized vectors have been developed to allow the genetic manipulation of a wide range of both prokaryotes and eukaryotes. One of the major goals of biotechnology in 1980 was to use host cells as 'factories' for the production of proteins that were only available in minute quantities from natural sources. This has already lead to a new generation of pharmaceutical products. Advances in our understanding of host-vector systems have defined new goals. The basic concepts of expression vector design will be illustrated. Some of the new goals are discussed with particular reference to the exploitation of novel host-vector systems to develop vaccines and anti-viral agents against AIDS.

Cloning, Molecular↗

PubMed2001

The effect of formulation and concentration of cholesteryl butyrate solid lipid nanospheres (SLN) on NIH-H460 cell proliferation.

Experimental factorial design was used to evaluate the influence of two factors involved in producing cholesteryl butyrate (chol-but) solid lipid nanospheres (SLN), microemulsion formulation and microemulsion/water ratio, on the effect of the SLN on the proliferation of NIH-H460, a non-small-cell lung carcinoma; six experimental settings were tested. The cells were treated with scalar concentrations of cholesteryl butyrate (from 0.008 to 1.000 mM) for each experimental condition; NIH-H460 cell growth was inhibited in all cases. The best experimental setting provided complete inhibition at 0.125 mM chol-but, while at the same concentration sodium butyrate provided only 38% inhibition.

Butyric Acid↗

PubMed2006

Live-cell imaging reveals replication of individual replicons in eukaryotic replication factories.

Faithful DNA replication ensures genetic integrity in eukaryotic cells, but it is still obscure how replication is organized in space and time within the nucleus. Using timelapse microscopy, we have developed a new assay to analyze the dynamics of DNA replication both spatially and temporally in individual Saccharomyces cerevisiae cells. This allowed us to visualize replication factories, nuclear foci consisting of replication proteins where the bulk of DNA synthesis occurs. We show that the formation of replication factories is a consequence of DNA replication itself. Our analyses of replication at specific DNA sequences support a long-standing hypothesis that sister replication forks generated from the same origin stay associated with each other within a replication factory while the entire replicon is replicated. This assay system allows replication to be studied at extremely high temporal resolution in individual cells, thereby opening a window into how replication dynamics vary from cell to cell.

Chromosomes, Fungal↗

PubMed2004

Reovirus nonstructural protein mu NS recruits viral core surface proteins and entering core particles to factory-like inclusions.

Mammalian reoviruses are thought to assemble and replicate within cytoplasmic, nonmembranous structures called viral factories. The viral nonstructural protein mu NS forms factory-like globular inclusions when expressed in the absence of other viral proteins and binds to the surfaces of the viral core particles in vitro. Given these previous observations, we hypothesized that one or more of the core surface proteins may be recruited to viral factories through specific associations with mu NS. We found that all three of these proteins--lambda 1, lambda 2, and sigma 2--localized to factories in infected cells but were diffusely distributed through the cytoplasm and nucleus when each was separately expressed in the absence of other viral proteins. When separately coexpressed with mu NS, on the other hand, each core surface protein colocalized with mu NS in globular inclusions, supporting the initial hypothesis. We also found that lambda 1, lambda 2, and sigma 2 each localized to filamentous inclusions formed upon the coexpression of mu NS and mu 2, a structurally minor core protein that associates with microtubules. The first 40 residues of mu NS, which are required for association with mu 2 and the RNA-binding nonstructural protein sigma NS, were not required for association with any of the three core surface proteins. When coexpressed with mu 2 in the absence of mu NS, each of the core surface proteins was diffusely distributed and displayed only sporadic, weak associations with mu 2 on filaments. Many of the core particles that entered the cytoplasm of cycloheximide-treated cells following entry and partial uncoating were recruited to inclusions of mu NS that had been preformed in those cells, providing evidence that mu NS can bind to the surfaces of cores in vivo. These findings expand a model for how viral and cellular components are recruited to the viral factories in infected cells and provide further evidence for the central but distinct roles of viral proteins mu NS and mu 2 in this process.

Animals↗

PubMed2001

Characterisation and high-resolution distribution of a matrix attachment region-binding protein (MFP1) in proliferating cells of onion.

