Molecular characterization of a corticotropin (ACTH) receptor

We have used a new methodology to generate a monospecific antiserum to the corticotropin (ACTH) receptor on mouse Y-1 adrenal cells. Using immunoaffinity chromatography the ACTH receptor was purified, and the molecular structure and 125I-ACTH binding characteristics were determined. A molecular weight (Mr) of 225 000 was determined for the complete ACTH receptor as analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The receptor was composed of 4 subunits with Mr 83 000, 64 000, 52 000 and 22 000. The 83 and 52 kDa subunits were disulfide linked and non-covalently associated with the 64 and 22 kDa subunits. The ability to specifically bind 125I-ACTH was localized to the 83 kDa subunit. The purified receptor possessed binding affinities of 3.4 X 10(10) M-1 and 1.0 X 10(9) M-1 as determined by Scatchard analysis.     

Ultrastructural analysis of membrane-bound polysomes in human myeloma cells

Summary We prepared ultra-thin sections of human myeloma cells, in which the rER was cut tangentially, and studied the make-up and distribution of membrane-bound polysomes electronmicroscopically. In IgG myeloma large and small polysomes were detected. The polysome distribution curve showed a high peak at 7 ribosomes and a lower peak at 17–18 ribosomes. IgA-, IgD- and IgE myeloma, as well as macroglobulinemia, showed peaks at 7 and 13 ribosomes. BJP myeloma manifested a sharp peak only at 7 ribosomes. Our results suggest that BJP myeloma has only small polysomes participating in L-chain synthesis, while the other myelomas exhibited large and small polysomes participating in H- and L-chain synthesis, respectively. The quantitative ratio of small and large polysomes was determined on the basis of an analytically corrected direct count.Supported in part by grants-in-aid for Scientific Research (748103, 867050, 167092, 267108, 777124) from the Ministry of Education, Science and Culture of Japan     

Ultrastructural evaluation of periodate-reactive glycoconjugates in human leukaemia cells

Periodate-reactive glycoconjugates in human leukaemic cells were examined electron microscopically by the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method. Granules in ALL cells were classified into 4 types based on PA-TCH-SP staining features. Abnormal granules containing glycogen were observed only in children with treatment-resistant ALL. Cytoplasmic granules in leukaemic cells of patients with AML and acute monocytic leukaemia exhibited moderate reactivity. The distribution pattern of glycogen in the cytoplasm of leukaemic cells was classified into 3 types, one lacking glycogen, one containing small glycogen particles scattered throughout cytoplasm, and one showing clusters of glycogen particles. Cells with glycogen clusters were observed in ALL cells and in erythroblasts from patients with erythroleukaemia. PA-TCH-SP reactivity was detected in the rough endoplasmic reticulum in acute promyelocytic leukaemia but not in ALL or other types of AML. Megakaryoblasts in megakaryocytic crisis of chronic myelogenous leukaemia exhibited characteristic PA-TCH-SP reactivity similar to that of normal megakaryocytes.     

Ultrastructural features of mast cells in human omental veins

Mast cells are relatively common in human omental veins where they generally occur in the tunica adventitia and among the peripheral smooth muscle cells of the tunica media. The distance between mast cells and adjacent muscle cells is often as short as 1 micrometer. The tunica intima does not contain mast cells. With few exceptions the typical mast cells were oval in shape, had numerous slender projections and contained granules (0.3-0.7 micrometer) with characteristic stacks of lamellae and scrolls. A few elongated cells with processes containing granules filled with dense homogeneous matrix material were also detected and could represent so-called chromaffin mast cells. Noradrenergic axons and terminals identified by their large (85 nm) and small (50 nm) dense-cored vesicles are present in the vicinity of many mast cells which could allow various interactions between the two cell types and their released amines and ATP. Electrical field stimulation which affects the nerve terminals does not change the ultrastrcutre of the mast cells unless the alpha-blocking agent phentolamine (7.5 X 10-7 M) is present during the stimulation, when degranulation results.     

