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The present study, using confocal laser scanning microscopy and immunoelectron microscopy, examined the intracellular localization of tyrosine-phosphorylated proteins in cultured mouse dorsal root ganglion neurons with special reference to their growth cones. The growth cone is the specialized structure formed at the growing tip of the axon; characteristically highly motile with filopodia on the surface, it is responsible for the extension and guidance of the neurites to the appropriate targets during nerve regeneration. It has been suggested that protein-tyrosine phosphorylation plays an important role in the intracellular signal transduction that regulates the extension and motility of growth cones. By fluorescence immunocyto-chemistry, phosphotyrosine immunoreactivity was found in the growth cones and neurites. Some of the filopodia exhibited strong immunoreactivity at their tips. By immunoelectron microscopy, a large number of immunogold particles (gold particles conjugated to the secondary antibody) were seen to be distributed in the cytoplasm and some were observed on the plasma membrane in the growth cones, whereas in the neurites the density of immunogold particles was the same in the axoplasm as on the plasma membranes. These findings suggest that in the growth cones phosphotyrosines might mainly be involved in intracellular signaling for maintaining their high motility whereas in the neurites they might mostly be associated with the receptor proteins at the plasma membrane for adhesion as well as for growth of neurites. Thus, tyrosine phosphorylation might contribute to different functions for growth cones and neurites.

In contrast to previous reports denying the occurrence of axonal regeneration of the dorsal column (DC) projections, here we demonstrate for the first time that marked regeneration occurs spontaneously after transection in infant rats. Transection was made sharply so as to produce edema-free lesions without subsequent formation of either scars or cysts. Transganglionic labeling of axons revealed that regenerated axons ascended in the normal tract in a manner similar to normal projections as a tightly-packed fasciculus and terminated densely in the nucleus gracilis. The present study indicates that failure of regeneration of DC axons is due to neither intrinsic deficiency of regrowth potential nor globally-inhospitable axonal environment but rather the local conditions of the lesion site.

In Caenorhabditis elegans, the vulval induction is mediated by tyrosine kinase receptor/Ras signal transduction pathway composed of the lin-3, let-23, and let-60 products. In addition to these gene products, the lin-2, lin-7, and lin-10 products are also implicated in this pathway. Lin-2 encodes a MAGUK and lin-7 encodes a small protein with one PDZ domain. The lin-10 product has no homology to known proteins. Here, we have cloned a rat homologue of the lin-10 product and characterized it. Rat lin-10 is ubiquitously expressed in various rat tissues and distributed in both the cytosol and membrane fractions. In brain, however, rat lin-10 is distributed only in the membrane fraction and enriched in the synaptic plasma membrane and postsynaptic density fractions. These results suggest that rat lin-10 is involved at least in synaptic functions in brain.

In a preceding paper, we reported a novel actin filament (F-actin)-binding protein, named neurabin, which was specifically expressed in neural tissue and implicated in neurite formation. We purified from rat brain another F-actin-binding protein, which had a domain organization similar to that of neurabin but was ubiquitously expressed, and named it neurabin-II. The original neurabin, renamed neurabin-I, had 1095 amino acids and a calculatedM r of 122,729, whereas neurabin-II had 817 amino acids and a calculated M r of 89,642. Both neurabin-I and -II had one F-actin-binding domain at the N-terminal region, one PDZ domain at the middle region, a domain known to interact with transmembrane proteins, and domains predicted to form coiled-coil structures at the C-terminal region. Both neurabin-I and -II bound along the sides of F-actin and showed F-actin-cross-linking activity. The subcellular distribution analysis indicated that neurabin-II was enriched at the postsynaptic density fraction in rat brain and the adherens junction fraction in rat liver. Immunofluorescence microscopic analysis revealed that neurabin-II was highly concentrated at the synapse in primary cultured rat hippocampal neurons and at the cadherin-based cell-cell adhesion sites in Madin-Darby canine kidney cells. Neurabin-II turned out to be the same as a recently reported protein phosphatase 1-binding protein named spinophilin. These results suggest that neurabin-II/spinophilin plays an important role in linking the actin cytoskeleton to the plasma membrane.

A novel actin filament (F-actin)–binding protein with a molecular mass of ∼205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin–binding domain at 1,631–1,829 aa residues and one PDZ domain at 1,016– 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009–1,093 aa residues but lacking the F-actin–binding domain. Homology search analysis revealed that the aa sequence of p190 showed 90% identity over the entire sequence with the product of the AF-6 gene, which was found to be fused to the ALL-1 gene, known to be involved in acute leukemia. p190 is likely to be a rat counterpart of human AF-6 protein. p205 bound along the sides of F-actin but hardly showed the F-actin–cross-linking activity. Northern and Western blot analyses showed that p205 was ubiquitously expressed in all the rat tissues examined, whereas p190 was specifically expressed in brain. Immunofluorescence and immunoelectron microscopic studies revealed that p205 was concentrated at cadherin-based cell-to-cell AJ of various tissues. We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin). These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.