The first matrix attachment region (MAR)-binding protein sequenced in plants, MFP1, has been characterised in two dicot species. Based on their antigenic relationship, we report here the conservation of MFP1-like proteins in proliferating root cells of onion (Allium cepa L). Two MFP1-like proteins with different molecular masses and solubilities were detected. The most abundant was a 90-kDa basic protein, presenting several separate spots in two-dimensional blots. The MFP1 was partially soluble and, similar to the proliferating cell nuclear antigen (PCNA)-labelled replication factories in the nucleus and nuclear matrix, was localised at discrete foci as detected by confocal microscopy. High-resolution immunolocalisation of MFP1 by electron microscopy identified the foci as nuclear structures, some of them containing PCNA, which are ultrastructurally similar to the replication factories described in animal cells. Our data provide the first report on MFP1-like proteins in the Alliaceae. In addition, we present evidence of the presence of AcMFP1 in the putative replication factories.

Arabidopsis Proteins↗

PubMed2004

Effect of ammonium chloride on the growth and metabolism of bovine ovarian granulosa cells and the development of ovine oocytes matured in the presence of bovine granulosa cells previously exposed to ammonium chloride.

Three experiments determined first, the effect of increasing ammonium chloride (NH(4)Cl) concentrations on the growth and metabolism of bovine granulosa cells isolated from small and medium-sized bovine ovarian follicles; secondly, whether the changes in granulosa cell growth and metabolism induced by NH(4)Cl were reversible; and thirdly, whether granulosa cells, previously conditioned with NH(4)Cl, were able to support maturation of oocytes in vitro. In Experiment 1, using a 2 (follicle size class) x 5 (NH(4)Cl concentration) factorial design, granulosa cells from small or medium-sized ovarian follicles were incubated for 96 h with 0, 0.2, 0.4, 0.8 or 1.6 micromol NH(4)Cl/ml. Experiment 2 used a split plot factorial design where granulosa cells were incubated for 96 h in the presence or absence of 1 micromol/ml NH(4)Cl and then incubated in the absence or presence of 1 micromol/ml NH(4)Cl for a further 48 h. Finally in Experiment 3, ovine oocytes were matured on layers of bovine granulosa cells which had not been conditioned with NH(4)Cl or conditioned with 0.5 or 1.0 micromol/ml NH(4)Cl and development of embryos to the blastocyst stage followed and blastocyst quality assessed. In Experiment 1, incubation of granulosa cells in increasing concentrations of NH(4)Cl reduced cell growth, increased cell protein concentrations and increased the amounts of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) oxidised and oestradiol and progesterone produced per 10(5) cells. Cells from medium-sized follicles were more sensitive to NH(4)Cl concentration and oxidised more MTT and produced less progesterone at high NH(4)Cl concentrations than cells from small-sized follicles. When, in Experiment 2, NH(4)Cl was removed from cell culture after 96 h incubation, cells previously exposed to NH(4)Cl grew at a slower rate during the subsequent 48 h, contained more cellular protein, oxidised more MTT and produced more oestradiol and progesterone than cells not previously exposed to NH(4)Cl. Maturation of ovine oocytes in coculture with bovine granulosa cells not exposed to NH(4)Cl (Experiment 3) increased egg cleavage rate and the proportion of cleaved eggs which developed to the blastocyst stage. Conditioning of granulosa cells with NH(4)Cl supported egg cleavage and development to the blastocyst stage at rates similar to those observed in the absence of granulosa cells. In conclusion, these experiments showed that the in vitro growth and metabolism of granulosa cells were altered by concentrations of NH(4)Cl similar to ammonium ion concentrations measured in follicular fluid and that these effects were not immediately reversible. Furthermore, the ability of granulosa cells conditioned with NH(4)Cl to support in vitro maturation of oocytes was impaired.

Ammonium Chloride↗

PubMed1979

Epidermal growth factor and the control of proliferation of Balb 3T3 and benzo[a]pyrene-transformed Balb 3T3 cells.