人肝癌细胞与LAK细胞凋亡的超微结构比较

目的比较观察人肝癌细胞株ＢＥＬ７４０２细胞与ＬＡＫ细胞体外凋亡的超微结构．方法将ＢＥＬ７４０２细胞、人血树突状细胞和人ＬＡＫ细胞（淋巴因子和ＰＨＡ激活的杀伤细胞）共同置于含１００ｍＬ／Ｌ新生牛血清的１６４０培养液内，在３７℃，５０ｍＬ／Ｌ，ＣＯ２，饱湿条件下培养６ｈ．另将ＬＡＫ细胞单独置于含有环磷酰胺的上述培养液内，在同样条件下培养６ｈ．收集沉淀细胞制备电镜样本．细胞经２５ｇ／Ｌ戊二醛和２０ｇ／Ｌ锇酸双固定，ＰＤＡＰ包埋，超薄切片，常规染色，透射电镜观察．结果凋亡的ＢＥＬ７４０２细胞与ＬＡＫ细胞，其细胞核染色质浓缩、边集，核碎裂，离散的碎片外包以双层膜，一些线粒体也变得致密．然而，在有些凋亡的ＢＥＬ７４０２细胞内，可见浓缩的细胞碎片向细胞外出芽隆起，形成膜包的凋亡小体；其线粒体和粗面内质网的变化也较为复杂．结论凋亡的ＢＥＬ７４０２细胞与ＬＡＫ细胞均具有凋亡细胞的基本特征，但是两种凋亡细胞具体的形态表现不尽一致．表明细胞内的主要细胞器主动参与了凋亡过程．     

Studies on immunoreactive ACTH from human term placenta. (I) Detection of a high molecular weight-immunoreactive ACTH in term placenta (author's transl)

Extracts of human term placenta were fractionated by Sephadex G-75 gel filtration and assayed for immunoreactive ACTH. Both high and low molecular weight protein fractions were detected to be immunologically reactive toward anti-human ACTH (1--39 alpha) antibody. For the extraction of low molecular weight ACTH from human term placenta (pl. -ACTH), a glacial acetic acid-acetone mixture was employed, while a pH 3.0-HCl solution was used for high molecular weight immunoreactive ACTH. The high molecular weight immunoreactive ACTH fraction (F-I), co-eluted with horse hemoglobin from a Sephadex G-75.column in 0.1M acetic acid, was essentially devoid of low molecular weight materials as revealed by polyacrylamide gel disc electrophoresis at pHs 9.5 and 4.3. Tryptic digestion of F-1 at pH 8.1 and 37 degrees C for 4 hr with E/S of 1/100, followed by fractionation with a Sephadex G-75, resulted in the formation of lower molecular weight fragments. One fragment was eluted at the same position as that of porcine ACTH with a recovery of 86% of immunoreactivity of F-I. Another fragment which was eluted last exhibited positive beta-endorphin receptor binding activity. These results suggest the presence of a common precursor protein to ACTH and beta-endorphin in human term placenta.     

Comparison of ACTH and ACTH precursor peptides secreted by human pituitary and lung tumour cells in vitro

The molecular forms of ACTH secreted by established human small cell lung cancer (SCLC) cells and primary cultures derived from a bronchial carcinoid tumour, a pituitary adenoma and hyperplastic pituitary tissue have been characterized by Sephadex G-75 chromatography and quantified with two novel immunoradiometric assays for ACTH and ACTH precursor peptides. Pro-opiomelanocortin (POMC; Mr 31,000) and pro-ACTH (Mr 22,000) were secreted by all cell types. No smaller peptides were identified in the culture media from SCLC and bronchial carcinoid cells, implying a deficiency in the enzymes and/or intracellular organelles required for extensive POMC processing. A more heterogeneous profile of ACTH-containing peptides was produced by cells of pituitary origin, indicating more extensive proteolytic processing of POMC. However, the major peptide secreted by cells from a large aggressive pituitary adenoma was unprocessed POMC (Mr 31,000). These results suggest that both lung and pituitary cells in vitro retain their in-vivo pattern of POMC processing and provide valuable models in which to study the regulation of ACTH synthesis and secretion.     

Ultrastructural characterisation of macrophages (type A cells) in the synovial lining

Summary The ultrastructural localisation of class II and macrophage antigens has been sought in the intimal layer of the human synovium. The cells described as type A in early morphological studies are shown to express both class II antigens and two markers of the macrophage/monocyte lineage of cells, OKM1 and MAB 24. The morphological type B cells were found to express none of these antigens. The findings are consistent with the idea that the synovial lining comprises two cell types, bone-marrow derived macrophages and mesenchymal fibroblasts.     

Multipotential pituitary cells that contain adrenocorticotropin (ACTH) and other pituitary hormones.

Anterior pituitary cells that contain adrenocorticotropin (ACTH) are morphologically and functionally heterogeneous. Immunolabeling has identified stellate subtypes with scattered or peripherally arranged granules, and ovoid or irregularly shaped cells with a denser accumulation of granules, which overlap morphologically with other cell types such as small gonadotropes or thyrotropes. Dual cytochemical labeling studies on the same or serial fields have identified cells that store ACTH with LH, FSH, TSH, or prolactin. Evidence is presented to suggest that they are multipotential cells with the capacity to augment corticotropes or one of the other cell populations.