Localization of synaptotagmin, a Ca2+-binding protein associated with synaptic vesicles, in regenerating axons was investigated by immunocytochemistry in the injured rat sciatic nerves. The early regenerating axonal sprouts emanating from nodes of Ranvier exhibited synaptotagmin immunoreactivity on vesicles, vacuoles and surface plasma membranes. In the well-developed regenerating sprouts extending through the space between Schwann cell basal lamina and myelin sheath of the parent axon, the growing tips, i.e., typical growth cones, exhibited an intense immunoreaction on vesicles, vacuoles and surface plasma membranes, while the stem regions where the sprouts were continuous with the parent axon exhibited almost no immunoreaction on any organelles including plasma membranes. These findings suggest that synaptotagmin-immunoreactive vesicles and vacuoles might be utilized for the supply of membrane components to the surface plasma membrane in the growth cone. Synaptotagmin which is known to regulate membrane fusion of synaptic vesicles with presynaptic plasma membranes in a Ca2+-dependent manner in synapses may participate in the regulation of the membrane fusion between the vesicles and plasma membranes in growth cones.
In the present work the haemocytes of mussels Mytilus galloprovincialis (Mollusca, Bivalvia) have been studied by enzyme cytochemistry in order to investigate the light and electron microscopical distribution of two lysosomal marker enzymes, acid phosphatase (AcPase) and arylsulphatase (ASase). Both hyalinocytes and granulocytes show positive reaction products for the two enzymes but granulocytes are far more reactive. In the hyalinocytes, AcPase and ASase activities are observed in a few pleomorphic lysosomes. In the granulocytes, the reaction product for the enzymes is found in Golgi bodies; AcPase is restricted to small trans-Golgi vesicles while ASase is localized in all the cisterns and vesicles. In addition, some but not all specific granules show both AcPase and ASase activities, mostly associated to their periphery. These results confirm that the granules of mussel granulocytes, although apparently similar in morphology, are functionally heterogeneous with regard to enzyme composition. Cortical or peripheral vesicular organelles are negative for AcPase but some are positive for ASase, indicating their endolysosomal nature. Larger vesicles containing remnants of algal cells do show strong AcPase activity and are thus considered as phagolysosomes. In controls incubated without substrates no precipitation of reaction products was detected either for AcPase nor for ASase.
Two monoclonal antibodies (mAbs) were raised as probes to study the specificity of a growth cartilage (GC). Immunized antigen was a homogenate of cultured GC cells from young rabbit ribs. Raised mAbs were investigated as to their reactivity with undecalcified or decalcified paraffin-embedded rabbit tissues. By immunohistochemical examination of the young rabbit tissues, one mAb, mAb-A, reacted with non-osteogenic cartilage, muscle, bone marrow cells, dermis, epidermis, gut, tendon, lung, liver, heart, spleen, and thymus, but not with the growth cartilage. The other mAb, mAb-B, however, reacted specifically with late hypertrophic and calcified growth cartilage zones, but not with other non-osteogenic cartilage or other tissues. These two mAbs are useful probes to distinguish osteogenic cartilage from nonosteogenic cartilage and other tissues by negative reactivity as in mAb-A, and by positive reactivity as in mAb-B. Further-more, these results imply the existence of certain molecules to distinguish osteogenic cartilage from non-osteogenic cartilage.
The localization of four heat shock proteins (HSPs), ubiquitin, HSP27, HSP72/73 and HSP90, was studied immunohistochemically in human tonsils. Ubiquitin was positive in follicular dendritic cells (FDCs) in the germinal centers (GCs), and interdigitating cells (IDCs) in the interfollicular areas. Fibroblastic reticulum cells (FRCs) in the mantle zones were weakly stained. Ubiquitin was localized in the cytoplasm and nucleus. HSP27 was expressed in FDCs, and in some IDCs and FRCs. HSP27 was localized mainly in the cytoplasm, showing a diffuse distribution. HSP72/73 was stained in FDCs in the light zones (LZs) of GCs. IDCs and FRCs also showed staining for HSP72/73. HSP72/73 was localized diffusely in the cytoplasm and/or granularly in the nucleus. HSP90 was positive in FDCs in the LZs. IDCs and FRCs were also stained for HSP90, which was localized heterogeneously in the cytoplasm. Our results show that HSP72/73 and HSP90 are strongly expressed in FDCs in the LZs, and the presence of HSP72/73 in the nucleus as well as cytoplasm of these FDCs suggests that this type of cell might be stressed. Furthermore, HSP27 appears to be reacted to only FDCs, and also some IDCs and FRCs.
