Platelet glycoproteins IIb and IIIa associated with blood monocytes are derived from platelets

Platelet glycoprotein IIb/IIIa (GP IIb/IIIa), the receptor complex for fibrinogen, has been regarded as a megakaryocyte/platelet lineage- restricted antigen. Recently, however, it has been reported that GP IIb/IIIa is expressed in blood monocytes. Studies were performed to establish the origin and immunological characteristics of monocyte- associated glycoproteins IIb and IIIa (GPs IIb and IIIa). Preparations of blood monocytes containing varying platelet-monocyte ratios were metabolically labeled with [35S]methionine with the expectation that any newly synthesized GPs IIb and IIIa would be monocyte-derived, since platelets have only rudimentary protein synthetic apparatuses. Analyses of sodium dodecyl sulfate (SDS) gels of homogenates of cell preparations containing from 200 to 5:1 platelet-monocyte ratios revealed that unlabeled GPs IIb and IIIa were readily immunoisolated using protein A-Sepharose immunobeads. However, fluorographic analyses of the same cell preparations pulse-labeled with [35S]methionine failed to demonstrate synthesis of GP IIb or IIIa. Additionally, no GP IIb or IIIa was detected when immunoisolation was carried out in pure preparations of monocytes containing less than 1:100 platelet-monocyte ratios and SDS acrylamide gels were stained by the sensitive silver stain method. Furthermore, heterologous polyspecific antisera and two monoclonal antibody preparations against GPs IIb and IIIa, which bound to platelets, failed to bind to monocyte membranes. Thus, evidence was presented that indicated that monocytes do not synthesize platelet GPs IIb and IIIa and that detection of these molecules in blood monocyte preparations reflects platelet contamination.     

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.     

Cytogenetic and molecular delineation of a region of chromosome 7 commonly deleted in malignant myeloid diseases

Loss of a whole chromosome 7 or a deletion of the long arm, del(7q), are recurring abnormalities in malignant myeloid diseases. To determine the location of genes on 7q that are likely to play a role in leukemogenesis, we examined the deleted chromosome 7 homologs in a series of 81 patients with therapy-related or de novo myelodysplastic syndrome or acute myeloid leukemia. Our analysis showed that the deletions were interstitial and that there were two distinct deleted segments of 7q. The majority of patients (65 of 81 [80%]) had proximal breakpoints in bands q11-22 and distal breakpoints in q31-36; the smallest overlapping deleted segment was within q22. The remaining 16 patients had deletions involving the distal q arm with a commonly deleted segment of q32-33. To define the proximal deleted segment at 7q22 at a molecular level, we used fluorescence in situ hybridization with a panel of mapped yeast artificial chromosome (YAC) clones from 7q to examine 15 patients with deletion breakpoints in 7q22. We determined that the smallest overlapping deleted segment is contained in a well- defined YAC contig that spans 2 to 3 Mb. These studies delineate the region of 7q that must be searched to isolate a putative myeloid leukemia suppressor gene, and provide the necessary cloned DNA for more detailed physical mapping and gene isolation.     

Association between physical performance characteristics and independence in activities of daily living in middle‐aged and elderly men

Aim: Functional status at one moment in time is a determinant of future functional status and survival. Physical deterioration tends to occur early in the disabling process; however, etiological questions remain. This study investigated the association between physical performance characteristics and functioning independently in middle‐aged and elderly men. Methods: A total of 400 independently‐living men aged 40–80 years were included in this cross‐sectional study. Preservation of function was measured using the Stanford Health Assessment Questionnaire. Physical characteristics were muscle strength and power by dynamometer, lung function, lower extremity function by physical performance score, and physical activity by Voorrips‐questionnaire. Logistic regression analysis was used to estimate the association between potential determinants and the dichotomized Health Assessment Questionnaire score. The odds ratios (OR) were adjusted for age, body mass index, education, socioeconomic status, smoking, alcohol and number of chronic diseases. Results: After adjustment for confounders, higher walking speed (OR = 2.96, 95% CI 1.31–6.72) and shorter time to carry out the chair stand test (OR = 0.84, 95% CI 0.76–0.94) were associated with a higher probability of being independent in activities of daily living (ADL). Borderline significant associations were found for higher lung function and higher leg strength with higher probability of being independent in ADL. No associations were found for grip strength, physical performance score, standing balance and physical activity. Conclusion: Lower body function and lung function were associated with a higher probability of being independent in ADL. Geriatr Gerontol Int 2013; 13: 274–280 .     

Isolation of an abnormal protein C molecule from the plasma of a patient with thrombotic diathesis

Protein C has been purified from the plasma of a patient with thrombotic diathesis. Both before and after isolation, the protein showed reduced capacity to hydrolyze synthetic substrates and to anticoagulate plasma. Proteolysis with the soluble thrombin- thrombomodulin complex proceeded normally and to completion as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Approximately one-third of the protein is functional, indicating a heterozygous defect. Indirect studies suggest that the abnormal component can bind to protein S and phospholipids. Both forms of activated protein C can also incorporate radiolabeled diisopropylfluorophosphate.     

Localization of pancreatic insulinoma: comparison of pre- and intraoperative US with CT and angiography

Methods of preoperative radiologic localization of insulinoma were compared in 52 patients, 44 of whom had solitary tumors. Examinations performed in these 44 patients were preoperative ultrasonography (US) in 28, angiography in 26, and computed tomography in 23. Prospective sensitivities were 61%, 54%, and 30%, respectively. Imaging sensitivities were lower for the eight patients with multiple insulinomas. In 28 of the 44 patients, intraoperative US was performed without the examiner being aware of the surgical findings. The sensitivity was 84%. Four insulinomas were not palpable but were visualized sonographically. The combined sensitivity of intraoperative US and surgical palpation for detecting solitary insulinomas was 100%. High-frequency intraoperative US is valuable for detecting occult solitary insulinomas and considerably useful for determining the proximity of insulinomas to the pancreatic and bile ducts.