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The axonal–myelin sheath interface of vertebrate myelinated axons possesses special structural complexities, and there may be an intercellular macromolecular traffic transversing the periaxonal cleft that spans the internodal axon. By conventional electron microscopy and serial sectioning, we observed a category of double-walled vesicles at the axonal–myelin sheath interface, which often contained ribosome-like particles or endoplasmic reticulum. Some of them were demonstrated to continue with the subjacent axon with a thin stalk. In addition, we described a special category of axonal invaginations, probably mediated by subsurface cisternae. The functional implications of these specialized structures were discussed.

The localization of calbindin-D28K (CB) was studied immunocytochemically in laminae I and II of the dorsal horn and in spinal ganglia in the chicken, and compared with the distribution of substance P (SP) using double immunolabeling. At the light microscopic level, CB immunoreactivity was observed most intensely in the lamina II using the avidin-biotinylated peroxidase complex (ABC) and immunofluorescence methods. At the electron microscopic level using the ABC method, CB immunoreactivity was observed in the following three neuronal elements: 1) the scalloped central terminal with many dense-cored vesicles (DCVs) in the synaptic glomerulus; 2) some vesicle-containing dendrites (VCDs) inside or outside the synaptic glomerulus; and 3) some axon terminals outside the synaptic glomerulus. The CB-immunoreactive (IR) VCDs in the synaptic glomerulus often formed reciprocal synapses with the central terminal. Strong immunoreactivity was observed at the postsynaptic membrane of CB-IR elements. Double immunofluorescence and immunolabeling methods at the electron microscopic level showed that CB and SP colocalized in the scalloped central terminal with DCVs of the synaptic glomerulus. Almost all SP-IR neurons in the spinal ganglion revealed the coexistence of CB in serial sections in the chicken. In light of previous biochemical and physiological reports, our findings suggest that CB - coexisting with SP - plays an important role in the control of pain transmission through its strong Ca(2+)-buffering action in the chicken.

Vesicular integral protein of 36 kDa (VIP36) is an intracellular lectin recognizing high-mannose type glycans and is highly expressed in salivary glands, especially the parotid gland, which secretes alpha-amylase in large quantities. Here immunoelectron microscopy demonstrated that VIP36 was primarily localized to secretory vesicles in the glandula parotis of the rat, where alpha-amylase also resided. A secretory vesicle fraction, prepared by Percoll density gradient centrifugation, contained both VIP36 and alpha-amylase. Moreover, alpha-amylase that was localized to these secretory vesicles contained high-mannose type glycans. In addition, VIP36 coprecipitated with alpha-amylase in an endo H treatment-sensitive manner. These results suggest that VIP36 is involved in the secretion of alpha-amylase in the rat parotid gland.

The vesicular integral membrane protein VIP36 belongs to the family of animal lectins and may act as a cargo receptor trafficking certain glycoproteins in the secretory pathway. Immunoelectron microscopy of GH3 cells provided evidence that endogenous VIP36 is localized mainly in 70-100-nm-diameter uncoated transport vesicles between the exit site on the ER and the neighboring cis-Golgi cisterna. The thyrotrophin-releasing hormone (TRH) stimulation and treatment with actin filament-perturbing agents, cytochalasin D or B or latrunculin-B, caused marked aggregation of the VIP36-positive vesicles and the appearance of a VIP36-positive clustering structure located near the cis-Golgi cisterna. The size of this structure, which comprised conspicuous clusters of VIP36, depended on the TRH concentration. Confocal laser scanning microscopy confirmed the electron microscopically demonstrated distribution and redistribution of VIP36 in these cells. Furthermore, VIP36 colocalized with filamentous actin in the paranuclear Golgi area and its vicinity. This is the first study to show the ultrastructural distribution of VIP36 in the early secretory pathway in GH3 cells. It suggests that actin filaments are involved in glycoprotein transport between the ER and cis-Golgi cisterna by using the lectin VIP36.

