Human herpesvirus 6 infection arrests cord blood mononuclear cells in G(2) phase of the cell cycle

We here report that after infection with human herpesvirus 6A, human cord blood mononuclear cells accumulate in G₂/M phase of the cell cycle. Experiments with foscarnet or ultraviolet (UV)-irradiated virus stocks pointed at an (immediate-)early, newly formed viral protein to be responsible for the arrest. At the molecular level, p53, cyclin B₁, cyclin A and tyrosine¹⁵-phosphorylated cdk1 accumulated after HHV-6A infection, indicating an arrest in G₂. However, no change was observed in the levels of downstream effectors of p53 in establishing a G₂ arrest, i.e. p21 and 14-3-3σ. We thus conclude that the HHV-6A-induced G₂ arrest occurs independently of p53 accumulation.     

Dehydroascorbate uptake activity correlates with cell growth and cell division of tobacco bright yellow-2 cell cultures

Recently, ascorbate (ASC) concentration and the activity of a number of enzymes from the ASC metabolism have been proven to correlate with differences in growth or cell cycle progression. Here, a possible correlation between growth and the activity of a plasma membrane dehydroascorbate (DHA) transporter was investigated. Protoplasts were isolated from a tobacco (Nicotiana tabacum) Bright Yellow-2 cell culture at different intervals after inoculation and the activity of DHA transport was tested with (14)C-labeled ASC. Ferricyanide (1 mM) or dithiothreitol (1 mM) was included in the test to keep the external (14)C-ASC in its oxidized respectively reduced form. Differential uptake activity was observed, correlating with growth phases of the cell culture. Uptake of DHA in cells showed a peak in exponential growth phase, whereas uptake in the presence of dithiothreitol did not. The enhanced DHA uptake was not due to higher endogenous ASC levels that are normally present in exponential phase because preloading of protoplasts of different ages did not affect DHA uptake. Preloading was achieved by incubating cells before protoplastation for 4 h in a medium supplemented with 1 mM DHA. In addition to testing cells at different growth phases, uptake of DHA into the cells was also followed during the cell cycle. An increase in uptake activity was observed during M phase and the M/G1 transition. These experiments are the first to show that DHA transport activity into plant cells differs with cell growth. The relevance of the data to the action of DHA and ASC in cell growth will be discussed.     

Fatty acid synthase drives the synthesis of phospholipids partitioning into detergent-resistant membrane microdomains

Fatty acid synthase (FAS) is a key metabolic enzyme catalyzing the synthesis of long-chain saturated fatty acids. It plays a central role in the production of surfactant in fetal lungs, in the supply of fatty components of milk, and in the conversion and storage of energy in liver and adipose tissue. Remarkably high levels of FAS expression are found in the majority of human epithelial cancers. As the role of FAS in cancer cells remains largely unknown, we have initiated studies to assess the fate of newly synthesized lipids in cancer cells and have estimated the contribution of FAS to the synthesis of specific lipid classes by treating the cells with small interfering RNAs targeting FAS. Here, we show that in cancer cells FAS plays a major role in the synthesis of phospholipids partitioning into detergent-resistant membrane microdomains. These are raft-aggregates implicated in key cellular processes including signal transduction, intracellular trafficking, cell polarization, and cell migration. These findings reveal a novel role for FAS, provide important new insights into the otherwise poorly understood mechanisms underlying the control of lipid composition of membrane microdomains, and point to a link between FAS overexpression and dysregulation of membrane composition and functioning in tumor cells.     

Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry

The applicability of LC–MS/MS in precursor ion scan mode for the detection of urinary stanozolol metabolites has been studied. The product ion at m/z 81 has been selected as specific for stanozolol metabolites without a modification in A- or N-rings and the product ions at m/z 97 and 145 for the metabolites hydroxylated in the N-ring and 4-hydroxy-stanozolol metabolites, respectively. Under these conditions, the parent drug and up to 15 metabolites were found in a positive doping test sample. The study of a sample from a chimeric uPA-SCID mouse collected after the administration of stanozolol revealed the presence of 4 additional metabolites. The information obtained from the product ion spectra was used to develop a SRM method for the detection of 19 compounds. This SRM method was applied to several doping positive samples. All the metabolites were detected in both the uPA-SCID mouse sample and positive human samples and were not detected in none of the blank samples tested; confirming the metabolic nature of all the detected compounds. In addition, the application of the SRM method to a single human excretion study revealed that one of the metabolites (4ξ,16ξ-dihydroxy-stanozolol) could be detected in negative ionization mode for a longer period than those commonly used in the screening for stanozolol misuse (3′-hydroxy-stanozolol, 16β-hydroxy-stanozolol and 4β-hydroxy-stanozolol) in doping analysis. The application of the developed approach to several positive doping samples confirmed the usefulness of this metabolite for the screening of stanozolol misuse. Finally, a tentative structure for each detected metabolite has been proposed based on the product ion spectra measured with accurate masses using UPLC–QTOF MS.     

