Nuclear translocation of angiogenin in proliferating endothelial cells is essential to its angiogenic activity.

The intracellular pathway of human angiogenin incalf pulmonary artery endothelial (CPAE) cells has been studied byimmunofluorescence microscopy. Proliferating CPAE cells specifically endocytosenative angiogenin and translocate it to the nucleus, where it accumulates in thenucleoli. Nuclear translocation of angiogenin does not occur innonproliferative, confluent CPAE cells. These cells were previously found toexpress an angiogenin-binding protein (AngBP) that was identified as smoothmuscle alpha-actin. Exogenous actin, an anti-actin antibody, heparin, andheparinase treatment all inhibit the internalization of angiogenin, suggestingthe involvement of cell surface AngBP/actin and heparan sulfate proteoglycans inthis process. It has been established that two regions of angiogenin areessential for its angiogenic activity, one is its endothelial cell binding siteand the other its catalytic site capable of cleaving RNA. CPAE cells do notinternalize four enzymatically active angiogenin derivatives whose cell bindingsite is modified, but they do internalize two enzymatically inactive mutantswhose cell binding site is intact. Thus, the putative cell binding site ofangiogenin is necessary for both endocytosis and nuclear translocation, but thecatalytic site is not. Three other angiogenic molecules are also translocated tothe nucleus of growing CPAE cells. Overall, the results suggest that nucleartranslocation of angiogenin and other angiogenic molecules is a critical step inthe process of angiogenesis.     

Contractile proteins participate in release of erythroid growth regulators from mononuclear cells

We have investigated the role of contractile proteins of circulating mononuclear cells in generation of membrane-associated, erythroid growth regulatory molecules. Lymphocytes and monocytes were incubated under serum-free conditions without and with cytochalasin B, cytochalasin D, or colchicine, and effects on positive and negative erythropoietic activities were determined in cell membranes and in surface membrane vesicle-rich pellets and supernatants of dialyzed medium conditioned by the cells. In serum-free cultures of human bone marrow, plasma membranes and exfoliated membrane-derived vesicles from cytochalasin-treated lymphocytes lost their capacity to support the formation of erythroid bursts, while monocyte membrane-associated inhibitory activity was abolished by preincubation with cytochalasin. In contrast, membrane-associated activities of colchicine-treated cells were unaffected. Cytochalasin-induced alterations of membrane regulatory molecules were observed in a dose-dependent fashion over a wide range of concentrations (1 to 100 micrograms/mL) tested. However, the capacity of membrane vesicle-free supernatants of medium conditioned by lymphocytes or monocytes was unaffected by cytochalasins, regardless of drug concentration used. Lysates of cytochalasin B-treated cells inhibited the activity of deoxyribonuclease I to a greater degree than did lysates of untreated cells, suggesting that the relative amount of monomeric actin is increased in the cytoplasm of treated cells. Furthermore, results of experiments with D-glucose and with cytochalasin D suggest that cytochalasin effects are independent of alterations in glucose metabolism. The data indicate that expression of plasma membrane- associated regulators is sensitive to agents that block polymerization of actin. They raise the possibility that changes in distribution of actin between unpolymerized and filamentous pools may influence the organization and/or function of mononuclear cell surface-associated erythroid regulatory molecules.     