VIP36, an intracellular lectin that recognizes high mannose-type glycans (Hara-Kuge, S., Ohkura, T., Seko, A., and Yamashita, K. (1999) Glycobiology 9, 833-839), was shown to localize not only to the early secretory pathway but also to the plasma membrane of Madin-Darby canine kidney (MDCK) cells. In the plasma membrane, VIP36 exhibited an apical-predominant distribution, the apical/basolateral ratio being approximately 2. Like VIP36, plasma membrane glycoproteins recognized by VIP36 were found in the apical and basolateral membranes in the ratio of approximately 2 to 1. In addition, secretory glycoproteins recognized by VIP36 were secreted approximately 2-fold more efficiently from the apical membrane than from the basolateral membrane. Thus, the apical/basolateral ratio of the transport of VIP36-recognized glycoproteins was correlated with that of VIP36 in MDCK cells. Upon overproduction of VIP36 in MDCK cells, the apical/basolateral ratios of both VIP36 and VIP36-recognized glycoproteins were changed from approximately 2 to approximately 4, and the secretion of VIP36-recognized glycoproteins was greatly stimulated. In contrast to the overproduction of VIP36, that of a mutant version of VIP36, which has no lectin activity, was of no effect on the distribution of glycoproteins to apical and basolateral membranes and inhibited the secretion of VIP36-recognized glycoproteins. Furthermore, the overproduction of VIP36 greatly stimulated the secretion of a major apical secretory glycoprotein of MDCK cells, clusterin, which was found to carry at least one high mannose-type glycan and to be recognized by VIP36. In contrast to the secretion of clusterin, that of a non-glycosylated apical-secretion protein, galectin-3, was not stimulated through the overproduction of VIP36. These results indicated that VIP36 was involved in the transport and sorting of glycoproteins carrying high mannose-type glycan(s).

The lamina propria of the large intestine is rich in macrophages, and they might be one of the first lines of the host defense in enterohemorrhagic Escherichia coli (EHEC) O157 : H7 infection. Although macrophages were infected with them, they can survive the EHEC O157 infection. We examined the structural rearrangements of the actin cytoskeleton during the microbial infection process. Macrophage actin filaments were rearranged in the following sequence ; 1) disappearance of the actin filament bundles in the cytoplasm, 2) accumulation of actin filaments under the cell surface, and 3) construction of actin networks underlying the endosome membrane. Before infection, actin filaments were distributed under the cell surface and in bundles located in the macrophage cytoplasm. Within 2 min, infection caused a rapid and marked loss of the actin filament bundles that had run parallel to the long axis of the cell. Concomitant with the loss, actin filaments became more markedly distributed under the cell surface. In the formation of the endosome, new networks of actin filaments were constructed below the phagosome membrane. The networks contained a large amount of actin as well as α fodrin-like immunoreactivity. The thickness of the networks reached about 400 nm under the phagosome membrane. The actin networks disappeared again after the bacterial digestion. The results of this study showed that actin filaments undergo three major rearrangements of the actin filaments during the infection in macrophages, and suggested that the third rearrangement is mediated by actin-binding proteins, such as α fodrin-like molecules. These morphological changes in macrophages were not clear after infection with other strains of Escherichia coli.

The basal ganglia and cerebral cortex have close relationship through the striato-pallido-thalamo-cortical loop. The loop circuit is composed of several sub-loops in connection with the modular organization in the basal ganglia, such as the putamino-pallidal and caudate-nigral systems. We sought to reveal functional similarities and differences of these sub-loop systems in the movement mechanisms. Single neuron activity in the primate putamen and caudate nucleus was examined during learned arm movements initiated in 3 different behavioral contexts: Monkeys depressed 3 push buttons laid out in a triangular form on a panel in an instructed sequence using its arm in visually guided, self-initiated and memory-guided modes

Two monoclonal antibodies, SPM-1 and SPM-2, raised against brain synaptic membrane specifically recognized two different ω-conotoxin receptor-associated proteins with molecular masses of 36 kDa and 28 kDa, respectively. In the anterior lobe of the rat pituitary, all secretory cells were immunostained with SPM-2, but not with SPM-1, although folliculo-stellate cells were stained with both SPM-1 and SPM-2. Castration or stimulation with hypothalamic hormones caused an increase in the intensity of the SPM-2 staining in hormone-secretory cells. In the intermediate lobe, all secretory cells showed a strong 28-kDa-protein immunoreactivity. In the posterior lobe, the immunoreactivity to both SPM-1 and SPM-2 was recognized in nerve endings