High levels of human immunodeficiency virus infection of CD8 lymphocytes expressing CD4 in vivo

Human immunodeficiency virus (HIV)-infected CD8 lymphocytes have been reported in vivo, but the mechanism of infection remains unclear. Experiments using the thy/hu mouse model support export of intrathymically infected CD8 precursors, while recent in vitro data suggest that mature CD8 lymphocytes upregulate CD4 upon activation (generating a CD8bright CD4dim phenotype) and are susceptible to HIV infection. To determine whether these mechanisms operate in vivo and to assess their relative importance in the generation of circulating HIV-infected CD8 lymphocytes, we quantified HIV long terminal repeat (LTR) DNA in CD8+ CD4- and CD8bright CD4dim lymphocytes isolated from HIV-infected individuals by fluorescence-activated cell sorting. HIV infection of CD8 lymphocytes was demonstrated in 17 of 19 subjects, with a significant inverse relationship between level of infection and CD4 lymphocyte count (R = -0.73; P < 0.001). The level of HIV infection of CD8bright CD4dim lymphocytes was significantly higher (median, 1,730 HIV LTR copies/10(6) cells; n = 9) than that of CD8+ CD4- lymphocytes (undetectable in seven of nine individuals; P < 0.01) and approached that of CD4 lymphocytes from the same individuals (median, 3,660 HIV LTR copies/10(6) cells). CD8bright CD4dim lymphocytes represented 0.8 to 3.3% of total CD8 lymphocytes and were most prevalent in the memory subset. Thus, HIV-infected CD8 lymphocytes commonly circulate in HIV-infected individuals and are generated through infection of activated CD8 lymphocytes rather than through export of intrathymically infected precursors. The high level of infection of CD8bright CD4dim lymphocytes could have a direct role in the decline in CD8 lymphocyte function that accompanies HIV disease progression.     

Control of gene expression and assembly of chromosomal subdomains by chromatin regulators with antagonistic functions

Epigenetic regulation of higher-order chromatin structure controls gene expression and the assembly of chromosomal domains during cell division, differentiation, and development. The proposed “histone code” integrates a complex system of histone modifications and chromosomal proteins that establish and maintain distinctive types of chromatin, such as euchromatin, heterochromatin, and centromeric (CEN) chromatin. The reversible nature of histone acetylation, phosphorylation, and (most recently discovered) methylation are mechanisms for controlling gene expression and partitioning the genome into functional domains. Many different regions of the genome contain similar epigenetic marks (histone modifications), raising the question as to how they are independently specified and regulated. In this review, we will focus on several recent discoveries in chromatin and chromosome biology: (1) identification of long-elusive histone “de-methylating” enzymes that affect chromatin structure, and (2) assembly and maintenance of chromatin domains, specifically heterochromatin and euchromatin, through a dynamic equilibrium of modifying enzymes, histone modifications, and histone variants identified biochemically and genetically. Review related to the 15th International Chromosome Conference (ICC XV), held in September 2004, Brunel University, London, UK     

The application of databases and PCR in the cloning of glycosidase genes from the protozoan Tritrichomonas foetus

Conserved sequence amplification (CSA) has been used to obtain sequence data for two glycosidase genes from the primitive eukaryote Tritrichomonas foetus. Few genes have been cloned from this organism, and there is little information concerning protein sequence. CSA is reliant on the use of database searches to identify short sequences of 3–9 amino acids conserved within a protein across a wide range of species. PCR primers are then constructed based on this sequence data and the DNA is amplified and sequenced. In the case of the β-galactosidase gene, N-terminal amino acid sequence data were used to construct a primer that replaced the upstream primer to ensure the amplified product was related to β-d galactosidase CSA was also applied to the gene encoding the enzyme β-N-acetyl-d-glucosaminidase from T. foetus, but in this case a segment of DNA was amplified, which, if correct, should contain a third conserved motif. The products of the CSA were sequenced, and the data obtained were compared to data in the SwissProt database. The results obtained suggest that this approach is useful for the cloning of genes to obtain novel sequence data from organisms where little genetic information is available.