Epstein-Barr virus lymphoproliferation after bone marrow transplantation

We review 15 cases of secondary B-cell lymphoproliferative disorders that occurred among 2,475 patients who received allogeneic bone marrow transplants (BMTs) at the Fred Hutchinson Cancer Research Center (Seattle) between 1969 and 1987. The histopathologic findings in 14 of the 15 patients spanned a wide spectrum of lymphoproliferative lesions. One patient had features characteristic of angioimmunoblastic lymphadenopathy. Epstein-Barr virus (EBV) genomic sequences were identified by Southern blot analysis in each of the 13 patients evaluated. Ten of the 12 lesions evaluated originated in donor cells. In two patients, who had mixed chimerism after transplantation, the lesions originated in host cells. The combined evidence from immunoglobulin light chain staining and the analysis of immunoglobulin heavy chain gene rearrangement indicated that the lesions in most patients represented polyclonal proliferations that gave rise to clonal subpopulations. The results indicate an overall actuarial incidence of 0.6% for this complication in BMT recipients. Anti-CD3 monoclonal antibody (MoAb) treatment of acute graft-v-host disease (GVHD) and T cell depletion of the donor marrow were statistically significant risk factors, and GVHD appeared to play a contributing role, particularly in the setting of human leukocyte antigen (HLA) disparity. Two patients had no identifiable risk factors. Prophylaxis or treatment with acyclovir had no detectable effect in the patients; all but two died with uncontrolled lymphoproliferation.     

Ex vivo expansion with stem cell factor and interleukin-11 augments both short-term recovery posttransplant and the ability to serially transplant marrow

The characterization of many cytokines involved in the control of hematopoiesis has led to intense investigation into their potential use in ex vivo culture to expand progenitor numbers. We have established the optimum ex vivo culture conditions that allow substantial amplification of transient engrafting murine stem cells and which, simultaneously, augment the ability to sustain serial bone marrow transplantation (BMT). Short-term incubation of unfractionated BM cells in liquid culture with stem cell factor (SCF) and interleukin-11 (IL- 11) produced a 50-fold amplification of clonogenic multipotential progenitors (CFU-A). Following such ex vivo expansion, substantially fewer cells were required to rescue lethally irradiated mice. When transplanted in cell doses above threshold for engraftment, BM cells expanded ex vivo resulted in significantly more rapid hematopoietic recovery. In a serial transplantation model, unmanipulated BM was only able to consistently sustain secondary BMT recipients, but BM expanded ex vivo has sustained quaternary BMT recipients that remain alive and well more than 140 days after 4th degree BMT. These results show augmentation of both short-term recovery posttransplant and the ability to serially transplant marrow by preincubation in culture with SCF and IL-11.     

Choosing identity-release sperm donors: the parents' perspective 13-18 years later

BACKGROUND: A growing number of donor insemination (DI) programmes offer 'open-identity' sperm donors, who are willing to have their identity released to adult offspring. We report findings from parents who chose such donors and whose children are now adolescents. METHODS: Using mail-back questionnaires, parents from 45 households (40% headed by lesbian couples, 38% by single women, 22% by heterosexual couples) reported their experience with using an open-identity donor and disclosure about it, as well as their child's plans for donor identity-release(sm). RESULTS: Almost no parents regretted using an open-identity donor. Almost all parents had told their child about his or her DI conception early on and reported a neutral to moderately positive impact. Finally, of those who had told, almost all expected their child to obtain the donor's identity. We also discuss differences found between birth mothers and co-parents and among single women, lesbian couples, and heterosexual couples. CONCLUSIONS: Families were relatively open and positive about their use of DI and that their child could obtain the donor's identity. Disclosure did not appear to have a negative impact on the families, regardless of parental sexual orientation and relationship status.     

Effects of manipulating sedentary behavior on physical activity and food intake

OBJECTIVES: Sedentary behaviors have been correlated with obesity. We investigated whether changes in sedentary behaviors relate to changes in energy intake and/or physical activity. STUDY DESIGN: Experimental within-subject crossover design in which children participated in three 3-week phases: baseline and increased and decreased targeted sedentary behaviors. PARTICIPANTS: Thirteen 8- to 12-year-old, nonobese children. MEASUREMENTS: Sedentary behaviors were measured through the use of daily activity logs, physical activity measured with accelerometers, and energy intake measured by means of repeated 24-hour recalls collected during each phase. Energy intake, energy expenditure, and energy balance per day were calculated. RESULTS: Children showed significant (P <.001) increases of 50% and decreases of 53% in targeted sedentary behaviors from baseline during the increase and decrease phases, respectively. There was a significant (P =.05) increase in energy balance per day (+350.7 kcal) when sedentary behaviors were increased, as the result of an increase in energy intake per day (+250.9 kcal) and a decrease in energy expenditure (-99.8 kcal). No significant changes in energy balance were observed when sedentary behaviors were decreased. CONCLUSIONS: Increasing sedentary behaviors had a greater influence on physical activity and energy intake than reducing sedentary behavior in nonobese youth. In some children, changes in sedentary behaviors may be important to modify energy balance and prevent obesity.     