Benzo[a]pyrene-transformed Balb 3T3 cells (BP3T3) exhibit "normal" growth controls at low concentrations of serum. Epidermal growth factor (EGF) stimulates DNA synthesis and cell division in both Balb 3T3 and BP3T3 cells at physiological concentrations. The growth response of BP3T3 cells to EGF is qualitatively the same as that of 3T3 cells, however, the transformed cells have a lower quantitative requirement. Both 3T3 and BP3T3 cells show a density-dependent response to EGF, but the shift in the dose response curve for BP3T3 cells at high cell density is smaller than that seen for 3T3 cells. One cause of the restricted growth of 3T3 cells at high cell density compared with BP3T3 cells is the increased concentration of growth factor needed for stimulation of 3T3 cells at higher cell densities. A lower rate of depletion of other growth factory by BP3T3 cells may also explain the smaller effect of cell density on the EGF response of these cells.

Animals↗

PubMed1992

One-trial backward excitatory fear conditioning transfers across contexts.

One path to human phobia may be one-trial backward fear conditioning. Human phobias transfer readily across contexts. However, animal studies of one-trial backward fear conditioning have yet to demonstrate such transfer. The present study sought to do so. It used a lick-suppression procedure with 84 naive male albino rats. Two conditioning contexts, designated O and V, were crossed factorially with two test contexts, O and V. Within each cell of the factorial design, rats received in the conditioning context either a single 12 sec tone backward paired with a single 4 sec 1 mA shock or the same tone explicitly unpaired with shock. Fear of context and fear of tone were subsequently assessed in terms of the suppression of licking that they evoked. Test results suggested that (1) the rats discriminated between contexts O and V, and (2) despite such discrimination, one-trial backward fear conditioning transferred across the two contexts. The results enhance the plausibility of one-trial backward fear conditioning as a source of human phobia.

Animals↗

PubMed1987

The evaluation of biological interactions using response surface methodology.

Response surface methodology was employed in the statistical analysis of the combination exposures of genotoxic agents, bischloroethylnitrosourea with cis-diamminedichloroplatinum (II) and cis-diamminedichloroplatinum (II) with X rays. The measured endpoint in each case was sister chromatid exchanges in V79 Chinese hamster cells. The combination experiments employed a factorial design in which cells were treated, in various concentration combinations, with two agents simultaneously. Bis-chloroethylnitrosourea and cis-diamminedichloroplatinum (II) each exhibited curvilinear concentration-related increases in sister chromatid exchanges. X rays exhibited a dose-dependent increase in sister chromatid exchanges. For the cis-diamminedichloroplatinum (II)/X ray combinations, response surface methodology indicates a less-than-additive interaction, suggested by the non-parallel concentration-response curves of one agent at varying concentrations of the other, and a slight dose-dependent increase in sister chromatid exchanges due to X rays alone. Both cis-diamminedichloroplatinum and bis-chloroethylnitrosourea exhibited concentration-related increases in sister chromatid exchanges, cis-diamminedichloroplatinum (II) being 8-10 times (dependent on what level of effect was compared) more potent than bis-chloroethylnitrosourea. For the cis-diamminedichloroplatinum (II)/bis-chloroethylnitrosourea combinations, an increasingly less-than-additive interaction was detected. The analysis of these combinations demonstrates the strength of response surface methodology, a collection of mathematical and statistical techniques for detecting, analyzing and describing the biological effects resulting from exposures to multiple cytotoxic agents. The descriptive ability of these procedures is shown to be useful in that it leads to the suggestion of hypotheses regarding mechanisms of action.

Animals↗

PubMed2004

Comparisons of the M1 genome segments and encoded mu2 proteins of different reovirus isolates.

