CFU-M-derived human megakaryocytes synthesize glycoproteins IIb and IIIa

Human megakaryocytes have been shown by immunofluorescent techniques to express platelet glycoprotein IIb/IIIa antigen. We report evidence that megakaryocytes derived from human committed megakaryocytic progenitor cells in vitro (CFU-M) synthesize glycoproteins IIb and IIIa. Nonadherent light-density human bone marrow cells were cultured in human plasma and methylcellulose using conditions that promote large megakaryocytic colonies. On day 13 the megakaryocytic colonies were picked, pooled, and pulsed with 35S-methionine in methionine-free media. Populations of approximately 100,000 cells with greater than or equal to 95% viability and containing 70% to 90% megakaryocytes were obtained reliably for study. After the radioactive pulse, the cell suspension was solubilized with nonionic detergent. To reduce nonspecific binding of 35S-labeled proteins to agarose, the lysate was chromatographed sequentially on glycine-quenched Affi-gel and antihuman factor X-Sepharose. The unbound material from these resins was then chromatographed on an antiglycoprotein IIb/IIIa monoclonal antibody resin (HP1-1D-Sepharose) or on a control monoclonal antibody resin. Bound fractions were eluted and analyzed by polyacrylamide gel electrophoresis and autoradiography. Autoradiograms of diethylamine eluates from HP1-1D-Sepharose revealed two labeled proteins with electrophoretic mobilities identical with those of human platelet membrane glycoproteins IIb and IIIa, isolated using similar conditions. Autoradiograms of material synthesized by control macrophages from the same donors revealed no significant labeling of proteins in the glycoprotein IIb/IIIa molecular weight range, nor were such proteins bound by HP1-1D-Sepharose. Our observations show that protein synthesis by CFU-M-derived human megakaryocytes can be readily studied using a small amount of bone marrow aspirate as starting material. This approach will allow the study of protein synthesis by megakaryocytes from normal subjects or from subjects with clinical disorders, and it will circumvent the need to obtain large amounts of bone marrow to prepare enriched populations of megakaryocytes.     

Idiotype as a tumor-specific marker in childhood B cell acute lymphoblastic leukemia

Immunoglobulin (Ig) or idiotype (Id) is a tumor-specific target in those B cell malignancies that express this molecule on their surface. We explored the biology of B cell acute lymphoblastic leukemia (B cell ALL) using Id as a tumor marker. In this report we describe the development of anti-Id monoclonal antibodies (MAB) for two children with B cell ALL. These reagents were used retrospectively to study tumor kinetics and to detect residual disease after chemotherapy. In both cases serum Id values were strikingly high at diagnosis (1.2 mg/mL and 10.8 mg/mL), suggesting that the tumor cells were relatively mature B cells capable of significant antibody production. In both patients the serum Id levels fell with the institution of therapy and confirmed that the patients were in remission. Increasing serum Id predicted relapse four months before conventional methods in patient 1, and Id proved to be a more sensitive measure of tumor burden than Southern blot analysis of rearranged Ig genes in bone marrow samples. Surprisingly, low levels of Id were redetected in the second patient just before completing therapy and have persisted for over a year despite the absence of clinical evidence of recurrent disease. Thus, serum Id levels reflect tumor burden during initial therapy but may not necessarily predict tumor progression after a complete clinical remission.     

Involvement of PKCα and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of μ-opioid receptors in mature brain neurons

Background and purpose:

The ability of an agonist to induce desensitization of the µ-opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist-specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high-efficacy agonist).

Experimental approach:

MOR function was measured in locus coeruleus neurons, by using whole-cell patch-clamp electrophysiology, in rat and mouse brain slices (both wild-type and protein kinase C (PKC)α knockout mice). Specific isoforms of PKC were inhibited by using inhibitors of the receptors for activated C-kinase (RACK), and in vivo viral-mediated gene-transfer was used to transfect neurons with dominant negative mutants (DNMs) of specific G-protein-coupled receptor kinases (GRKs).

Key results:

Morphine-induced desensitization was attenuated by using RACK inhibitors that inhibit PKCα, but not by other isoform-specific inhibitors. Further, the PKC component of morphine-induced desensitization was absent in locus coeruleus neurons from PKCα knockout mice. The PKC-enhanced morphine-induced desensitization was not affected by over-expression of a GRK2 dominant negative mutant (GRK2 DNM). In contrast, DAMGO-induced MOR desensitization was independent of PKC activity but was reduced by over-expression of the GRK2 DNM but not by that of a GRK6 DNM.

Conclusions and implications:

In mature mammalian neurons, different MOR agonists can induce MOR desensitization by different mechanisms, morphine by a PKCα-mediated, heterologous mechanism and DAMGO by a GRK-mediated, homologous mechanism. These data represent functional selectivity at the level of receptor desensitization.     

Bisphosphonates and atrial fibrillation: Bayesian meta-analyses of randomized controlled trials and observational studies

Background  

Occurrence of atrial fibrillation (AF) amongst bisphosphonate users has been increasingly reported but results are conflicting. We performed a Bayesian meta-analysis to address the possible association between the occurrence of AF and bisphosphonate use and estimated the posterior probability of development of AF with bisphosphonate use.     

Viscoelastic properties of bovine articular cartilage attached to subchondral bone at high frequencies

Background  

Articular cartilage is a viscoelastic material, but its exact behaviour under the full range of physiological loading frequencies is unknown. The objective of this study was to measure the viscoelastic properties of bovine articular cartilage at loading frequencies of up to 92 Hz.     

红花油乳液生产中的质量控制条件探索

建立红花油乳液的生产质量控制条件。方法：检测乳滴大小、离心效果及卫生学指标变化。结果：在640倍显微镜下观察,红花油乳滴直径小于5μm;使用高速离心机,以4000r／min离心10min后无明显分层,若有少许分层但振摇后立即恢复均相;卫生学符合国家标准。     

四种萆薢类药材原植物叶表面扫描电镜观察

目的：通过扫描电镜观察叶表面特征鉴别绵萆Ｘｉｅ、粉萆Ｘｉｅ、龙草Ｘｉｅ等４种萆Ｘｉｅ类药材原植物。方法：取上述４种原植物叶用扫描电镜常规制片法制片,置扫描电镜中观察摄影。结果：上述４种原植物表面特征有明显差异。结论：用扫描电镜观察叶表面特征的方法可鉴别上述４种药材的原植物。