Palmitate-TLR4 signaling regulates the histone demethylase,JMJD3, in macrophages and impairs diabetic wound healing

Chronic macrophage inflammation is a hallmark of type 2 diabetes (T2D) and linked to the development of secondary diabetic complications. T2D is characterized by excess concentrations of saturated fatty acids (SFA) that activate innate immune inflammatory responses, however, mechanism(s) by which SFAs control inflammation is unknown. Using monocyte-macrophages isolated from human blood and murine models, we demonstrate that palmitate (C16:0), the most abundant circulating SFA in T2D, increases expression of the histone demethylase, Jmjd3. Upregulation of Jmjd3 results in removal of the repressive histone methylation (H3K27me3) mark on NFκB-mediated inflammatory gene promoters driving macrophage-mediated inflammation. We identify that the effects of palmitate are fatty acid specific, as laurate (C12:0) does not regulate Jmjd3 and the associated inflammatory profile. Further, palmitate-induced Jmjd3 expression is controlled via TLR4/MyD88-dependent signaling mechanism, where genetic depletion of TLR4 (Tlr4^−/−) or MyD88 (MyD88^−/−) negated the palmitate-induced changes in Jmjd3 and downstream NFκB-induced inflammation. Pharmacological inhibition of Jmjd3 using a small molecule inhibitor (GSK-J4) reduced macrophage inflammation and improved diabetic wound healing. Together, we conclude that palmitate contributes to the chronic Jmjd3-mediated activation of macrophages in diabetic peripheral tissue and a histone demethylase inhibitor-based therapy may represent a novel treatment for nonhealing diabetic wounds.     

Cryptic chromosome rearrangements detected by subtelomere assay in patients with mental retardation and dysmorphic features

The regions near telomeres of human chromosomes are gene rich. Chromosome subtelomere rearrangements occur with a frequency of 7-10% in children with mild-to-moderate mental retardation (MR) and approximately 50% of cases are familial. Clinical investigation of subtelomere rearrangements is now prompted by fluorescence in situ hybridization (FISH) analysis using specific DNA probes from all relevant chromosome ends. In our study, 40 children were selected for subtelomere assay using either the Chromophore Multiprobe-T Cytocell device or the VYSIS TelVision probes. Inclusion criteria were: developmental delay or MR; a normal 550 G-band karyotype; FRAXA negative; and at least one other clinical criterion. Exclusion criteria included an identified genetic or environmental diagnosis. Of the 40 patients analysed, four (10%) were found to have subtelomere rearrangements. Three of 40 (7.5%) were found to have an unbalanced subtelomere rearrangement and one of 40 (2.5%) was found to have an apparently normal variant subtelomere deletion. The first of the three with an unbalanced karyotype was the result of a familial translocation, the second was a de novo finding, and the origin of the third could not be determined. The subtelomere FISH assay detected almost twice the frequency of unbalanced karyotypes as those detected by 550 G-banding in our cytogenetics laboratory (4.7%). In addition, subtelomere screening was eight times more likely than fragile X screening in our DNA laboratory (1%) to detect genetic abnormalities in mentally handicapped individuals. Our findings support the view that screening for subtelomere rearrangements has a greater positive yield than other commonly used genetic investigations and, if cost and resources permit, should be the next diagnostic test of choice in a child with unexplained MR/dysmorphisms and a normal 550 G-band karyotype.