The effects of an insect insulin-related peptide, bombyxin, on carbohydrate metabolism were investigated in the silkworm Bombyx mori. Bombyxin lowered the concentration of the major hemolymph sugar, trehalose, in a dose-dependent manner when injected into neck-ligated larvae. Bombyxin also caused elevated trehalase activity in the midgut and muscle, suggesting that bombyxin induces hypotrehalosemia by promoting the hydrolysis of hemolymph trehalose to glucose and thereby facilitating its transport into tissues. In addition, bombyxin reduced the glycogen content in the fat body and concurrently raised the percentage of active glycogen phosphorylase in this tissue. Because hemolymph trehalose is also a major storage form of carbohydrate in insects, our results indicate that bombyxin reduces the amount of both principal storage carbohydrates in B. mori larvae. It is therefore suggested that although bombyxin is involved in the control of carbohydrate metabolism like insulin, the physiological role of bombyxin in insects is different from that of insulin in mammals.

In the peripheral nerve, regenerating axonal sprouts usually emanate at nodes of Ranvier, and extend as growth cones along the inner surface of Schwann cells and/or through Schwann cell columns in the distal nerve segment. In order to elucidate the significance of Ca(2+)-independent protein kinase C in nerve regeneration, localizations of delta, epsilon and zeta subtypes were examined immunocytochemically in sprouts and growth cones of regenerating axons, as well as in normal intact nerves in the rat sciatic nerve. In normal nerves, intense immunoreactivities of delta, epsilon and zeta subtypes were present in axons of both myelinated and unmyelinated fibres. Subcellularly, the distribution of these subtypes in the axoplasm was patchy, and discontinuous in the axolemma and subaxolemmal peripheral zones of myelinated nerves. Some thin myelinated axons showed no immunoreactivity for epsilon subtype. Schwann cells of both myelinated and unmyelinated fibres had moderate immunoreactivities for each subtype. In areas of nerve regeneration, axonal sprouts at nodes of Ranvier, and growth cones extending along Schwann cell basal laminae, had intense immunoreactivities for delta, epsilon and zeta subtypes which are distributed diffusely throughout the axoplasm, and on the entire axolemma. In the sprouts, immunoreactivity for epsilon subtype was strong on the axolemma, but weak or almost absent in the axoplasm. These data, together with those of our previous study, indicate that Ca(2+)-independent protein kinase C subtypes (delta, epsilon and zeta) have basically the same distribution patterns as those of Ca(2+)-dependent subtypes in sprouts and growth cones of regenerating axons, as well as in normal intact axons; albeit epsilon subtype is somewhat different in distribution and intensity from delta and zeta subtypes. It is suggested that Ca(2+)-independent subtypes are involved in maintaining growth cone activities along with the Ca(2+)-dependent subtypes.

The dynamic rearrangement of cell–cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2, and adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell–cell contacts. We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6) as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell–cell contact sites. We investigated roles of Ras and AF-6 in the regulation of cell–cell contacts and found that AF-6 accumulated at the cell–cell contact sites of polarized MDCKII epithelial cells and had a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy revealed a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacted with ZO-1 in vitro. ZO-1 interacted with the Ras-binding domain of AF-6, and this interaction was inhibited by activated Ras. AF-6 accumulated with ZO-1 at the cell–cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells resulted in the perturbation of cell–cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell–cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell–cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras.

Schwann cell basal lamina tubes serve as attractive conduits for regeneration of peripheral nerve axons. In the present study, by using basal lamina tubes prepared by in situ freeze-treatment of rat saphenous nerve, the effects of exogenously applied basic fibroblast growth factor (bFGF) on peripheral nerve regeneration was examined 2 and 5 days after bFGF administration. Regenerating axons were observed by light and electron microscopy using PGP9.5-immunohistochemistry for specific staining of axons. In addition, the localizations of bFGF and its receptor (FGF receptor-1) were examined by immunohistochemistry using anti-bFGF antibody and anti-FGF receptor-1 antibody, respectively. Regenerating axons extended further in the bFGF-administered segment than in the bFGF-untreated control segment. Electron microscopy showed that regenerating axons grew out unaccompanied by Schwann cells. Findings concerning angiogenesis and Schwann cell migration were very similar between the bFGF treated and control nerve segment. bFGF-immunoreactivity was not detected in the control nerve segment. In contrast, bFGF-immunoreactivity was detected on the basal lamina tubes as well as on the plasmalemma of regenerating axons facing the basal lamina in the bFGF treated nerve segment up to 5 days after administration, suggesting that exogenous bFGF can be retained in the basal lamina for several days after administration. FGF receptor was detected on the plasma membrane of regenerating axons where they abutted the basal lamina. These results indicate that bFGF could promote the extension of early regenerating axons by directly influencing the axons, but not via Schwann cells or angiogenesis.

Rab guanosine triphosphatases in eukaryotic cells are key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab7 regulates traffic from early to late endosomes and from late endosomes to vacuoles/lysosomes. The Rab7-interacting lysosomal protein (RILP) was extracted from the silkworm, Bombyx mori (B. mori), and expressed in Escherichia coli (E. coli), followed by its purification. The glutathione sulfotransferase pull-down assay revealed that Rab7 of B. mori interacted with RILP of B. mori. We then produced antibodies against RILP of B. mori in rabbits for their use in Western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue for RILP revealed a single band, at approximately 50 kD. RILP-like immunohistochemical reactivity (RILP-ir) was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Furthermore, RILP-ir was colocalized with the eclosion hormone-ir and bombyxin-ir. However, RILP-ir was not colocalized with prothoracicotropic hormone-ir. These results were similar to those of Rab7 from our previous study. These findings suggest that RILP and Rab7 are involved in the neurosecretion in a restricted subtype of neurons in B. mori. Thus, our study is the first to report of a possible relationship between an insect Rab effector and neurosecretion.

