Exercise affects the gene expression profiles of human white blood cells.

White blood cells (WBCs) express tens of thousands of genes, whose expression levels are modified by genetic and external factors. The purpose of the present study was to investigate the effects of acute exercise on gene expression profiles (GEPs) of WBCs and to identify suitable genes that may serve as surrogate markers for monitoring exercise and training load. Five male participants performed an exhaustive treadmill test (ET) at 80% of their maximal O(2) uptake (Vo(2 max)) and a moderate treadmill test (MT) at 60% Vo(2 max) for exactly the same time approximately 2 wk later. WBCs were isolated by the erythrocyte lysis method. GEPs were measured using the Affymetrix GeneChip technology. After scaling, normalization, and filtering, groupwise comparisons of gene expression intensities were performed, and several measurements were validated by real-time PCR. We found 450 genes upregulated and 150 downregulated (>1.5-fold change; ANOVA with Benjamini-Hochberg correction, P < 0.05) after ET that were closely associated with the gene ontology lists "response to stress" and "inflammatory response". Analysis of mean expression levels after MT showed that the extent of up- and downregulation was workload dependent. The genes for the stress (heat shock) proteins HSPA1A and HSPH1 and for the matrix metalloproteinase MMP-9 showed the most prominent increases, whereas the YES1 oncogene (YES1) and CD160 (BY55) were most strongly reduced. Despite different methodological approaches used, the consistency of our results with the expression data of another study (Connolly PH, Caiozzo VJ, Zaldivar F, Nemet D, Larson J, Hung SP, Heck JD, Hatfield GW, Cooper DM. J Appl Physiol 97: 1461-1469, 2004) suggests that expression fingerprints are useful tools for monitoring exercise and training loads and thereby help to avoid training-associated health risks.     

Identification of proteins specific for human herpesvirus 6-infected human T cells.

Proteins specific for human herpesvirus 6 (HHV-6)-infected human T cells (HSB-2) were examined by using polyclonal rabbit antibodies and monoclonal antibodies against HHV-6-infected cells and human sera. More than 20 proteins and six glycoproteins specific for HHV-6-infected cells were identified from [35S]methionine- and [3H]glucosamine-labeled total-cell extracts. Polyclonal rabbit antibodies immunoprecipitated 33 [35S]methionine-labeled HHV-6-specific polypeptides with approximate molecular weights ranging from 180,000 to 31,000. In immunoprecipitation and Western immunoblot reactions, a patient's serum also recognized more than 30 HHV-6-specific proteins and seven glycoproteins. In contrast, sera from individuals with high-titered antibodies against other human herpesviruses reacted with fewer HHV-6-infected cell proteins, and only a 135,000-Mr polypeptide was prominent. Monoclonal antibodies to HHV-6-infected cells reacted with single and multiple polypeptides specific for virus-infected cells and immunoprecipitated three distinct sets of glycoproteins, which were designated gp105k and gp82k, gp116k, gp64k, and gp54k, and gp102k.     

Identification of DNA binding proteins from human cells

Eukaryotic DNA binding proteins have been observed indirectly by means of filter-binding assays, mobility shifts on nondenaturing gel electrophoresis, nucleolytic protection studies, and functional analyses. Transacting factors, presumably proteins, are implicated in regulation of gene expression at the promoter and enhancer. The identification of the polypeptide or polypeptides involved in DNA recognition and binding is an important, challenging problem. A general method is presented herein for the identification of proteins that bind DNA, based directly on the property of DNA binding. A nuclear protein extract, fractionated by ion-exchange chromatography, is assayed across the column for binding activity using nondenaturing polyacrylamide gel electrophoresis. Samples of column eluate that display binding activity are then subjected to nondenaturing gel electrophoresis in the presence or absence of substrate DNA. The nondenaturing gel strips are cut out and run orthogonally on discontinuous sodium dodecyl sulfate gels for the identification of proteins. A protein that undergoes a first-dimension mobility shift to the position of DNA bound to protein is the protein that bound the DNA. We have identified a pair of polypeptides from leukemic human cells of apparent molecular weights 70 and 85 kd that bind DNA as a complex.     

Hyperthermia enhances bortezomib-induced apoptosis in human white blood cancer cells

At present, the current therapeutic strategy for apoptosis induction mainly relies on the administration of pharmacological apoptotic modulators. Apart from that, apoptosis can be induced by various external stimuli such as hyperthermia, ionizing radiation, and electric fields. Despite advantages, both physical and pharmacological approaches bear some limitations as well. The rationale of this study was to overcome the limitations by combining hyperthermia and apoptotic modulator ‘bortezomib’ (Velcade). Two types of human blood cancer cell lines were utilized: human leukemic monocyte lymphoma cell U937 line and peripheral blood mononuclear cells (PMBCs) derived from the patient diagnosed with acute myeloid leukemia. Prior to apoptosis experiments, cytotoxicity tests were performed at three types of temperature regimes (40 °, 42 ° and 44 °C). We observed a gradual inhibition of cell viability correlating with an increase of temperature and drug concentration in both cell lines. However, there was no significant difference between sham group and groups of leukemic PMBCs treated by high temperature (44 °C) and bortezomib. In U937 cells, combined treatment by heat shock and bortezomib led to an increase the number of cells underwent the late apoptosis stage. At the same time, similar treatment of PMBCs resulted in the stimulation of early apoptosis. Our data suggest that combination of bortezomib and hyperthermia enhances apoptosis induction in human cancer white blood cells, indicating a therapeutic potential for blood cancer therapy.     

