An endogenous glycosylphosphatidylinositol-specific phospholipase D releases basic fibroblast growth factor-heparan sulfate proteoglycan complexes from human bone marrow cultures

Basic fibroblast growth factor (bFGF) is a hematopoietic cytokine that stimulates stromal and stem cell growth. It binds to a glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycan on human bone marrow (BM) stromal cells. The bFGF- proteoglycan complex is biologically active and is released by addition of exogenous phosphatidylinositol-specific phospholipase C. In this study, we show the presence of an endogenous GPI-specific phospholipase D (GPI-PLD) that releases the bFGF-binding heparan sulfate proteoglycan and the variant surface glycoprotein (a model GPI-anchored protein) from BM cultures. An involvement of proteases in this process is unlikely, because released proteoglycan contained the GPI anchor component, ethanol-amine, and protease inhibitors did not diminish the release. The mechanism of release is likely to involve a GPI-PLD and not a GPI-specific phospholipase C, because the release of variant surface glycoprotein did not reveal an epitope called the cross- reacting determinant that is exposed by phospholipase C-catalyzed GPI anchor cleavage. In addition, phosphatidic acid (which is specifically a product of GPI-PLD-catalyzed anchor cleavage) was generated during the spontaneous release of the GPI-anchored variant surface glycoprotein. We also detected GPI-PLD-specific enzyme activity and mRNA in BM cells. Therefore, we conclude that an endogenous GPI-PLD releases bFGF-heparan sulfate proteoglycan complexes from human BM cultures. This mechanism of GPI anchor cleavage could be relevant for mobilizing biologically active bFGF in BM. An endogenous GPI-PLD could also release other GPI-anchored proteins important for hematopoiesis and other physiologic processes.     

Analysis of optimal conditions for retroviral-mediated transduction of primitive human hematopoietic cells

We sought to define optimal conditions for retroviral-mediated transduction of long-lived human hematopoietic progenitors from bone marrow and peripheral blood. CD34+ cells were transduced by the LN and G2 retroviral vectors in the presence or absence of stromal support and with or without cytokine addition. After transduction, a portion of the cells was plated in methylcellulose colony-forming assay, with or without G418, to assess the extent of gene transfer into committed progenitors. The remaining cells from each experiment were transplanted into immunodeficient mice to allow analysis of transduction of long- lived progenitors. Human colony-forming cells contained within the murine bone marrow were analyzed after engraftment periods of 2 to 11 months. Cells were plated in a human-specific colony-forming assay with and without G418 to assess the extent of transduction of primitive progenitors. Individual human colonies were also analyzed by polymerase chain reaction for the presence of provirus. Bone marrow progenitors were efficiently transduced only when stroma was present, whereas mobilized peripheral blood progenitors were effectively transduced in the presence of either stroma or cytokines. Inclusion of the cytokines interleukin-3, interleukin-6, and stem cell factor did not further augment the extent of gene transfer in the presence of a stromal support layer. Additionally, human CD34+ progenitors from bone marrow or mobilized peripheral blood that had been transduced for 3 days in the absence of stroma failed to produce sustained, long-term engraftment of bnx mice. Mice transplanted with the same pools of human progenitors that had been transduced in the presence of stroma for 3 days had significant levels of human cell engraftment at the same timepoints, 7 to 11 months after transplantation. Our data show loss of long-lived human progenitors during 3-day in vitro transduction periods in the absence of stromal support. Therefore, the presence of bone marrow stroma has dual benefits in that it increases gene transfer efficiency and is essential for survival of long-lived human hematopoietic progenitors.     

Assessment of testicular metabolic integrity with P-31 MR spectroscopy

To evaluate the reliability of phosphorus-31 magnetic resonance (MR) spectroscopy in the assessment of acute testicular ischemia, vascular integrity, and spermatogenesis, the authors studied in vivo canine and primate testicles grouped as follows: group 1 testes (n = 8), in situ canine controls; group 2 (n = 11), canine testes subjected to warm ischemia; group 3, canine (n = 4) and primate (n = 4) testicles from hormone-treated animals. Group 1 control testicles showed high monophosphoester (MP) levels; low levels of inorganic phosphate (Pi), phosphodiester (PD), and phosphocreatine; and high levels of adenosine triphosphate (ATP). Group 2 testes revealed a time-dependent decay of MP/Pi ratios (from 2.1 to 0.70). Regeneration of ATP was noted in the acute reperfusion period. After 6 weeks of pituitary gonadotropin suppression, group 3 testes showed a significant decrease (P less than .05) in MP/PD ratios from a control level of 2.6 +/- 0.3 and a decrease in the MP/beta-ATP ratio from 2.4 +/- 0.1 to 1.8 +/- 0.3. P-31 MR spectroscopy appears to be a potential method for noninvasively assessing testicular ischemic injury and the metabolic integrity of spermatogenesis.     

Neurological and adrenal dysfunction in the adrenal insufficiency/alacrima/achalasia (3A) syndrome

Review of 20 patients with glucocorticoid deficiency (three cases also with salt loss) associated with absent tear secretion (19 cases) and achalasia of the cardia (15 cases) revealed neurological abnormalities in 17 including hyper-reflexia, muscle weakness, dysarthria, and ataxia together with impaired intelligence and abnormal autonomic function, particularly postural hypotension. These findings indicate that significant neurological problems are common in this multisystem disorder.     

Diverticular abscesses: percutaneous drainage

Percutaneous catheter drainage was performed in 16 patients with diverticulitis complicated by abscesses. Each patient had resolution of fever within 72 hours. Eleven patients subsequently underwent simultaneous sigmoid resection and operative anastomosis 10-40 days after percutaneous drainage. One patient required a three-stage procedure after percutaneous drainage, and one patient was too unstable for operation at any time during her course and eventually died of respiratory failure. Three patients did not undergo resection after catheter drainage and have remained asymptomatic for 1-2 1/2 years. Ten of 16 patients had fistulas, eight of which closed spontaneously. Experience with percutaneous drainage of diverticular abscesses suggests that it obviates surgical abscess drainage and permits a single operation (sigmoid resection and closure) to be performed safely. Percutaneous abscess drainage has cost-saving implications, since one or two operations may be avoided in most patients, and in some high-risk elderly patients all operations may be obviated.     

Corpus callosum and limbic system: neuroanatomic MR evaluation of developmental anomalies

Agenesis of the corpus callosum is a complex malformation of the brain that has been associated with varying degrees of limbic system maldevelopment. We retrospectively reviewed the records of 11 patients with callosal agenesis (seven total, four partial) who underwent magnetic resonance (MR) imaging, with particular attention to the associated malformations of the limbic system. Comparison was made with selected images from MR examinations of healthy volunteers and with necropsy specimens from other patients with callosal agenesis. Ten of 11 patients demonstrated limbic anomalies (severe motion artifact precluded evaluation of these structures in one patient). MR depicted not only the abnormalities intrinsic to callosal agenesis but also the frequently associated malformations of the limbic system.     

伊贝碱甙B的分离和结构测定

本文报道自吉林栽培的伊贝母(Fritillaria pallidiflora Schrenk)中分得一种新的C-去甲-D-异甾体生物碱甙,命名为伊贝碱甙B(yibeinoside B)。根据红外、质谱、氢谱和碳谱确定了结构。