A quarter of a century has passed since bombyxin, the first insulin-like peptide identified in insects, was discovered in the silkmoth Bombyx mori. During these years, bombyxin has been studied for its structure, genes, distribution, hemolymph titers, secretion control, as well as physiological functions, thereby stimulating a wide range of studies on insulin-like peptides in other insects. Moreover, recent studies have identified a new class of insulin family peptides, IGF-like peptides, in B. mori and Drosophila melanogaster, broadening the base of the research area of the insulin-related peptides in insects. In this review, we describe the achievements of the studies on insulin-like and IGF-like peptides mainly in B. mori with short histories of their discovery. Our emphasis is that bombyxins, secreted by the brain neurosecretory cells, regulate nutrient-dependent growth and metabolism, whereas the IGF-like peptides, secreted by the fat body and other peripheral tissues, regulate stage-dependent growth of tissues.

Sevoflurane is the most widely used volatile anesthetic of general anesthesia. In children and neonates, it is commonly used alone or in combination with thiopental or propofol. A few recent studies reported that sevoflurane induced neuronal death in the developing rodent brain. We measured the neurotoxicity of these anesthetics at clinical doses, alone and in combination, in the developing mouse brain.Seven-day-old C57BL/6 mice were randomly assigned to 6 treatment groups. Three groups were exposed to 3% sevoflurane for 6 h after injection of saline, thiopental (5 mg/kg), or propofol (10 mg/kg), whereas three groups were exposed to room air for 6 h after injection of equal doses of saline, thiopental, or propofol. Apoptosis in the hippocampal CA1 region (CA1) and retrosplenial cortex (RC) was assessed using caspase-3 immunostaining.Sevoflurane alone caused significantly higher apoptosis in the CA1 compared with saline plus air (P = 0.04). Sevoflurane in combination with propofol resulted in significantly greater numbers of apoptotic neurons than sevoflurane alone in both the CA1 and the RC (P = 0.04). However, there was no significant difference in apoptotic neuron density in both the regions between the groups treated with sevoflurane alone and in combination with thiopental (P = 0.683).Sevoflurane alone can induce neuronal apoptosis, and this effect is enhanced by propofol. Thiopental did not exacerbate the neurotoxicity of sevoflurane. There is the possibility that the combination of sevoflurane and propofol is a more harmful anesthetic technique than sevoflurane alone in pediatric patients.

The Ca(2+)-dependent intercellular adhesion molecule cadherin is known to be linked to the cytoskeleton by the protein catenin, an association of which appears to be important for the cell-adhesion function of cadherin. Catenin consists of three subtypes-alpha, beta, and gamma. In our previous study, N-cadherin was shown to be localized on the plasmalemma of normal and regenerating chick peripheral nerve. Thus, as alpha N-catenin is a subtype of alpha-catenin (which is specifically associated with N-cadherin), we investigated the immunolocalization of alpha N-catenin in normal and regenerating chick sciatic nerve. In normal nerve, unmyelinated axons exhibited either intense or weak alpha N-catenin immunoreactivity throughout the axoplasm, whereas myelinated axons were completely immunonegative. Regenerating axons, including those derived from parent myelinated axons, showed alpha N-catenin immunoreactivity of variable intensities in growth cones and axon shafts. Schwann cells were invariably devoid of immunoreactivity. Thus alpha N-catenin is not necessarily bound to the surface plasmalemma, but is distributed throughout the cytoplasm, suggesting that most alpha N-catenin molecules are dissociated from N-cadherin.

We produced mouse antisera against synthetic peptides corresponding to the sequences of the Samia cynthia ricini homologues of the Bombyx mori PTTH and bombyxin. Immunohistochemical analyses of the Samia cephalic neuroendocrine system using these antisera were performed to identify the neurosecretory cells (NSC) containing the PTTH and bombyxin homologues and to examine the developmental changes in their amounts in the NSC. The results show that the PTTH and bombyxin homologues are produced by two pairs of dorsolateral and 16 pairs of dorsomedial NSC of Samia brain, respectively, and both are transported to, and released from, the corpora allata. No clear-cut correlation was found between the fluctuation in the amount of immunoreactive substances in the brain NSC and the endocrinologically anticipated timings of PTTH secretion. From Samia brain extract, two forms of PTTH activity (∼30 kDa and ∼5 kDa) were resolved through Sephadex gel filtration. The ∼30 kDa and ∼5 kDa PTTH seem to represent the PTTH and bombyxin homologues, respectively. We discuss that the ∼30 kDa PTTH homologue is the true PTTH of Samia.