Differential apoptotic response to ionizing radiation in subpopulations of human white blood cells.

The purpose of this paper is to characterize the apoptotic response of various subpopulations of human white blood cells after in vitro exposure to ionizing radiation using the modified neutral comet assay (MNCA). White blood cells, isolated from human whole blood, were fractionated into granulocytes and mononuclear cells which were further separated into B-cells, natural killer (NK) cells, and CD4(+) and CD8(+) T-cells. The separated fractions were exposed to low doses of X-rays and then MNCA was used to measure the apoptotic fraction (AF) at different time points in irradiated and unirradiated aliquots of sorted cultures. The spontaneous AF in unirradiated control cells was the most critical determinant of whether an apoptotic response could be detected in irradiated cells. When cultured in isolation granulocytes and B-cells had the highest background AF, with NK cells having the next highest. CD4(+) and CD8(+) T-cells had a low, stable, spontaneous AF which gave them the highest signal-to-noise ratio. Although B-cells demonstrated the highest radiation-induced apoptotic response to 1Gy of X-rays, CD8(+) T-cells were the most radiation-responsive lymphocytes due to their low spontaneous AF. By generating dose response curves for CD4(+) and CD8(+) T-cells, the sensitivity of the MNCA for detecting apoptosis in these two cell types was also examined.     

Efficient identification of phosphatidylserine-binding proteins by ORF phage display

To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in ∼300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressed for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.     

Time domain dielectric spectroscopy study of human cells. II. Normal and malignant white blood cells.

The dielectric properties of human lymphocyte suspensions were studied by time domain dielectric spectroscopy (TDDS). Nine populations of malignant and normal lymphocytes were investigated. Analysis of the dielectric parameters of cell structural parts were performed in the framework of Maxwell-Wagner mixture formula and the double-shell model of cell. The specific capacitance of the cell membranes was estimated by the Hanai-Asami-Koisumi formula. It was shown that the dielectric permittivity, capacitance and conductivity values of cell membranes are higher for normal lymphocytes than for the malignant ones. The difference of the same parameters for normal B- and T-cells is also discussed.     

DNA methylation in white blood cells

Alterations in DNA methylation patterns, both at specific loci and overall in the genome, have been associated with many different health outcomes. In cancer and other diseases, most of these changes have been observed at the tissue level. Data on whether DNA methylation changes in white blood cells (WBC) can serve as a useful biomarker for different health outcomes are much more limited, but rapidly emerging. Epidemiologic studies have reported associations between global WBC methylation and several different cancers including cancers of the colon, bladder, stomach, breast and head and neck, as well as schizophrenia and myelodysplastic syndrome. Evidence for WBC methylation at specific loci and disease risk is more limited, but increasing. Differences in WBC DNA methylation by selected risk factors including demographic (age, gender, race), environmental exposures (benzene, persistent organic pollutants, lead, arsenic, and air pollution), and other risk factors (cigarette smoke, alcohol drinking, body size, physical activity and diet) have been observed in epidemiologic studies though the patterns are far from consistent. Challenges in inferences from the existing data are primarily due to the cross-sectional and small size of most studies to date as well as the differences in results across assay type and source of DNA. Large, prospective studies will be needed to understand whether changes in risk factors are associated with changes in DNA methylation patterns, and if changes in DNA methylation patterns are associated with changes in disease endpoints.     

In vitro triiodothyronine binding to cytoplasmic proteins from human red blood cells.

In vitro incubations of cytosol proteins from human red blood cells with [¹²⁵I] labelled L-3,5,3′ triiodothyronine demonstrated the existence of high affinity and limited capacity binding sites for T₃. At 4°C, the rate constant of association was 3 × 10⁷ M^?1h^?1, and the rate constant of dissociation was 9.10^?3h^?1. The dissociation constant K_d was calculated from these data or measured by Scatchard analysis and found to be between 3 and 7.10^?10M. The maximum binding capacity was 1.4 f moles of L-3,5,3′ triiodothyronine per mg cytosol proteins. A close parallel between the biological pontency of the analogs of L-T₃ was observed.     