The short-term effects after experimental partial and total denervation on histochemical fiber type profiles were studied in rat soleus muscles by the myofibrillar ATPase stain. In 58 control soleus muscles, a total of 155788 fibers were counted, comprising type 1 (75.1%), type 2A (13.3%), type 2B (1.4%) and type 2C (10.3%), respectively. In 34 animals with total denervation, including 17 with soleus nerve resection at 1cm before entering the soleus muscle and 17 with total sciatic neurectomy, the proportion of type 1 fiber was decreased in denervated muscles to 39.1% 5 weeks after the procedures. In parallel with a decrease in type 1 fiber proportion, undifferentiated type 2C fibers increased, thereby suggesting muscle fiber type dedifferentiation taking place in totally denervated muscle. In contrast, 24 muscles with partial denervation showed a decreased proportion of type 1 fibers at the initial stage followed by an increase in type 1 fiber to 89.0% 5 weeks after denervation, resulting from an active reinnervating process.
Carcinoembrionic antigen (CEA) is a glycosyl-phosphatidylinositol (GPI) anchored protein which has N-glycosylation sites. To determine whether it acts as a cell surface adhesion molecule or functions as molecular transportation, we studied the stability and regulation of CEA expression in a human gastric cancer cell line (HPE-GAC-T) using immunological techniques. Amounts of CEA in the cytoplasm of GAC-T cells were increased by stimulation with 12-O-tetra-decanoylphorbol-13-acetate (TPA), with or without calcium ionophore A23187. Synergistic effects of TPA with A23187 (TPA/A23187) on the cell surface expression of CEA were observed dose- and time-dependently. Tunicamycin, an inhibitor of N-linked glycosylation, decreased the total cellular CEA level of mature form of 170kD. Monensin, an inhibitor of intracellular transport, also decreased the amount of high molecular weight (170, 000) CEA, but the smaller molecular weight (140, 000) CEA that appeared by TPA/A23187 treatment remained unaffected. Both drugs suppressed GAC-T cell growth moderately at high doses used, but did not suppress the increased cell surface expression of CEA induced with TPA/A23187. Up-regulation and stability of the membrane expression of CEA may involve the physiological role CEA plays on the cell surface.
Mouse intra-acrosomal antigens, recognized by monoclonal antibodies, MN7 and MC41, were analyzed by SDS-PAGE and Western blotting. Both the MN7- and MC41-antigens were glycoproteins of 90kDa and 200kDa, respectively. However, the two antigens exhibited different biochemical properties; the MN7-antigen was extracted only in the acidic condition (pH 3.0), while the MC41-antigen was detectable in both acidic and neutral conditions. The releasing process during the acrosome reaction was examined by immunoelectron microscopy. The MN7-antigen, which was initially distributed uniformly in the entire acrosome, gradually disappeared from the acrosome, being localized once at the cortex-medulla border region at the middle stage, and dispersed without close contact with the vesiculated membrane. By contrast, the MC41-antigen, which was initially restricted to the cortical region of the anterior acrosome, was localized on the electrondense materials lining closely to the fenestrated membrane at the middle stage, and dispersed keeping in close contact with the vesicles. These findings indicate that the antigens originally segregated into different subdomains can be differentially released during the acrosome reaction.
An in vitro study employing dissociated matrix cells obtained from the rostral part of mouse neural tube or forebrain vesicle was done in an attempt to elucidate their cellular characteristics. We found a reaggregate formation with an organoid architecture. The organization of the reaggregate was studied by transmission as well as scanning electron microscopy, and by immunocytochemistry. After three days in a stationary culture, dissociated matrix cells formed a spherical, organoid reaggregate having a central lumen. The size of the reaggregate varied from approximately 100μm to 200μm. The transmission electron microscopy revealed that the reaggregate wall is composed of two types of cells; one has an electron-lucent cytoplasm with few organelles except for polysomes, whereas the other contains an electron-dense cytoplasm with many microtubules and mitochondria. The latter type of cell was located in the marginal portion of the wall intermingled with a large number of cellular processes. These cells were immunostained with anti-neurofilament antibodies and regarded as neuroblasts. The luminal surface was composed predominantly of the former cell type showing well-developed junctional complexes in between them and having cilia on its luminal side. Mitotic figures were frequently demonstrable adjacent to the lumen.
P70, a 70kDa protein found in the cobalt-induced epileptogenic cortex of the rat brain, has been implicated in epileptogenesis. In the gerbil brain, we found that an antibody directed against rat P70 reacts with a similar protein which is localized mainly in the nucleus and Golgi apparatus of the neurons of the superior colliculus and substantia nigra. Surprisingly, similar immunoreactivity is seen in young pups which have not yet experienced motor seizures, and in animals from seizure-resistant group, indicating that this protein may have a role other than direct participation in epileptogenesis as assumed to be the case for rat P70.