d-生物素的立体专一性全合成研究

对confaione的d-生物素(1)全合成进行了改进,从半胱氨酸计算,总收率3.7%。5与4-溴代三苯膦丁酸甲酯进行Wittig-Schlosser缩合反应可立体专一性地转化成反式-烯(6)。以叠氮三甲基硅烷代替叠氮化锂亲核进攻9分子中C3-溴原子,可显著地提高顺式-叠氮内酰胺10的收率。     

锌酞菁脂质体光动力作用引起小鼠肿瘤的细胞程序性死亡

电镜观察了锌酞菁脂质体光动力作用引起小鼠ＭＳ－２纤维肉瘤的形态学变化。发现其作用很强，并对肿瘤细胞有明显的直接影响。肿瘤细胞的结构表现出明显的程序性细胞死亡（ａｐｏｐｔｏｓｉｓ，ｐｒｏｇｒａｍｍｅｄｃｅｌｄｅａｔｈ）的特点：胞核染色质凝聚边集、核固缩、核破裂、染色质凝块流失、胞质内吞噬现象、胞膜表面肿胀粗钝的胞突形成、细胞碎裂等。加深了对锌酞菁脂质体光敏作用机理的认识，但其详细的发生机制和调节途径有待阐明。     

Effect of priming polymorphonuclear leukocytes with cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF] and G-CSF) on the host resistance to Streptococcus pneumoniae in chinchillas infected with influenza A virus

Patients infected with influenza A virus (IAV) are at increased risk for bacterial superinfections, and this occurs in association with depressed polymorphonuclear leukocyte (PMNL) function. Recently, we reported that in vitro exposure of human PMNL to granulocyte-macrophage colony-stimulating factor (GM-CSF) reverses IAV-induced cell dysfunction. The present study used an established animal model of IAV infection to examine whether G-CSF and/or GM-CSF can overcome IAV- induced PMNL dysfunction and thereby prevent secondary infections. Preliminary studies determined a dosing schedule of these cytokines that caused significant priming of chinchilla PMNL. In subsequent studies, animals were inoculated intranasally with IAV (day 1) followed 3 days later by Streptococcus pneumoniae, and administered daily intraperitoneal injections with a cytokine or placebo on days 3 through 9. Animals had blood obtained on multiple occasions for PMNL studies, and were followed-up for evidence of pneumococcal disease. Both cytokines caused significant priming of the PMNL chemiluminescence response and this was associated with reversal of the IAV-induced PMNL dysfunction. However, neither cytokine decreased the incidence of pneumococcal disease.     

Studies on levamisole--induced agranulocytosis

Widespread clinical trials of leavo-tetramisole (levamisole) as an immunopotentiating agent in rheumatoid arthritis, metastatic carcinoma, and immunodeficiency states have been complicated by agranulocytosis (AGC) in 2.5%-13% of patients. Other than a relationship with prolonged high dosage, very little is known regarding the pathogenesis of levamisole-induced AGC. Whereas leukoagglutination was negative, fluorochromatic microgranulocytotoxicity (GCY) tests were positive with serum from 10 of 10 acutely neutropenic patients. The antibody was IgM, reacted with 100% of unrelated granulocytes, but not with T or B lymphocytes. Some sera also reacted with monocytes and the myeloid cell line, K-562. Tests for antigen-antibody complexes or cold autoantibodies were negative. Although clinical evidence strongly suggests a haptene (drug) mechanism, in vitro mixing experiments were also negative. An alternative choice parallels the model of aldomet- induced Coombs'-positive hemolytic anemia. Finally, GCY first became positive 2-3 mo prior to the onset of AGC on two patients, suggesting the possibility of identifying those at risk well before the onset of neutropenia.     