Correlative morphological and physiological analysis was carried out in order to clarify the role of somatostatin in the inhibition of the secretion of growth hormone (GH) from somatotrophs of the rat anterior pituitary gland in vivo. Transmission electron microscopy combined with immunogold labelling showed an increased number of exocytotic GH-containing secretory granules in somatotrophs fixed between 2 and 10 min after injection of GH-releasing factor (GRF). Injection of GRF also induced the appearance of immunopositive material in cisternae of the Golgi apparatus, many coated vesicles and multivesicular bodies. Microtubules were observed more frequently throughout the cytoplasm, particularly in and near the Golgi region. At 2 and 10 min after injection of somatostatin (SRIF), both the number of exocytotic figures in the somatotrophs previously stimulated by GRF and the amount of radioimmunoassayable GH in the plasma were clearly decreased. Undulation of the plasma membrane (PM) induced by GRF rapidly disappeared, and the number of granules just beneath the plasma membrane was significantly reduced. After injection of SRIF, parallel bundles of microfilaments were often observed in the space between the granules and the plasma membrane. SRIF did not cause a noticeable decrease in the amount of immunopositive material, coated vesicles and multivesicular bodies in the Golgi areas or any significant changes in the distribution of microtubules. SRIF therefore appears to inhibit hormone release mainly at the level of the plasma membrane, probably through changes in the distribution of microfilaments.

Mast cells are known to be involved in type I allergy and to be localized in almost all tissues in the body. However, they have slightly different properties depending on their tissue of residence. Although mast cells are found in skeletal muscle tissue, there have been no reports of their appearance in cultured skeletal muscles. We report here that mast cells appear in long-term cultures of skeletal muscles from neonatal rats and rat fetuses. When muscle cells were disseminated and cultured in minimum essential medium with 10% fetal calf serum and 10% horse serum, oval cells containing large granules started to appear on myotube sheets at 5 days of culture. These oval cells continued to proliferate for 2–3 months, and showed immunoreactivity for histamine, tryptase, FceRI, and c-kit. They showed metachromatic staining with 0.5% toluidine blue at pH 0.5 and were stained with both Alcian blue and safranin. Biochemically measured histamine content per dish was significantly higher in 2-month than in 5-day culture. From these results, we concluded that these oval cells were mast cells. Because proteases from mast cells have been reported previously to affect myoblast proliferation, the present findings suggest that there may be some interaction between mast cells and muscle cell proliferation or differentiation. The present finding that mast cells are easily obtained from ordinary skeletal muscle cultures provides a useful method for the study of the diverse functions of mast cells. Anat Rec, 1424-1430, 2007. © 2007 Wiley-Liss, Inc.

