Two types of secretory pathways are present in mammalian cells : constitutive secretion and regulated secretion (1). In the constitutive secretory pathway, which is found in all types of cells, secretory products are packed in small vesicles. Most of the proteoglycans and glycoproteins of the extracellular matrix are secreted in this way. The regulated secretory pathway, found in the more differentiated secretory cells, is mediated by specialized secretory granules. Hormones and neuropeptides are secreted in this way. Both the constitutive and regulated secretory pathways emanate from the trans-Golgi network (TGN).
Recent studies have indicated that the granin family (secretogranins/chromogranins) plays an important role in the sorting and aggregation of secretory products in the trans-Golgi network (TGN), and in the subsequent formation of secretory granules. The granin family is thought to be one of the mediators of the regulated secretory pathway (2-6). This family is widely expressed in endocrine (7-9) and neuronal cells (10-12), where they are stored in secretory granules together with various peptide hormones and neuropeptides. Three acidic sulfated proteins, chromogranin A (Cg A), secretogranin I (Sg I ; also called chromogranin B ; Cg B), and secretogranin II (Sg II) are well known as the major proteins in the family. Recently, three acidic secretory proteins, 1B1075 gene product (13), HISL-19 antigen (14) and 7B2 (15), have been thought to be members of the granin family, accordingly termed as Sg III, Sg IV and Sg V, respectively. However, it still remains open to discussion whether 1B1075 gene product, the HISL-19 antigen and 7B2 are true members of the granin family.
In this review, the possible roles of granin family, especially Cg A, Sg I (Cg B) and Sg II in the regulated secretory pathway are discussed, focusing on the sorting, aggregation of proteins in the TGN, and subsequent secretory granule formation.

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Animal cells synthesize cholesterol from acetyl CoA through a series of more than 20 enzymatic reactions. In addition, cells obtain cholesterol from plasma in the form of low-density lipoprotein (LDL), which is internalized via the LDL receptor and hydrolyzed to free cholesterol in lysosomes. Each cell must balance these internal and external sources while avoiding sterol shortage or overaccumulation. Both the biosynthetic and uptake pathways are well-regulated through feedback control. When cells are cultured in the presence of LDL, the activity of both 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase decline by more than 90% and the number of LDL receptors also decreases (1). In the absence of LDL, the cells maintain high activities of these two enzymes, which are rate-limiting enzymes of the biosynthetic pathway, and also maintain a large numberof LDL receptors on their surface. In this review we assess recent progress in understanding the mechanisms involved in transcriptional and posttranscriptional regulation of intracellular cholesterol metabolism.

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Human endothelial cells express antithrombotic properties by producing prostacyclin, heparan sulfate and plasminogen activator (PA). Using an established cell line, TKM-33, from human umbilical vein endothelial cells, the pericellular urokinase-type PA (u-PA) activity and expression of u-PA receptor (u-PAR) were investigated. The endothelial cells produced and secreted large amounts of u-PA and low levels of tissue-type PA (t-PA) and of PA inhibitor-1 (PAI-1), which were identified by immunohistochemical study and electrophoretic enzymography. Diisopropylfluoro-phosphate-treated ¹²⁵I-u-PA bound specifically to acid-treated monolayered endothelial cells with a K_d of 3.46±1.17 nM, and B_max of (0.09±0.04)×10⁶ sites/cell. mRNA of u-PAR was detected by using Northern blot analysis. Thus, these endothelial cells express u-PAR which bounds u-PA specifically. Phorbol myristate acetate (PMA) stimulation to the endothelial cells altered the K_d value to 3.18 ± 0.64 nM, and B_max value to (0.19±0.10)×10⁶ sites/cell, respectively. PMA treatment of endothelial cells increased u-PAR mRNA. Similarly, H7-treated endothelial cells showed a dose-dependent increase of u-PAR mRNA. However, PMA and H7 did not stimulate the expression of u-PA and t-PA mRNAs significantly. The expression of PAI-1 mRNA was increased by both PMA and H7. These findings suggest that the established endothelial cell line, TKM-33, possesses the character of endothelial cells and expresses u-PAR on their cell surface which is occupied by intrinsic u-PA secreted from the cells. The pericellular u-PA activity and the expression of u-PAR were regulated by protein kinase pathway.

