Identification and properties of two methyltransferases in conversion of phosphatidylethanolamine to phosphatidylcholine.

Two methyltransferases involved in the methylation of phosphatidylethanolamine to form phosphatidylcholine were demonstrated in a microsomal fraction of bovine adrenal medulla. The first methyltransferase catalyzes the methylation of phosphatidylethanolamine to form phosphatidyl-N-monomethylethanolamine. This enzyme has an optimum pH of 6.5, a low Km for S-adenosyl-L-methionine (1.4 micron), and an absolute requirement for Mg2+. The second methyltransferase catalyzes the two successive methylations of phodphatidyl-N-monomethylethanolamine to phosphatidyl-N,N-dimethylethanolamine and phosphatidylcholine. In contrast to the first methyltransferase, it has an optimum pH of 10 and a high Km for S-adenosyl-L-methionine (0.1 mM) and does not require Mg2+.     

A diffusible stimulator of eosinophilopoiesis produced by lymphoid cells as demonstrated with diffusion chambers

Previous experiments have indicated that eosinophilopoiesis is stimulated in lymphoid cell-dependent eosinophil responses to certain antigens. In order to study if the potential for this stimulation of eosinophilopoiesis is a function of lymphoid cells and can be expressed on challenge with the eosinophilia-inducing antigen, the diffusion chamber technique for the culture in vivo of murine hemopoietic cells has been modified. A quadrachamber diffusion assembly allows for the simultaneous maintenance in the same host of four cell populations, pairs of which are separated by a cell-impermeable Millipore diffusion membrane of defined porosity. Spleen cells for chambers were from normal mice and mice primed with tetanus toxoid; secondary challenge induces eosinophilia. These spleen cells were placed transfilter from isogeneic bone marrow cells and cultured in vivo for 6 days in normal mice that received tetanus toxoidintraperitoneally following chamber- assembly implant. The marrow cell transfilter from spleen cells of primed-donor origin exhibited significantly greater eosinophilopoiesis than contiguous-chamber marrow transfilter from normal spleen cells. Such stimulated eosinophilopoiesis was independent of total chamber marrow cellularity. The data indicated that antigen-stimulated lymphoid cells may be the source of an eosinophilopoietic factor.     

Characterization of human skeletal stem and bone cell populations using dielectrophoresis

Dielectrophoresis (DEP) is a non‐invasive cell analysis method that uses differences in electrical properties between particles and surrounding medium to determine a unique set of cellular properties that can be used as a basis for cell separation. Cell‐based therapies using skeletal stem cells are currently one of the most promising areas for treating a variety of skeletal and muscular disorders. However, identifying and sorting these cells remains a challenge in the absence of unique skeletal stem cell markers. DEP provides an ideal method for identifying subsets of cells without the need for markers by using their dielectric properties. This study used a 3D dielectrophoretic well chip device to determine the dielectric characteristics of two osteosarcoma cell lines (MG‐63 and SAOS‐2) and an immunoselected enriched skeletal stem cell fraction (STRO‐1 positive cell) of human bone marrow. Skeletal cells were exposed to a series of different frequencies to induce dielectrophoretic cell movement, and a model was developed to generate the membrane and cytoplasmic properties of the cell populations. Differences were observed in the dielectric properties of MG‐63, SAOS‐2 and STRO‐1 enriched skeletal populations, which could potentially be used to sort cells in mixed populations. This study provide evidence of the ability to characterize different human skeletal stem and mature cell populations, and acts as a proof‐of‐concept that dielectrophoresis can be exploited to detect, isolate and separate skeletal cell populations from heterogeneous bone marrow cell populations. Copyright © 2012 John Wiley & Sons, Ltd.     

Short-term fluoxetine treatment induces neuroendocrine and behavioral anxiogenic-like responses in adolescent male rats

Fluoxetine (FLX) is prescribed to treat depression and anxiety in adolescent patients. However, FLX has anxiogenic effects during the acute phase of treatment, and caution has been raised due to increased suicidal thinking and behavior. Herein, we sought to study in adolescent (35-day-old) male rats, the effects of short-term FLX treatment (10 mg/kg/day, i.p. for 3–4 days) on hypothalamic–pituitary–adrenal axis activity, serotonin (5-hidroxytriptamine, 5-HT) transporter (SERT) mRNA expression in the dorsal raphe nucleus (DRN), energy balance-related variables and behavioral profiles in the holeboard. Our results revealed that daily FLX administration increased plasma corticosterone (B) concentrations without affecting basal gene expression of corticotrophin releasing hormone in the hypothalamic paraventricular nucleus (PVN) nor of pro-opiomelanocortin in the anterior pituitary. However, FLX had significant effects increasing the mRNA expression of PVN arginine vasopressin (AVP) and reducing SERT mRNA levels in the dorsolateral subdivision of the DRN. In the holeboard, FLX-induced anxiety/emotionality-like behaviors. As expected, FLX treatment was endowed with anorectic effects and reduced body weight gain. Altogether, our study shows that short-term FLX treatment results in physiological, neuroendocrine and behavioral stress-like effects in adolescent male rats. More importantly, considering that the AVP- and 5-HTergic systems: (1) are intimately involved in regulation of the stress response; (2) are regulated by sex hormones and (3) are related to regulation of aggressive behaviors, our results highlight the potential significance of these systems mediating the anxiogenic/emotionality/stress-like responses of adolescent male rats to short-term FLX treatment.     