VIP36 is an intracellular lectin that cycles between the endoplasmic reticulum (ER) and the Golgi apparatus, and is thought to act as a cargo receptor in the transport and sorting of glycoproteins. Here we sought to identify the proteins that interact with VIP36 during the quality control of secretory proteins. VIP36 was crosslinked and immunoprecipitated from HEK293 cells that expressed Myc-tagged VIP36. An approximately 80 kDa protein coprecipitated with VIP36 and LC/MS/MS analysis revealed it to be immunoglobulin-binding protein (BiP), a major protein of the Hsp70 chaperone family. A VIP36 mutant with defective lectin activity was also proficient for the coimmunoprecipitation of an equivalent amount of BiP, indicating that the interaction between VIP36 and BiP was carbohydrate-independent. Immunoelectron microscopy experiment demonstrated that the interaction between VIP36 and BiP occurred in the ER. However, the VIP36 coprecipitated with BiP was resistant to endo beta-N-acetylglucosaminidase H treatment. A pulse-chase experiment revealed that the amount of BiP interacting with VIP36 did not change over more than 2 h. These results suggest that the interaction of VIP36 and BiP is not due to chaperone-substrate complex. Surface plasmon resonance analysis using recombinant proteins confirmed these binding characteristics of VIP36 and BiP in vitro. The interaction between recombinant soluble VIP36 and BiP is dependent on divalent cations but not on ATP. This mode of interaction is also different from that observed between BiP and its chaperone substrates. These observations suggest a new role for VIP36 in the quality control of secretory proteins.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in a variety of tumor cells through two of its receptors: TRAIL-R1 and TRAIL-R2. We investigate the susceptibility of human renal cell carcinoma (RCC) cells to TRM-1 and HGS-ETR2, 2 human monoclonal agonistic antibodies specific for TRAIL-R1 and TRAIL-R2, respectively. HGS-ETR2 effectively induced apoptotic cell death in 10 of 11 cell cultures, including 2 human RCC cell lines and 9 human primary RCC cell cultures, with a more pronounced effect after preincubation with anti-human IgG Fc. In contrast, TRM-1 was effective in only 1 primary RCC cell culture. The increased effectiveness of HGS-ETR2 for inducing cell death might have been affected by differences in the cell-surface expression of the 2 TRAIL receptors, namely that TRAIL-R2 but not TRAIL-R1 was frequently expressed in most of the RCC cells tested. The activities of caspase-9, -8, -6, and -3 were increased with HGS-ETR2-induced apoptosis, and cell death could be blocked by specific caspase inhibitors for caspase-9, -8, and -3, and the general caspase inhibitor. In vivo administration of HGS-ETR2 with or without cross-linker significantly suppressed tumor growth of subcutaneously inoculated human RCC xenografts in immunodeficient mice. These results suggest the potential utility of TRAIL-R2 antibody as a novel therapeutic agent in RCC.

The axonal initial segment is the initiation site of action potentials and is characterized morphologically by a dense undercoating and fascicles of microtubules connected by cross-bridges. In order to analyze subcellular structures in the initial segment, we made serial transverse sections of initial segments of identified chicken motoneurons by retrograde transport of horseradish peroxidase (HRP) injected into the muscle. The mean (+/-SD) length of the initial segment was 28.1+/-2.3 microm (n=6). Mitochondria accumulated in the distal part of the initial segment, which was 1.4-6.9 microm in length (5-23% of the total length of the initial segment). In the transverse section of the distal part, mitochondrial density was 15.8+/-6.2% (n=5), while in the middle and proximal parts it was 6.1+/-1.6% and 5.6+/-1.4%, respectively. Mitochondrial accumulation was observed in common in phasic and tonic motoneurons in the chicken, and also observed in the distal part of the initial segment of the large ventral horn neurons of the chicken without HRP injection. These findings suggest that accumulated mitochondria play an important role in maintaining the physiological function of the distal part of the motoneuron initial segment.

VIP36 (36-kD vesicular integral membrane protein), originally purified from Madin-Darby canine kidney (MDCK) epithelial cells, belongs to a family of animal lectins and may act as a cargo receptor. To understand its role in secretory processes, we performed morphological analysis of the rat parotid gland. Immunoelectron microscopy provided evidence that endogenous VIP36 is localized in the trans-Golgi network, on immature granules, and on mature secretory granules in acinar cells. Double-staining immunofluorescence experiments confirmed that VIP36 and amylase co-localized in the apical regions of the acinar cells. This is the first study to demonstrate that endogenous VIP36 is involved in the post-Golgi secretory pathway, suggesting that VIP36 plays a role in trafficking and sorting of secretory and/or membrane proteins during granule formation.

High levels of deoxyribonuclease I (DNase I) were detected in the cytoplasm of hormone-secreting cells in anterior and intermediate lobes of human and rat pituitary glands. Tissue homogenate fractions of anterior and intermediate pituitary lobes showed strong DNase I-specific enzymatic activity. Immunofluorescence, with several specific antibodies against human and rat DNase I, indicated strong immunoreactivity in the cytoplasm of hormone-secreting cells within the gland. In situ hybridization demonstrated high levels of DNase I mRNA in the cells. By immunoelectron microscopy, immunogold particles were found in the rough endoplasmic reticulum, Golgi apparatus and secretory granules. Exocytotic secretory granules were also immunopositive. These results indicate that endocrine cells in the pituitary gland synthesize and secrete DNase I together with their respective pituitary hormones.