Platelet-derived growth factor promotes polymorphonuclear leukocyte activation

The platelet-derived growth factor (PDGF) has several well defined important biologic activities. Platelet-derived growth factor is the major mitogen in human serum for cells of mesenchymal origins; it is a potent chemoattractant protein for human monocytes, neutrophils, fibroblasts, and smooth muscle cells; and has been implicated in transformation by simian sarcoma virus and perhaps in transformation by other agents as well. In this article, PDGF has been shown to stimulate activation of human peripheral blood neutrophils defined by loss of membrane associated calcium as reflected by loss of chlortetracycline fluorescence, release of superoxide anion and specific granule enzymes, and enhanced neutrophil adherence and aggregation. These responses occurred in a dose-dependent fashion at concentrations of PDGF between 10 ng/mL (0.4 nmol/L) and 40 ng/mL (1.5 nmol/L) and were comparable to effects obtained with optimal concentrations of fMLP and C5a. Degranulation induced by PDGF was selective for secondary (specific) granules and not primary (azurophil) granules. Platelet-derived growth factor thus is ideally suited for a pivotal role in attracting inflammatory cells locally and initiating neutrophil activation at sites of blood vessel injury. Platelet-derived growth factor or a closely related protein also may play an important role in attracting and activating neutrophils in association with inflammatory tumors.     

The binding of ouabain to normal and chronic lymphocytic leukemic lymphocytes

The binding of the cardiac glycoside, ouabain, to cells had been used to quantify the number of active cation pumps. In this study, lymphocytes were incubated with 3H-ouabain and the equilibrium binding analyzed for the maximal number of specific binding sites. Lymphocytes from normal peripheral blood bound 44,200 +/- 9920 molecules/cell, compared with 29,200 +/- 8370 molecules/cell for the lymphocytes of chronic lymphocytic leukemia (CLL) subjects. This difference was significant (p less than 0.01) and did not reflect a lower number of sites on B cells than T cells, since B-cell-enriched lymphocytes from normal peripheral blood showed the same ouabain binding characteristics as the standard T-cell-rich preparation. Although monocytes bind threefold more ouabain than lymphocytes, the small monocyte contamination (3.0%) in normal lymphocyte preparations could not account for the difference between normal and CLL. The fewer ouabain binding sites on CLL lymphocytes may reflect both their smaller size (by 10%) and lower mitotic activity compared with lymphocytes from normal peripheral blood.     

Improved hematopoiesis in anemic Sl/Sld mice by splenectomy and therapeutic transplantation of a hematopoietic microenvironment

The ability of a clonal hematopoiesis-supportive bone-marrow stromal cell line GBlneor to engraft and alter the microenvironment-induced anemia of Sl/Sld mice was studied. Prior to stromal cell transplantation, Sl/Sld mice received 1 Gy total body irradiation (TBI) and 13 Gy to the right hind limb. Two months after intravenous (IV) injection of 5 x 10(5) GBlneor cells, 54.4% +/- 17.0% donor origin (G418r) colony-forming cells were recovered from the right hind limb of Sl/Sld mice. Long-term bone marrow cultures (LTBMCs) established from GBlneor-transplanted mice produced 189.5 CFU-GEMM-forming progenitors/flask over 10 weeks compared with 52.7 +/- 6.2 CFU-GEMM forming progenitors/flask from irradiated nontransplanted Sl/Sld mice. A partial correction of macrocytic anemia was detected 2 months after GBlneor transplantation in splenectomized, irradiated Sl/Sld mice (HgB 7.2 +/- 0.4 g/dL; MCV 68.3 +/- 7.0 fL) compared to splenectomized, irradiated, nontransplanted Sl/Sld mice (HgB 5.5 +/- 1.1 g/dL; MCV 76 +/- 8.5 fL) or control Sl/Sld mice (HgB 5.4 +/- 0.5 g/dL; MCV 82.4 +/- 1.3 fL). Mean RBC volume distribution analysis showed a 2.5-fold increase in percentage of peripheral blood RBCs with MCV less than or equal to 45 fL and confirmed reduction of the MCV in splenectomized- GBlneor-transplanted mice compared to control Sl/Sld mice. A hematopoiesis-suppressive clonal stromal cell line derived from LTBMCs of Sl/Sld mice (Sldneor) engrafted as effectively (43.5% +/- 1.2% G418r CFU-F/limb) as did GBlneor cells (38.3% +/- 0.16% G418r CFU-F/limb) to the irradiated right hind limbs of C57Bl/6 mice. LTBMCs established after 2 or 6 months from Sldneor-transplanted mice showed decreased hematopoiesis (182 +/- 12 [2 months] and 3494.3 +/- 408.1 [6 months] CFU-GEMM forming progenitors/flask over 10 weeks) compared to those established from GBlneor-transplanted mice (5980 +/- 530 [2 months] and 7728 +/- 607, [6 months] CFU-GEMM progenitors forming/flask). Thus, transplantation of clonal bone-marrow stromal cell lines in vivo can stably transfer their physiologic properties to normal or mutant mice.     