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Non-excitable mutants of Paramecium caudatum, CNR (caudatum non-reversal) are unable to show the avoiding reaction due to the malfunction of voltage dependent Ca²⁺ channels. CNR mutants are divided into four complementation groups and are controlled by four genes at different loci : cnrA, cnrB, cnrC, and cnrD.
When CNR mutants are mated with the wild type, the mutant cell temporarily expresses the wild-type phenotype (called conjugation rescue). Transfer of diffusible wild-type gene products to the mutant mate through cytoplasmic connections is thought to be the cause of this phenomenon. Excitability of mutant cells during conjugation with wild type or other mutants controlled by different loci, and immediately after pair separation was examined by K⁺ test solution. Restorations of excitability in CNR mutants show distinguishable characters depending upon the different loci. cnrC showed wild type character indistinguishable from wild-type cells soon after pair formation. cnrD showed clear conjugation rescue but never reached the level of wild-type cells. cnrA showed only slight conjugation rescue immediately after pair separation. cnrB never showed conjugation rescue.

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A monoclonal antibody (mAbG3A5) recognizing p138 antigen was used to identify the Golgi cisternal membrane and determine behavior of Golgi fragments during mitosis in HeLa cells. At the start of mitosis, Golgi stacks identified with the mAbG3A5 antibody were fragmented into fine membrane vesicles which were distributed throughout the cytoplasm leaving only the region of the chromosome cluster unoccupied. On Western immunoblotting analysis, p138 was found associated with the membrane fraction prepared from mitotic HeLa cells having a buoyant density the same as that of interphase Golgi membranes. In addition to the fine Membrane vesicles, clusters labeled with mAbG3A5 antibody were frequently observed in mitotic cells. They Numbered 11 on average per mitotic cell and consisted of fine membrane vesicles of which membrane region was labeled with the mAbG3A5 antibody. This fact indicates that the membrane vesicles in mitotic Golgi clusters were also part of the fragments of Golgi cisternae. The number of mitotic Golgi clusters per mitotic cell was constant from prophase to anaphase, increasing twofold at telophase, although the average size of mitotic Golgi cluster remained unchanged throughout mitosis. The increase in number of mitotic Golgi clusters at telophase was accompanied by decrease in immunofluorescence of fine membrane vesicles. Treatment with nocodazole caused the disappearance of the mitotic Golgi clusters from prophase cells; however upon removal of it, they were reformed. These results suggest that during mitosis the Golgi apparatus were fragmented to fine membrane vesicles leaving only a part as mitotic Golgi clusters and were reassembled through tentative clustering of the fine membrane vesicles at the end of mitosis.The Golgi apparatus of mammalian cells is comprised of several stacks arranged in a circumscribed juxtanuclear region to which the microtubule organizing center localizes (Thyberg and Moskalewski, 1985). Studies using electron microscopy and immunocytochemical procedures have shown that Golgi apparatus are fragmented and dispersed throughout the cytoplasm in mitotic cells (Zeligs and Wollman, 1979; Lucocq et al, 1987). The fragmentation of Golgi apparatus during mitosis.