Uncoupling in the expression of platelet GP IIb/IIIa in human endothelial cells and K562 cells: absence of immunologic crossreactivity between platelet GP IIb and the vitronectin receptor alpha chain [see comments]

Platelet glycoproteins (GP) IIb and IIIa exist as noncovalently associated Ca++-dependent heterodimer complexes within the platelet membrane and express the major platelet alloantigens Leka (Baka) and PIA1 (Zwa), which are genetic markers of GP IIb and GP IIIa, respectively. Since heterodimers immunologically related to platelet GP IIb/IIIa have been identified in a number of nucleated cell types, we tested anti-Leka and anti-PIA1 antiserum, polyclonal anti-platelet GP IIb/IIIa IgG, as well as a panel of 28 monoclonal anti-GP IIb, GP IIIa, or complex dependent anti-GP IIb/IIIa antibodies on endothelial cells, peripheral blood mononuclear cells, and the erythroleukemic cells HEL and K562 in order to determine whether nucleated cell GP IIb/IIIa related proteins and platelet GP IIb/IIIa are immunologically related. Using immunofluorescence, immunoblotting, and immunoprecipitation experiments, evidence is presented that (1) the alloantigen Leka is not expressed in endothelial cells of an individual whose platelets are of the Leka/PIA1 phenotype, whereas the PIA1 alloantigen is readily detectable in these cells, (2) that in contrast to HEL cells, which express platelet GP IIb/IIIa and are of the Leka/PIA1 phenotype, platelet GP IIb is immunologically undetectable in 12-O-tetradecanoyl- phorbol-13-acetate (TPA)-treated K562 cells despite the presence of platelet GP IIIa, and (3) that peripheral blood mononuclear cells do not express platelet GP IIb or GP IIIa on their cell surface.     

Human herpesvirus 6: infection and disease following autologous and allogeneic bone marrow transplantation

Human herpesvirus 6 activity (HHV-6) was studied in 15 allogeneic and 11 autologous marrow transplantation patients. After transplantation, HHV-6 was isolated from the peripheral blood mononuclear cells of 12 of 26 patients (6 allogeneic and 6 autologous). All isolates were variant B. Eleven of 26 and 12 of 19 patients showed salivary shedding of HHV-6 DNA before and after transplantation, respectively. The antibody titer increased in 7 of 26 patients. Thus, 23 of 26 patients showed evidence of active HHV-6 infection either by virus isolation, salivary shedding, or increases in antibody titers. The fraction of saliva specimens positive in 19 patients was negatively associated with their antibody titers (P= .005). The proportion of cultures positive increased after transplantation (P = .007). Sinusitis was associated with HHV-6 isolation in autologous recipients (P= .002). In allogeneic patients, active human cytomegalovirus infection was associated with HHV-6 isolation (P = .04). No association was observed between HHV-6 infection and GVHD, pneumonia, delay in engraftment, or marrow suppression. Of the 120 clinical events analyzed in 26 patients, HHV-6 was defined as a probable cause of 16 events in 9 patients based on the propinquity of HHV-6 activity and the clinical event plus the absence of other identified causes of the event.     

Procoagulant activity of lymphocyte-macrophage populations in rabbits: selective increases in marrow, blood, and spleen cells during Shwartzman reactions

The present experiments examine leukocyte procoagulant activity using mononuclear cell populations purified or enriched from rabbit bone marrow, blood, spleen, lymph node, thymus, and pulmonary alveoli. Cells from these six sites, obtained from control and endotoxemic animals and assayed without an intermediate culture step, were found to have procoagulant activity identified as tissue factor. Under control conditions, tissue factor activity was found to be at low levels in marrow and blood populations compared to median activities 3- and 11- fold higher in populations from spleen and lymph node, and 33- and 45- fold higher in thymus and alveolar populations. By contrast to respective controls, significantly increased amounts of tissue factor (35-, 15-, and 12-fold at median levels) were found in marrow, blood, and spleen populations from endotoxemic animals. The types of leukocytes in these latter three populations were morphologically and histochemically indistinguishable from respective controls, indicating that endotoxin induced increases of activity in cells with relatively low amounts under control conditions. Activity did not change significantly in lymph node, thymus, or alveolar populations after endotoxemia. These studies show that tissue factor is present in a range of leukocyte populations not previously reported to have procoagulant activity. In addition, the finding of widespread gains of tissue factor in the marrow-blood-spleen pool due to endotoxemia provides new evidence supporting the importance of leukocyte procoagulants in Shwartzman-like reactions.