The distribution of protein kinase C (alpha, beta, gamma subtypes) was studied using immunocytochemical techniques in normal nerve fibers and in regenerating sprouts (growth cones) from the nodes of Ranvier following crush injuries to the rat peripheral nervous system. In normal nerves, for each protein kinase C subtype, immunoreactivity was present in both myelinated and unmyelinated axons. In myelinated axons, immunoreactivity for all three subtypes was patchy in the axoplasm and diffuse in the subaxolemmal peripheral zones. No immunoreactivity was found in the microtubule and neurofilament (cytoskeletal) domain. In contrast, in unmyelinated axons, immunoreactivity was distributed diffusely in the axoplasm. Schwann cells of myelinated fibers exhibited protein kinase C immunoreactivity, but those of unmyelinated fibers did not. In regenerating nerves, early sprouts and growth cones extending through the crushed site along Schwann cell basal laminae exhibited intense immunoreactivity for all three subtypes. Immunoreactivity was distributed diffusely throughout the axoplasm of the regenerating sprouts (growth cones), in which microtubules and neurofilaments were very rare. Thus, the subcellular localization of the protein kinase C immunoreactivity in growth cones of early regenerating nerves differed from that of normal parent axons. These findings suggest that protein kinase C (alpha, beta and gamma subtypes), whose subcellular distribution becomes more extensive in regenerating axons, may have important functional roles in axonal sprouting and in the regulation of growth cone activity in the peripheral nervous system.

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.

The PAR complex targets Tiam1 to adhesions, where it interacts with talin to promote adhesion-induced Rac1 activation, cell spreading, and migration., Migrating cells acquire front-rear polarity with a leading edge and a trailing tail for directional movement. The Rac exchange factor Tiam1 participates in polarized cell migration with the PAR complex of PAR3, PAR6, and atypical protein kinase C. However, it remains largely unknown how Tiam1 is regulated and contributes to the establishment of polarity in migrating cells. We show here that Tiam1 interacts directly with talin, which binds and activates integrins to mediate their signaling. Tiam1 accumulated at adhesions in a manner dependent on talin and the PAR complex. The interactions of talin with Tiam1 and the PAR complex were required for adhesion-induced Rac1 activation, cell spreading, and migration toward integrin substrates. Furthermore, Tiam1 acted with talin to regulate adhesion turnover. Thus, we propose that Tiam1, with the PAR complex, binds to integrins through talin and, together with the PAR complex, thereby regulates Rac1 activity and adhesion turnover for polarized migration.

The small GTP-binding protein Rho plays a crucial role in a wide variety of cellular functions through various effector proteins. Rho-kinase is a key effector protein of Rho, which is composed of two isoforms, ROCK1 and ROCK2. To clarify the site of action of ROCK1 and ROCK2, we performed immunofluorescence and immunoelectron microscopic analyses using isoform-specific antibodies in mouse tissues. In the large and small intestines, ROCK1 immunoreactivity was predominantly identified in epithelial cells, and ROCK2 immunoreactivity was negligible. In these epithelial cells, ROCK1 immunoreactivity was distributed on plasma membranes, while ROCK1 immunogold signals were localized at cell-cell contacts and cell adhesion sites, especially at the adherens junctions at the ultrastructural level. In the bladder epithelium, however, ROCK1 and ROCK2 signals were identified at intermediate filaments, and ROCK2 signals were also observed in nuclei. In the three types of muscular cells-smooth, cardiac, and skeletal muscle cells-ROCK1 and ROCK2 also showed differential distribution. ROCK1 signals were localized at actin filaments, plasma membranes, and vesicles near plasma membranes in smooth muscle cells; at the lysosomes in skeletal muscle cells; and were undetectable in cardiac muscle cells. ROCK2 signals were localized at actin filaments and centrosomes in smooth muscle cells, at intercalated discs in cardiac muscle cells, and at Z-discs and sarcoplasmic reticulum in skeletal muscle cells. In the brain, ROCK1 immunoreactivity was distributed in glia, whereas ROCK2 immunoreactivity was observed in neurons. These results indicate that the two isoforms of Rho-kinase distribute differentially to accomplish their specific functions.

The myoinhibiting peptides (MIPs), also designated as allatostatin-Bs or prothoracicostatic peptides in some insects, are neuropeptides that are characterized by two tryptophan (W) residues at the C-terminal, denoted as the W(X(6))Wamide motif. They are believed to be the ancestral ligands for the Drosophila sex peptide (SP) receptor. Physiological functions of MIPs include the inhibition of contraction of insect visceral muscles, in addition to allatostatic and prothoracicostatic activities. The MIP precursor in Rhodnius prolixus encodes MIPs that have an unusual W(X(7))Wamide motif. In the present study, MIP-like immunoreactivity was detected within neurons in the central nervous system and within the innervation to the salivary glands, hindgut, and female and male reproductive systems of adult R. prolixus. The effects of peptides with the unusual W(X(7))Wamide motif (Rhopr-MIP-4) and with the typical W(X(6))Wamide motif (Rhopr-MIP-7) were tested for physiological activity on R. prolixus hindgut contractions. Both peptides reduce the frequency and amplitude of hindgut contractions in a dose-dependent manner. In addition, both peptides activate the Drosophila SP receptor. The MIP/SP receptors are therefore activated by peptides with the unusual W(X(7))Wamide motif. ispartof: Frontiers in Neuroscience vol:6 issue:JULY ispartof: location:Switzerland status: published