BACKGROUND: The reovirus M1 genome segment encodes the mu2 protein, a structurally minor component of the viral core, which has been identified as a transcriptase cofactor, nucleoside and RNA triphosphatase, and microtubule-binding protein. The mu2 protein is the most poorly understood of the reovirus structural proteins. Genome segment sequences have been reported for 9 of the 10 genome segments for the 3 prototypic reoviruses type 1 Lang (T1L), type 2 Jones (T2J), and type 3 Dearing (T3D), but the M1 genome segment sequences for only T1L and T3D have been previously reported. For this study, we determined the M1 nucleotide and deduced mu2 amino acid sequences for T2J, nine other reovirus field isolates, and various T3D plaque-isolated clones from different laboratories. RESULTS: Determination of the T2J M1 sequence completes the analysis of all ten genome segments of that prototype. The T2J M1 sequence contained a 1 base pair deletion in the 3' non-translated region, compared to the T1L and T3D M1 sequences. The T2J M1 gene showed approximately 80% nucleotide homology, and the encoded mu 2 protein showed approximately 71% amino acid identity, with the T1L and T3D M1 and mu2 sequences, respectively, making the T2J M1 gene and mu2 proteins amongst the most divergent of all reovirus genes and proteins. Comparisons of these newly determined M1 and mu2 sequences with newly determined M1 and mu2 sequences from nine additional field isolates and a variety of laboratory T3D clones identified conserved features and/or regions that provide clues about mu2 structure and function. CONCLUSIONS: The findings suggest a model for the domain organization of mu2 and provide further evidence for a role of mu2 in viral RNA synthesis. The new sequences were also used to explore the basis for M1/mu2-determined differences in the morphology of viral factories in infected cells. The findings confirm the key role of Ser/Pro208 as a prevalent determinant of differences in factory morphology among reovirus isolates and trace the divergence of this residue and its associated phenotype among the different laboratory-specific clones of type 3 Dearing.

Animals↗

PubMed1991

In vitro-matured/in vitro-fertilized bovine oocytes can develop into morulae/blastocysts in chemically defined, protein-free culture media.

Development of bovine embryos derived from in vitro-matured/in vitro-fertilized (IVM/IVF) oocytes was examined in two culture media: hamster embryo culture medium (HECM), a relatively simple, chemically defined, protein-free medium containing 20 amino acids; and tissue culture medium (TCM)-199, a more complex medium designed for culture of somatic cells. The first experiment studied (1) effects of glucose and/or phosphate (Pi) using HECM and (2) the development of bovine IVM/IVF embryos in four different conditions: HECM, TCM-199, TCM-199 + 10% unheated bovine calf serum (BCS), and oviduct cell-conditioned TCM-199 + 10% BCS. After IVF, 45% of the inseminated oocytes developed to the morula/blastocyst stages (M&B) when cultured in HECM; blastocyst development was depressed in the presence of glucose and Pi when compared to Pi alone. When the four culture conditions were compared, there was no significant difference in M&B development (42-51% of inseminated oocytes). However, blastocyst development in TCM-199 supplemented with 10% BCS (29.7%) or with BCS + oviduct cell-conditioned medium (21.6%) was significantly greater than in nonsupplemented HECM (9.7%) or TCM-199 (13.8%). In the second experiment, the effects of serum supplementation and/or oviduct cell conditioning on HECM and TCM-199 were examined in a 2 x 2 x 2 factorial experiment. Oviduct cell conditioning of either HECM or TCM-199 without serum supplementation did not enhance bovine embryo development. Serum supplementation exhibited a biphasic effect, with inhibition at the first cleavage and stimulation of morula compaction and blastocyst formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1999

DNA vaccines: basic mechanism and immune responses (Review).

DNA vaccines raise immune responses by expressing proteins in vaccinated hosts. Responses are raised by nanogram levels of protein expression. Popular methods of DNA delivery include intramuscular (i. m.) injections of DNA in saline and gene gun delivery of DNA-coated gold beads to the epidermis. Professional antigen-presenting cells derived from the bone marrow present DNA-expressed antigens to T-cells. Following gene gun immunizations directly transfected dendritic cells present antigens, whereas following i.m. immunizations both directly transfected dendritic cells and macrophages can present antigen. For both methods of DNA delivery, non-lymphoid cells can serve as factories of antigen for professional antigen presenting cells. Gene gun immunizations depend on antigen expression at the skin target whereas i.m. immunizations are largely independent of DNA expression in the muscle target. For both methods, antigen expression capable of initiating an immune response persists for about one month. intramuscular deliveries of DNA tend to raise type 1 T-cell help for intracellular and plasma membrane antigens but type 2 T-cell help for secreted antigens. Gene gun immunizations tend to raise type 2 T-cell help for both cell-associated and secreted antigens. In mice, DNA-raised immune responses can be equivalent to those raised by viral infections for both the height and longevity of antibody and cytotoxic T-cell responses.

