Separation and analysis of subcellular organelles in a human promyelocytic leukemia cell line, HL-60: application to the study of myeloid lysosomal enzyme synthesis and processing

We describe a system for analysis of the intracellular pathways in the biosynthesis and packaging of functionally important proteins in human myeloid cells. The human promyelocytic cell line HL-60 was used since peripheral blood neutrophils are terminally differentiated and do not actively synthesize protein. Cells were disrupted by nitrogen cavitation and subcellular organelles in postnuclear supernatant separated on a discontinuous gradient of Percoll modified to resolve organelles important in protein synthesis. This Percoll gradient separated azurophilic granules from less dense organelles and partially separated the less dense organelles from one another. Approximate densities of organelles identified by electron microscopy and by biochemical markers are azurophilic granules, 1.102 g/mL; endoplasmic reticulum, 1.039 g/mL; Golgi apparatus, 1.032 g/mL; and plasma membrane, 1.027 g/mL. We validated the utility of this method of subcellular fractionation by examining intracellular transport of myeloperoxidase, a myeloid lysosomal enzyme present in azurophilic granules. The subunits of mature myeloperoxidase (molecular weight [mol wt] = 59,000 and 13,500) cosediment with biochemical markers for lysosomes, whereas the large-mol wt (89,000) precursor forms cosediments with biochemical markers of less dense organelles. Within the limits of assay sensitivity, the 89,000-mol wt precursor is enzymatically inactive and has no spectral evidence for a heme group, suggesting that precursors of myeloperoxidase may undergo proteolytic maturation in a prelysosomal compartment with concomitant incorporation of a heme group and acquisition of enzymatic activity. This system of analysis should be suitable for the identification, subcellular localization, and maturational analysis of other myeloid lysosomal enzymes as well as functionally important membrane proteins.     

Fodrin and band 4.1 in a plasma membrane-associated fraction of human neutrophils

Erythrocytes possess a well-characterized submembranous filamentous network which interacts with transmembrane glycoproteins and is composed primarily of spectrin, ankyrin, band 4.1, and short actin filaments. An analogous structure was recently described in platelets. Human polymorphonuclear leukocytes (PMNs) were examined for the presence and plasma membrane association of similar proteins. Isolated PMNs, free of contamination with erythrocytes or platelets, were disrupted by nitrogen cavitation and separated into subcellular organelles on a discontinuous Percoll gradient. Detergent lysates of plasma membrane vesicles, but not azurophilic or specific granules, contained insoluble actin filaments and associated proteins. Immunoblots of detergent-insoluble plasma membrane fractions contained proteins recognized by antibodies to brain fodrin and erythrocyte band 4.1, whereas blots probed with antibodies to erythrocyte spectrin and ankyrin were negative. Fodrin and band 4.1 were not detected in granule fractions, but some fodrin was present in the cytosol. The association of proteins related to fodrin and band 4.1 with the plasma membrane suggests that PMNs contain a submembranous skeleton structurally analogous to that of erythrocytes and platelets. The specific function of these proteins and their structural organization in human PMNs await further study.     

Do positive or negative experiences of social support relate to current and future health? Results from the Doetinchem Cohort Study

Background  

Cross-sectional studies have reported associations between social support and health, but prospective evidence is less conclusive. This study aims to investigate the associations of positive and negative experiences of social support with current and future lifestyle factors, biological risk factors, self-perceived health and mental health over a 10-year period.     

Human ovarian granulosa cells and follicular fluid indices: the relationship to oocyte maturity and fertilization in vitro

The study investigates the correlation between oocyte maturity and fertilization and a variety of hormonal parameters in follicular fluid and ovarian granulosa cells. A methodology for purification of granulosa cells from contaminating blood cells is also established. A total of 63 follicular aspirates were collected at oocyte retrieval from 30 women superovulated using the long luteinizing hormone- releasing hormone (LHRH analogue)/human menopausal gonadotrophin regimen. Oestradiol, progesterone, testosterone and human chorionic gonadotrophin (HCG) were quantified in follicular fluid and granulosa cells were immunostained for human chorionic gonadotrophin. Immunopurification of granulosa cells from contaminating blood cells was performed. HCG in follicular fluid was significantly high in follicles yielding immature (grade 3) oocytes (P=0.002); there was no correlation with fertilization. Aspirates from follicles containing mature (grade 1) oocytes and oocytes that subsequently fertilized had significantly more granulosa cells immunobound to HCG (P < 0.001, P=0.02). Moreover, the immunomagnetic purification technique provided >98% pure population of granulosa cells. The data demonstrate that HCG in follicular fluid and on granulosa cells may help to predict oocyte maturity and fertilization. Furthermore, immunomagnetic beads provide a reliable procedure for the purification of ovarian granulosa cells.      

Patterns of contrast enhancement of benign and malignant hepatic neoplasms during bolus dynamic and delayed CT

Bolus dynamic and delayed computed tomographic (CT) scans of the liver were evaluated in 43 patients with 54 hepatic hemangiomas and 111 patients with primary or secondary malignant hepatic neoplasms. Twelve patterns of contrast enhancement were recognized during the bolus dynamic phase and delayed scanning. A "typical" CT pattern for hemangiomas (present in 29 of 54 hemangiomas [53.7%]) was established: (a) diminished attenuation prior to intravenous contrast medium administration (excluding lesions arising in a liver with diffuse fatty infiltration), (b) peripheral contrast enhancement during the bolus dynamic phase, and (c) complete isodense fill-in on delayed scan images. Using these criteria, we distinguished hemangiomas from malignant neoplasms in most patients. Only one of 63 (1.6%) malignant neoplasms manifested these typical CT criteria of hemangioma. There is an 86% chance that a lesion with the typical CT appearance of hemangioma is actually a hemangioma, even when found in a patient with a known nonhepatic primary neoplasm.     

平胃散对湿阻中焦模型大鼠血浆抗利尿激素及红细胞内钠、钾浓度的影响

目的:探索湿阻中焦证的病理机制,并探讨平胃散对湿阻中焦证的作用机理。方法:选用湿阻中焦证大鼠模型,给予平胃散配伍利水药,观测各组大鼠血浆抗利尿激素(ADH)的浓度及红细胞内电解质Na~+、K~+浓度。结果:湿阻造模组大鼠与正常组相比ADH显著升高(P<0.01);细胞内的Na~+增高,K~+显著降低(P<0.05)。给予平胃散后,高、中、低剂量组及加泽泻组大鼠ADH基本恢复正常;细胞内的Na~+下降至接近于正常,K~+尤明显变化;不造模给药组与正常组比较ADH显著升高(P<0.05),Na~+、K~+明显下降(P均<0.01)。结论:(1)血浆ADH浓度升高、细胞内Na~+增多、K~+降低在湿阻中焦证形成中起重要作用。(2)平胃散治疗湿阻中焦证的作用机理与调节ADH和细胞内Na~+、K~+浓度有关,且可能存在双向调节机制。