Two-photon laser scanning microscopy (TPLSM), relying on two-photon excitation restricted to the focal plane, has become the gold standard in biomedical research because of its ability to produce high-resolution, higher penetrating imaging of biological materials over an extended duration without an significant photobleaching. Intravital (in vivo) imaging using TPLSM for intra-abdominal organs has long been a technical challenge because of the difficulty of achieving high-quality and higher magnification imaging with acceptable cardiac and respiratory motion artifacts. A method of intravital TPLSM was developed using an organ-stabilizing system for imaging the intra-abdominal organs of green fluorescent protein transgenic mice. This method was further refined for the time-series imaging of intra-abdominal organs in the same mouse model using intravital TPLSM. These procedures allow the observation of not only a single cell or tissue microenvironment at a higher penetrating depth for a longer period of time but also to observe the same organ of the same mouse at multiple time points in preclinical models. This report presents the general principles and properties of TPLSM for biomedical research. In addition, the methods and the usefulness of time-series intravital TPLSM imaging for preclinical gastrointestinal diseases is discussed.

Summary A monoclonal antibody against a synthetic pentadecapeptide corresponding to the N-terminal end of Bombyx-prothoracicotropic hormone (PTTH: 1–15) and a monoclonal antibody against a synthetic decapeptide corresponding to the N-terminal portion of the A-chain of bombyxin-1, bombyxin (1–10), were used as parallel probes to localize both molecules at the cellular and subcellular levels in the brain-retrocerebral complex of Manduca sexta. Immunohistochemical analysis revealed that molecules immunochemically similar to Bombyx-PTTH were localized in the cytoplasm of two pairs of dorso-lateral neurosecretory cells (L-NSC III) and could be traced along their axons to the corpora cardiaca (CC) and corpora allata (CA), whereas bombyxin-like material was absent from these cells and was detected only in the cytoplasm of four pairs of median neurosecretory cells (M-NSCs). Immunocytochemical labelling at the ultrastructural level using the PTTH antibody showed high specificity with the 5 nm immunogold particles ...

The localization of protein kinase C (PKC) α, β and γ subspecies in sensory axon terminals of muscle spindles in the plantar lumbrical muscles of rat was investigated by light and electron microscopic immunocytochemistry using monoclonal and polyclonal antibodies. Immunoreactivity for these subspecies was detected specifically in sensory axon terminals which wound spirally around the intrafusal muscle fibres of the muscle spindle. Immunostaining was found to be stronger with polyclonal than with monoclonal antibodies. By electron microscopy, immunoreactivity for α, β and γ subspecies was almost diffusely distributed in the cytoplasm of the axon terminal, and the overall pattern of distribution of immunoreactivity was similar for all three subspecies. In the cases of a and β subspecies, some intensely immunostained regions were found in the cytoplasm, but no definite subcellular structures corresponding to such regions could be identified. Considering that PKC plays a crucial role in the regulation of ion channels, it is suggested that PKC might be involved in the control of mechanoelectric transduction in sensory axon terminals.

Rabphilin-3A is a putative target protein for Rab3A small GTP-binding protein which is implicated in neurotransmitter release. Rabphilin-3A is expressed mainly in brain, but its subcellular localization remains to be clarified. Immunohistochemical analysis has revealed that Rabphilin-3A is most abundant in the synaptic area of the rat cerebellum, retina, and neuromuscular junction. Ultrastructural analysis of the neuromuscular junction using the immunogold method indicates that Rabphilin-3A is localized on the synaptic vesicle. Subcellular fractionation analysis of rat brain has shown that Rabphilin-3A is most highly concentrated in the purified synaptic vesicle fraction. These results indicate that Rabphilin-3A is localized on the synaptic vesicle in the presynapse.

The disulfide bond location of a homodimeric peptide, prothoracicotropic hormone (PTTH) of the silkworm, Bombyx mori, was determined by a combination of partial reduction and sequence analysis of peptide fragments generated through a partial reduction of PTTH followed by alkylation and enzyme digestion. The partial reduction and S-alkylation broke the interchain disulfide bond but did not affect the intrachain disulfide bonds, generating monomeric PTTH whose intrachain disulfide bonds were kept intact. This monomeric PTTH has about one-half the biological activity of intact PTTH. Sequence analysis of the fragments generated by lysyl endopeptidase digestion of this monomeric PTTH after complete reduction and S-alkylation by another S-alkylating reagent showed that only the Cys15 residue was reduced and S-alkylated by the foregoing partial reduction, indicating that this residue formed the interchain disulfide bond. The other disulfide bonds which formed intrachain bridgings were determined by sequence and mass analyses of the fragments generated by two successive enzyme digestions of the monomeric PTTH. In conclusion, the disulfide bond location of PTTH was assigned to Cys15-Cys15' as an interchain disulfide linkage and Cys17-Cys54, Cys40-Cys96, and Cys48-Cys98 as intrachain disulfide linkages.

A series of ethylene and methanimine derivatives possessing both electron-donor and -acceptor groups were synthesized in order to obtain frequency doublers of infrared semiconductor lasers. Their second-harmonic-generation (SHG) was investigated by means of a powder method and an electric-field-induced second-harmonic method. Three ethylene derivatives were found to be SHG active without any absorption in the blue wavelength region. X-Ray crystallographic analyses were made for two of them. It was found that strong donor and acceptor substituents tend to twist the central C=C double bond, thereby resulting in smaller β-values than those expected for non-twisted molecular structures. Based on the molecular and crystal structures, some strategies are presented regarding the synthesis of SHG materials of these push-pull ethylenes and methanimines.