Animals↗

PubMed2006

Development of a human mitochondria-focused cDNA microarray (hMitChip) and validation in skeletal muscle cells: implications for pharmaco- and mitogenomics.

Mitochondrial research has influenced our understanding of human evolution, physiology and pathophysiology. Mitochondria, intracellular organelles widely known as 'energy factories' of the cell, also play fundamental roles in intermediary metabolism, steroid hormone and heme biosyntheses, calcium signaling, generation of radical oxygen species, and apoptosis. Mitochondria possess a distinct DNA (mitochondrial DNA); yet, the vast majority of mitochondrial proteins are encoded by the nuclear DNA. Mitochondria-related genetic defects have been described in a variety of mostly rare, often fatal, primary mitochondrial disorders; furthermore, they are increasingly reported in association with many common morbid conditions, such as cancer, obesity, diabetes and neurodegenerative disorders, although their role remains unclear. This study describes the creation of a human mitochondria-focused cDNA microarray (hMitChip) and its validation in human skeletal muscle cells treated with glucocorticoids. We suggest that hMitChip is a reliable and novel tool that will prove useful for systematically studying the contribution of mitochondrial genomics to human health and disease.

Adolescent↗

PubMed1992

Adipokinetic hormones: cell and molecular biology.

Adipokinetic hormones AKH I (pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2) and AKH II (pGlu-Leu-Asn-Phe-Ser-Trp-Gly-Thr-NH2) are synthesized by neurosecretory cells (NSC) of the corpora cardiaca (CC) in the locust, Schistocerca gregaria. These NSC constitute a homogeneous 'peptide factory' as each cell synthesizes both AKH I and AKH II. This homogeneity makes the CC an excellent system in which to study aspects of neuropeptide biosynthesis. This report summarizes recent findings on AKH inactivation and metabolism, as well as on AKH prohormone processing and biosynthesis.

Amino Acid Sequence↗

PubMedDate not supplied

Hybridoma and CHO cell partitioning in aqueous two-phase systems.

The partitioning of mouse/mouse hybridoma cell line BIF6A7, mouse/rat hybridoma PFU-83, and CHO DUKX B11-derived cell line BIC-2 in aqueous two-phase systems (ATPSs) of poly(ethylene glycol) (PEG) and dextran was studied. The partitioning of BIF6A7 was investigated systematically by using a statistical experimental design. The aims were to identify the key factors governing cell partitioning and to select ATPSs with suitable cell partitioning for extractive bioconversions with animal cells. The influence of five factors, i.e., the poly(ethylene glycol) molecular weight (PEG MW), dextran molecular weight (Dx MW), tie-line length (TLL), pH, and the ratio of potassium phosphate to potassium chloride, defined as the fraction KPi/(KPi + KCl), on BIF6A7 cell partitioning was characterized by using a full factorial experimental design. The cell partitioning ranged from complete partitioning into the interface to an almost complete partitioning to the lower phase. In all cases less than 1% of the cells partitioned to the top phase. The potassium phosphate fraction had the largest effect on cell partitioning. Low potassium phosphate fractions increased the proportion of cells in the lower phase. To a lesser extent the other factors also played a role in the cell partitioning. The best partitioning for the BIF6A7 cell line was obtained in ATPSs with PEG MW = 35,000, Dx MW = 40,000, TLL = 0.10 g/g, pH 7.4, and KPi/(KPi + KCl) = 0.1, where 93% of the cells were present in the lower phase. The previously reported partitioning of BIF6A7 cells in ATPS culture medium, corresponded well with the current findings. The partitioning of mouse/rat hybridoma cell line PFU-83 and CHO cell line BIC-2 was studied in an ATPS culture medium with PEG 35,000, dextran 40,000, TLL = 0.12 g/g, and hybridoma culture medium. Both cell lines partitioned almost completely into the lower phase. Moreover, the PFU-83 cell line was able to grow in the ATPS hybridoma culture medium. This ATPS hybridoma culture medium therefore seems to be suitable for extractive bioconversions with a wide range of hybridoma cell lines, provided that their product can be partitioned into the upper PEG-rich phase.

Animals↗