Rapid flow cytometric quantitation of reticulated platelets in whole blood

Young or reticulated platelets contain some residual mRNA, which is rapidly degraded after platelet release into the circulation. In order to minimize platelet activation and possible loss of large platelets during sample handling a whole blood method has been developed utilising the RNA fluorochrome thiazole orange (TO) in combination with an antibody to anti-glycoprotein Ib (GpIb) directly conjugated to phycoerythrin (PE), to specifically stain reticulated platelets via flow cytometric analysis. In this study whole blood analysis of platelet mRNA was undertaken in healthy normal subjects and a variety of patients with haematological abnormalities. The percentage of Gp Ib positive platelets containing mRNA in normals (n = 22) was 11.61% with a two SD range of 3.19-20.01%. The percentage of reticulated platelets was significantly increased (mean mRNA content ± one SD) in sickle cell disorders (n = 22) 38.12% ± 18.42 (P < 0.001); thalassaemia (major, intermedia and trait) (n = 24) 29.76 ± 19.15 (P < 0.001); ITP (N = 20) 23.53% ± 13.04 (P < 0.02) and essentialthrombocythemia (N = 32) 37.12 ± 19.84 (P < 0.001). Platelets from patients with reactive thrombocytosis (N = 15) were only 12.23% positive (±6.95) and not significantly different from the normal range (P = 0.95). This method offers a rapid and simple procedure for assessment of reticulated platelets in whole blood and suggests that there may be an increased platelet turnover in certain haemoglobinopathies.     

Stimulation of tyrosine phosphorylation after ligation of beta7 and beta1 integrins on human B cells

B lymphocytes express several members of the integrin family of adhesion molecules that mediate cell-cell and cell-extracellular matrix interactions. In addition to beta1 integrins, predominantly alpha4 beta1, mature B cells also express alpha4 beta7, which is a receptor for vascular cell adhesion molecule-1 and fibronectin, and is also involved in the homing of B cells to mucosal sites through binding to a third ligand, mucosal address in cell adhesion molecule-1. Here we describe that crosslinking of alpha4 beta7 integrins on B cell lines and normal tonsillar B cells, induces tyrosine phosphorylation of multiple substrates of 105-130 kD, indicating that beta7 integrin plays a role as signaling molecule in B cells. This pattern of phosphorylated proteins was very similar to that induced following ligation of alpha4 beta1. Interestingly, ligation of alpha5 beta1 or alpha6 beta1 also stimulated the 105-125 kD group of phosphorylated proteins, whereas ligation of beta2 integrins did not. The focal adhesion tyrosine kinase p125FAK was identified as one of these substrates. Beta1 or beta7 mediated tyrosine phosphorylations were markedly decreased when the microfilament assembly was inhibited by cytochalasin B. These results suggest that intracellular signals initiated by different integrins in B cells may converge, to similar cytoskeleton-dependent tyrosine phosphorylated proteins.