The cDNAs encoding allatotropin (AT) and allatotropin-like peptides (ATLPs) were isolated from the silkworm, Bombyx mori. Similar to those of the tobacco hornworm, Manduca sexta, four peptides (AT, ATLP1, ATLP2, and ATLP3) are present in three different variants generated by alternative splicing. RT-PCR analyses showed that these splice variants are expressed in the central nervous system with differing expression patterns in each ganglion. Immunohistochemistry using an anti-AT antibody confirmed that AT-expressing cells were located in these central nervous ganglia as well as in two large anterior cells of the frontal ganglia. Injection of synthetic AT and ATLP-1 into B. mori larvae increased the latency to feed, indicating that AT and ATLP might function in the regulation of feeding behavior in B. mori.

In vertebrates, insulin-like growth factors (IGFs) play important roles in the regulation of growth and development. Although the principal source of circulating IGFs is the liver, IGFs are also secreted by many other tissues, functioning locally through paracrine/autocrine mechanism. In the silkmoth Bombyx mori, Bommo-IGF-like peptide (BIGFLP) is the functional counterpart of vertebrate IGFs and is mainly produced by the fat body, a functional equivalent of the vertebrate liver and adipocytes. However, its production by other tissues has not yet been analyzed. In this study, we systematically surveyed the BIGFLP-producing tissues and stages by means of immunohistochemistry, in situ hybridization and real-time quantitative RT-PCR, showing that BIGFLP is also produced by the neurosecretory cells in the brain, ovariole sheath and testis sheath, in a stage-specific manner. The BIGFLP-producing cells in the brain were identical to the cells that produce bombyxins, insulin-like peptides of B. mori, but the temporal expression patterns of both peptides were totally different. The BIGFLP gene expression in the sheaths of ovariole and testis were induced by ecdysteroid in vitro, similar to the expression in the fat body. A very high BIGFLP immunoreactivity was also found in the pupal nephrocytes, a functional equivalent of the glomerular podocytes in the vertebrate kidney, without the expression of the gene, suggesting that circulating BIGFLP is taken up and degraded by these tissues. Based on the present observations, the physiological functions of BIGFLP in B. mori development are discussed.

Intravital three-dimensional (3D) visualization of treatment efficacy in experimental colitis in living mice using two-photon laser scanning microscopy (TPLSM) has not been described.Colitis was induced with dextran sulfate sodium (DSS) in green fluorescent protein (GFP) transgenic mice. The 3D tomographic image of DSS-induced colitis with or without prednisolone was obtained intravitally using TPLSM. A serosal-approaching method was developed, by which we could observe all layers of the cecum from serosa to luminal mucosa without opening and everting the cecum. The dynamic pathology and treatment efficacy were assessed in the same mouse on several occasions.The time-lapse 3D tomographic movie of DSS-induced colitis was obtained in living mice at a magnification of greater than ×600, which demonstrated irregularity of crypts, disappearance of crypts, inflammatory cell infiltrates in the lamina propria, and abscess formation at the bottom of crypts. Intravital TPLSM in the same mice demonstrated fewer infiltrating leukocytes and crypt abscesses on day 14 in the steroid group compared with the nonsteroid group.Intravital 3D tomographic visualization of experimental colitis using TPLSM in combination with the serosal-approaching method can provide dynamic pathology at a high magnification, which may be useful in evaluating treatment efficacy in the same living mice.

Following crush injury of rat sciatic nerve, strong synaptophysin immunoreactivity was demonstrated in the regenerating sprouts that emerged from the proximal nodes of Ranvier and in their growth cones that extended through the space between Schwann cell basal lamina and myelin sheath of the parent axon. These findings suggest that synaptophysin is involved in the growth regulation of regenerating sprouts.

Ultrastructural localization of protein kinase C (PKC) beta-subspecies in neuromuscular junctions of the rat lumbrical muscle was investigated by the immunoperoxidase and immunofluorescence methods. By light microscopy, PKC beta-like immunoreactivity (PKC beta-LIR) was found in the axon terminal expansions as well as in the preterminal axons. By confocal laser scanning microscopy, the staining for PKC beta-like immunoreactivity was more intense in the presynaptic regions just in contact with the acetylcholine receptor stained by FITC-alpha-bungarotoxin. By electron microscopy, PKC beta-like immunoreactivity was distributed non-uniformly in the terminal expansions. In the terminal expansions, PKC beta-like immunoreactivity was accumulated in the presynaptic regions in contact with the post-synaptic folds. This accumulation was approximately 0.1-0.2 microns in diameter, which comprised a part of the presynaptic plasma membrane and a group of synaptic vesicles adjacent to it. Weak immunoreactivity was also found diffusely in the axoplasmic matrix. The discrete presynaptic accumulation of PKC beta-subspecies may represent the strategical localization specialized for the effective regulation of neurotransmitter release.

PDA-3BCMU monomer was epitaxially grown on KCl substrate by vacuum-deposition method. Followed by solid phase polymerization, the PDA-3BCMU thin film containing square crystals was obtained. The PDA main chains in the crystals were parallel to KCl[110] or [110] direction. THG anisotropy of the thin film was explained the orientation of PDA main chains and enhancement of χ(3) THG was observed against un-oriented PDA-3BCMU thin film.