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Apoptosis is a prominent mechanism of programmed cell death in the immune system. In the thymus apoptosis is responsible for the deletion of autoreactive T-cells during thymic differentiation. The typical features of apoptosis are characterized by nuclear and cytoplasmic morphologic changes, along with cleavage of chromatin at regularly spaced sites. Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerizing enzyme found at an early stage of T and B lymphocyte differentiation, which generates diversity in the DNA sequence of immunoglobulin (Ig) or T cell receptor (TCR). The combined evaluations of thymocyte morphological features, immune phenotype and thymic topography associated to TdT expression allow the recognition of three different thymocyte subpopulations, characterized by small-size, intermediate-size and large-size. The results of this study show that dexamethasone (Dx)-treatment induces cell death via apoptosis involving distinct transformations related to differentiation stages of thymic subpopulations. Intermediate and small-size thymocytes that are TdT-negative or weakly positive at nuclear level are Dx sensitive. In contrast the large-size thymocytes, highly TdT positive, corresponding to the undifferentiated cells, do not show significant morphological modifications and TdT positivity to Dx-treatment. Immunocytochemical analysis shows that Dx-treatment does not affect TdT synthesis but morphological changes, occurring during apoptotic process, are responsive to intracellular movement and intranuclear arrangement of the TdT.

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Under starvation conditions, the yeast S. cerevisiae sequesters its owncytoplasmic components by forming autophagosomes with double membrane in the cytoplasm. The autophagosome then fuses with the vacuolar membraneand delivers its owncytoplasmic componentsinto the vacuole in the form of an autophagic body with a single membrane (Baba, M., Takeshige, K., Baba, N., and Ohsumi, Y. 1994. J. Cell. Biol., 124: 903-913). Weexamined membrane structures involved in the autophagy induced by nitrogen-starvation by using freeze-replica method. The most conspicuous characteristic of the autophagic body is that the intramembrane particles were rarely detected on either the protoplasmic or exoplasmic face of its fracture membrane. This morphological feature of the fractured face was clearly different from other intracellular organelles. Next we examined the autophagosomal membrane. The inner membrane of autophagosome was also intramembrane particlefree, and its morphological feature was identical to the membrane of autophagic body. At the fusion site between autophagosome and vacuole we obtained direct evidence that two differnt membranes, the outer membrane of autophagosome and vacuolar membrane, became continuous by using freeze-etching technique. From these results we concluded that the autophagic body originated from the inner membrane of the autophagosome, and its membrane reflects an intrinsic feature of autophagosomal membrane. The outer membrane of autophagosome had only a few intramembrane particles and may be differentiated from the inner membrane. In cells under nitrogen-starvation condition, the density of intramembrane particles of vacuolar membrane decreased beyond that in control cells.

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Fusion regulatory proteins (FRPs) regulate virus-mediated cell fusion and multinucleated giant cell formation of monocytes. Anti-FRP-1 mAbs immunoprecipitated 80 kDa and 38 kDa proteins from HeLa cells. After long exposure other bands were detected, suggesting the presence of molecule(s) associated with FRP-1. To identify the molecule(s), we prepared monoclonal antibodies against immunoaffinity-purified FRP-1 complex derived from membrane fractions of HeLa cells. Immunofluorescence microscopy revealed that these monoclonal antibodies recognized the intracytoplasmic molecules in HeLa cells. Using immunoblotting, the antibodies reacted with 200 kDa, 70 kDa, 55 kDa and 35 kDa molecules, so we designated these molecules as FRP related molecules (FRMs). Subsequently, we performed gene cloning from a HeLa λgt11 cDNA library using anti-FRM mAbs and immunoblotting analysis with either purified cytoskeletal proteins or specific antibodies against various cytoskeletal proteins. Three kinds of positive clone were obtained, which encoded partial sequences of vimentin, tropomyosin, and heat shock cognate protein 70 (hsc70). The 200 kDa molecule was expected to be a myosin heavy chain, judging from the immunoblotting pattern. Immunoblotting confirmed that these purified proteins were readily recognized by anti-FRM mAbs. Furthermore, anti-vimentin and anti-myosin mAbs reacted with the precipitates by anti-FRP-1 mAb, indicating a physical association between FRP-1 molecules and these cytoskeletal proteins. Whenanti-FRP-1 mAb was added to culture fluids of HeLa cells, the cell-shape and immunofluorescence-pattern stained with anti-FRM mAbs changed. Taken together, the fusion regulatory molecular complex is suggested to consist of at least FRP-1, hsc70, actomyosin and vimentin systems.

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