The expression and distribution of deoxyribonuclease I (DNase I) in human duodenum, jejunum and ileum were examined by DNase I activity assay and the reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, in situ hybridization, and immunocytochemical ultrastructural analyses. High levels of DNase I were detected in the cytoplasm of Paneth cells in human small intestine. A tissue homogenate fraction rich in Paneth cells showed strong DNase I-specific enzymatic activity. Immunofluorescence analysis using several specific anti-human DNase I antibodies showed very strong immunoreactivity in the cytoplasm of every Paneth cell. In situ hybridization demonstrated high levels of DNase I mRNA in Paneth cells. Immunogold electron microscopy revealed gold particles localized along the secretory pathway, with the exocrine secretory granules mostly labeled. Our findings strongly suggest that Paneth cells synthesize and secrete DNase I into the intestinal lumen.

A large asymptomatic anterior mediastinal thymolipoma, discovered by chest radiograph during a regular check-up for company employees, was excised from a 33-year-old Japanese man. On immunohistochemical and electron-microscopic examination, clusters of myoglobin-positive cells with cytoplasmic Z band structures were found scattered in the medulla. Myoid cells have been previously seen in the normal thymus as well as in thymic hyperplasia, thymoma, and in the thymus of patients with myasthenia gravis. To our knowledge, this is the first reported instance of myoid cells in a thymolipoma.

An audio decoding apparatus decodes high frequency component signals using a band expander that generates multiple high frequency subband signals from low frequency subband signals divided into multiple subbands and transmitted high frequency encoded information. The apparatus is provided with an aliasing detector and an aliasing remover. The aliasing detector detects the degree of occurrence of aliasing components in the multiple high frequency subband signals generated by the band expander. The aliasing remover suppresses aliasing components in the high frequency subband signals by adjusting the gain used to generate the high frequency subband signals. Thus occurrence of aliasing can be suppressed and the resulting degradation in sound quality can be reduced, even when real-valued subband signals are used in order to reduce the number of operations.

The distribution of somatostatin-containing neurons in mice of both sexes was immunohistochemically examined and compared with that in rats. In radioimmunoassay the relative somatostatin content in the mouse brain was 2-3 times higher than that in the rat. The overall immunohistochemical staining for somatostatin was much stronger and more prominent in the mouse than in the rat. Although the distribution pattern of somatostatin immunoreactivity was basically the same between the two animals, several regions, especially the nucleus anterior hypothalami and the nucleus interpeduncularis, were found to contain large aggregates of somatostatin-immunoreactive neurons in the mouse brain but not in the rat. The electrolytic lesions to the nucleus anterior hypothalami caused a marked decrease in somatostatin immunoreactivity of the outer layer of the median eminence in the mouse. This suggests that the nucleus anterior hypothalami is an additional source of somatostatin for the median eminence in the mouse. The differences recognized between the species are interesting from functional and evolutionary points of view.

The ultrastructure of GH release from the somatotrophs of the rat anterior pituitary was examined in vivo by immunogold electron microscopy. Under pentobarbital anesthesia, injection of GH-releasing factor clearly induced an increase in both plasma GH content and the number of exocytotic GH-immunopositive granules in the cells. The exocytotic events occurred from a part of the plasma membrane facing endocrine cells, including other somatotrophs, and other portions facing folliculo-stellate cells or the walls of blood vessels. When released from the plasma membrane, the GH-immunopositive secretory granules sometimes appeared to aggregate with each other and showed an irregular shape surrounded by a single unit membrane. The exocytotic secretory granules were released into the extracellular space, and then flowed into the sinusoids as irregularly shaped GH-immunopositive electron-dense masses. After reaching the vascular space via the intercellular spaces between the endothelial cells, the contents of each mass became diffusely dispersed into the blood stream, with concomitant disappearance of immunopositivity. The present study thus revealed the morphological aspects of the process of GH secretion from somatotrophs into the blood vessels.