We have been paying attention to the development of the nondestructive physical analysis (NDPA) technology. We think that NDPA is a technology which doesn't depend on the worker's capability or experience. There are many NDPA techniques, and analysis using X-ray imaging is one of the principal techniques. Due to the progress of the image analysis using computers in recent years, X-ray imaging have been evolving from two dimensional images to three dimensional imaging. We have been applying X-ray CT imaging to actual failure analysis and reliability evaluation since 2008. At ISTFA 2009, we reported on the effectiveness of X-ray Computed Tomography (CT) images in the failure analysis. [1] We confirmed that the X-ray CT image had various applications, for example, screening for counterfeit parts, the detection of the defect of the multi-layers printed wiring boards (multi-layers PWB), the structure confirmation of caulking contacts, and the detection of cracks or voids of the solder joint. This paper discusses the effectiveness of X-ray CT imaging in failure analysis and discusses the effectiveness of applying X-ray CT imaging to the propagation of cracks occurring at solder joints during temperature cycling test.

Insulin secretion is essential for maintenance of glucose homeostasis, but the mechanism of insulin granule exocytosis, the final step of insulin secretion, is largely unknown. Here, we investigated the role of Rim2alpha in insulin granule exocytosis, including the docking, priming, and fusion steps. We found that interaction of Rim2alpha and Rab3A is required for docking, which is considered a brake on fusion events, and that docking is necessary for K(+)-induced exocytosis, but not for glucose-induced exocytosis. Furthermore, we found that dissociation of the Rim2alpha/Munc13-1 complex by glucose stimulation activates Syntaxin1 by Munc13-1, indicating that Rim2alpha primes insulin granules for fusion. Thus, Rim2alpha determines docking and priming states in insulin granule exocytosis depending on its interacting partner, Rab3A or Munc13-1, respectively. Because Rim2alpha(-/-) mice exhibit impaired secretion of various hormones stored as dense-core granules, including glucose-dependent insulinotropic polypeptide, growth hormone, and epinephrine, Rim2alpha plays a critical role in exocytosis of these dense-core granules.

~~ Monoclonal antibodies were raised against pure, native bombyxin-ll (bombyxin-ll antibody) and against a synthetic nonapeptide corresponding to the amino-terminus of the C-peptide of the bombyxin precursor protein (C-peptide antibody). The bombyxin-ll antibody recognized both bombyxin and probombyxin. A radioimmunoassay for bombyxin using the bombyxin-ll antibody was developed, and developmental change in the titer of bombyxin immunoreactivity in the Bombyx hemolymph was investigated. The titer was low and almost constant during the fourth and early fifth instars. In the male, the titer rose abruptly 3 days after the beginning of wandering. One day after pupation it rose again steeply to reach the maximal level which lasted until the middle of the developing adult stage. The titer decreased thereafter and increased again at adult emergence. In the female, the pattern of titer fluctuation was similar to that in the male, but the female titers during pupal-adult development were 2-3 times higher than the male titers.

2,6-Di-tert-butylphenol moiety was found to be effective to control molecular packing in crystals for SHG materials. Many derivatives having compact acceptor group at 4-position of hindered phenols showed SHG responses depending on the electron withdrawing effect of an acceptor. The X-ray structural analyses revealed that molecular packing was controlled both by the steric requirements and the intermolecular hydrogen bonding.

Immunocytochemical demonstration of protein kinase C (PKC) subspecies (α, β, γ) was carried out in Pacinian corpuscles of rat hind feet using monoclonal or polyclonal antibodies against each of these subspecies. The inner core cells and lamellae and the Schwann cell cytoplasm of the nerve fiber innervating the corpuscle were strongly positive for PKC α-immunoreactivity (IR). In contrast, the axon terminal and the outer core did not display any positive α-IR. Very weak PKC β-IR was detected in the ultraterminal region of the axon terminal, while the trunk region showed no immunoreactivity. Very faint PKC β-IR was found also in the lamellar cells located at the periphery of the inner core and the endoneurial fibroblasts in the intermediate layer. PKC γ-IR was not detected in any part of the corpuscle. The strong PKC α-IR in the inner core and the presence of absence of PKC α-, β-, and γ-IR in the axon terminal are discussed from the point of view of the functional aspects of each part.

Traumatic degeneration of myelinated fibers was studied by electron microscopy over 5 days following transection of mouse sciatic nerve. Special attention was paid to the mechanism which separates the degenerating part, while preserving the viable part of the axon. Immediately after transection, the opened end of the proximal stump revealed extensive subcellular changes including the disorganization of neurofilaments, and disruption of mitochondria and axonal endoplasmic reticulum (SER). Subsequently, vesicles of round and tubular profiles filled up the whole area of the stump end, and proximal to it appeared a neurofilament-predominant area characterized by randomly oriented neurofilaments and normally appearing mitochondria and SER. Characteristic membranous demarcations occurred in early periods at the border between the vesicle accumulation and the neurofilament-predominant areas, and later also within these areas. The demarcation membranes formed both by invagination of the surface plasma membrane and, probably, by fusion of the large vesicles. These became prominent with time, dividing the axoplasm into compartments of varying sizes, which gradually underwent degeneration and were liberated from the parent axon. Occurrence of autophagic vacuoles was characteristic of the degenerating portions of the parent axon. Thus, by the function of demarcation membranes, the parent axon to be preserved could remain membrane-bound, while the degenerating parts were shed off.