Differential apoptotic response to ionizing radiation in subpopulations of human white blood cells

The purpose of this paper is to characterize the apoptotic response of various subpopulations of human white blood cells after in vitro exposure to ionizing radiation using the modified neutral comet assay (MNCA). White blood cells, isolated from human whole blood, were fractionated into granulocytes and mononuclear cells which were further separated into B-cells, natural killer (NK) cells, and CD4⁺ and CD8⁺ T-cells. The separated fractions were exposed to low doses of X-rays and then MNCA was used to measure the apoptotic fraction (AF) at different time points in irradiated and unirradiated aliquots of sorted cultures. The spontaneous AF in unirradiated control cells was the most critical determinant of whether an apoptotic response could be detected in irradiated cells. When cultured in isolation granulocytes and B-cells had the highest background AF, with NK cells having the next highest. CD4⁺ and CD8⁺ T-cells had a low, stable, spontaneous AF which gave them the highest signal-to-noise ratio. Although B-cells demonstrated the highest radiation-induced apoptotic response to 1 Gy of X-rays, CD8⁺ T-cells were the most radiation-responsive lymphocytes due to their low spontaneous AF. By generating dose response curves for CD4⁺ and CD8⁺ T-cells, the sensitivity of the MNCA for detecting apoptosis in these two cell types was also examined.     

Identification of membrane proteins of human blood platelets with a hydrophobic photolabel

A photoactivable glycolipid probe, 12-(4-azido-2-nitrophenoxy)stearoyl[1-14C]glucosamine, was used to label proteins and lipids of platelet membranes. The proteins were analyzed by two-dimensional high-resolution gelelectrophoresis. The labeling patterns showed that three membrane proteins were labeled which were not previously identified by ectolabeling (Sixma, J.J. and Schiphorst, M.E. (1980) Biochim. Biophys. Acta 603, 70-83). Analysis of the lipid fraction showed that phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine were labeled by the probe. The distinct labeling of phosphatidylserine strongly suggests that the probe redistributes between the two halves of the bilayer.     

Egg white proteins.

1. Egg white proteins are the principal solutes present in egg white, making up approximately 10% of its weight. 2. They are globular proteins and most have acidic isoelectric points. 3. Many are glycoproteins with carbohydrate contents ranging from 2 to 58%. 4. Of the major egg white proteins, lysozyme is the only one having catalytic activity, but many have specific binding sites, e.g. for vitamins such as biotin, riboflavin and thiamin, or for metal ions such as FeIII. 5. A major group are those showing proteinase inhibitory activity, and they include ovomucoid, ovoinhibitor, cystatin and ovostatin. 6. The synthesis of egg white protein occurs in the oviduct, and is hormonally controlled either by oestrogens or progesterone. 7. Extensive studies have been carried out in the genes coding for egg white proteins.     

Flow cytochemical patterns of white blood cells in human hematopoietic malignancies: III. Miscellaneous hemopoietic diseases

Peripheral blood samples from 48 untreated and 20 treated patients with disease entities that directly or indirectly affect hematopoiesis [dys-myelopoietic syndrome (DMS), refractory anemia with excess blasts (RAEB) or in transformation (RAEBIT), lymphoma, myeloma, acquired immunodeficiency syndrome (AIDS), and solid tumors with uninvolved bone marrow] were measured with the Technicon H-6000 automated hematology analyzer; this instrument provides a differential count on 10(4) white blood cells (WBC) effected by means of flow cytochemistry (peroxidase content) and volume (light scatter) discrimination. Cases with DMS and RAEB showed statistically significantly lower WBC counts than normal, whereas cases with lymphoma showed significantly higher values. No disease entity demonstrated changes in mean peroxidase activity (MPA) that were significantly different from normal, although all disease entities, including cases with solid tumors, showed significantly higher (two to severalfold) proportions of cells with high peroxidase (HPX) content, probably as a reflection of a disturbance of normal hemopoiesis with the emergence of younger granulocytic forms. All cases with paraleukemia (DMS, RAEB, and RAEBIT) showed significantly higher values of large unstained cells (LUC), whereas cases with lymphoma showed significantly lower LUC values. There were no statistically significant differences for any parameter (WBC counts, MPA, HPX, or LUC) among the paraleukemia subtypes. However, based on the displayed trends, a case presenting with dyserythropoiesis, relatively low WBC counts, abnormal HPX values, and LUC below 10% should be suspected for RAEB, whereas the presence of greater than 10% LUC and almost normal or even slightly elevated WBC counts should suggest a more accelerated phase of RAEB. Unless complicated by a leukemic phase, cases of lymphoma or myeloma did not display changes in any of the parameters analyzed by the H-6000. Similarly, patients with AIDS had no overt changes other than a trend to lower WBC counts with occasionally higher or lower absolute lymphocyte counts than normal. The peripheral blood of patients with solid tumors displayed a slight increase in HPX, suggesting an indirect effect on hemopoiesis since careful workup failed to demonstrate bone marrow involvement. Our data demonstrates that an H-6000 analysis has a role in the evaluation and follow-up of all these entities particularly to document leukemic transformation of either lymphoma, myeloma, or RAEB.