Existence and distribution of small guanine nucleotide binding proteins (G proteins) such as smg p25A, smg p21s, and ras p21s were examined in the rat gastric mucosa. By immunoblot analysis using the specific monoclonal antibodies against smg p25A and ras p21 and the anti-smg p21 antiserum, smg p25A and smg p21s were detected in the gastric mucosa, while ras p21s were not detected. In immunocytochemical studies, moderate fluorescence of smg p25A was homogenously seen in the cytoplasmic portions of chief cells and surface epithelial cells. smg p21 immunoreactivity was detected in glandular cells and submucosal muscle layer in the mucosa. On the other hand, the cells constituting gastric glands were unstained with the anti-ras p21 monoclonal antibody, RASK-4. These results suggest that smg p25A and smg p21s exist as in other tissues and might exert their specific actions in the rat gastric mucosa.

Oxidative stress plays an important role in the pathogenesis of asthma via the upregulation of local inflammatory mediators and/or promoting Th2-skewing during Ag sensitization. Thioredoxin (TRX), a 12 kDa redox-active protein with antioxidative property, has been recently shown to play a protective role in various inflammatory diseases. Using a mouse model of asthma, we show here that IL-13 and eotaxin production are decreased in TRX-Tg mice leading to reduced eosinophils recruitment and mucus metaplasia. The reduction in airway inflammation occurs without the attenuation of systemic Th2 immunity in that comparable levels of Th2-type cytokines and Ig were detected in LN and serum, respectively, from TRX-Tg and WT mice. Likewise, CD4(+) T cells from both strains of mice developed similar Th1 and Th2 responses in vitro. Asthmatic lungs of TRX-Tg and WT mice contained similar amounts of GATA-3(+) and Foxp3(+) T cells. Finally, production of MIF, an upstream modulator of airway inflammation, was significantly reduced in the lungs of TRX-Tg mice. Our data suggest that TRX suppresses airway inflammation by inhibiting MIF production thereby limiting the downstream recruitment of eosinophils to the lung independently of modulating systemic Th1/Th2 immunity.

Now we are attempting to apply non destructive analysis from evaluation tests or failure analysis to acceptance tests or production tests. Needless to say non destructive analysis has an advantage of conserving the state of samples and the reducing the time of analysis as compared to conventional methods with destructive physical analysis. Moreover, we are paying attention to the following reasons for nondestructive physical analysis. It is difficult to keep the reproducibility of the analysis because of the high skill level required for destructive physical analysis. On the other hand, high reproducibility can be easily achieved by fixing the condition or parameters of the device during nondestructive analysis when performed by tools like X-ray. Moreover, we expect that neither the analytical result nor the quality of the nondestructive analysis depends upon the worker's capability. In this paper we will discuss the following two items from the viewpoint of quality assurance. 1. The method of the screening for fake parts (1) The procedure flow for the production discontinued parts (2) The comparison and examination between the diagnostic using X-ray computed tomography (X-ray CT) images and various examinations (3) Other observation cases using X-ray CT images 2. Effectiveness and consideration in reliability evaluation test using X-ray CT image (1) Comparison of observation cases with a variety of jointing points in parts (2) Consideration of application of nondestructive observation technique in reliability test Use of X-ray CT images is effective in diagnosing the quality of the product or the process. Moreover, we find that use of X-ray CT images is effective for the improvement of the reproducibility of the evaluation examination. Then we find that use of X-ray CT images can reduce the time of evaluation examination too.

We identified that whirlin is localized to chromosome 9q32-33, and is up-regulated in colorectal cancer tissues by using oligonucleotide array techniques and the Sosui system (http://www.tuat.ac.jp/~mitaku/sosui/). The deduced 920-amino acid protein encoded by the whirlin gene contains three PDZ domains and a proline-rich region that separates PDZ2 from PDZ3, which is located at the C terminus. As previously reported, human whirlin gene is alternatively spliced to form a long and a short transcript in situ hybridization. The sequence of the encoded protein shows that the short C-terminal isoform contains one PDZ domain and the proline-rich domain (whirlin isoform 2), whereas the long isoform is composed of all three PDZ domains and the proline-rich domain (whirlin isoform 1). The gene expression of whirlin was found to be up-regulated in colorectal cancer tissues compared with matched normal colon tissues by semi-quantitative RT-PCR (P

Two-photon laser-scanning microscopy (TPLSM) is a powerful diagnostic tool for real-time, high-resolution structural imaging. However, obtaining high-quality in vivo TPLSM images of intra-abdominal organs remains technically challenging. An organ-stabilizing system was applied to high-quality TPLSM imaging. Real-time imaging of visceral organs, such as the liver, spleen, kidney and intestine, of transgenic green fluorescent protein (GFP) mice was performed in vivo using TPLSM. The bacterial translocation model using dextran sodium sulfate (DSS)-induced colitis was also investigated in prepared GFP mice following simple surgery. This allowed the capture of morphological real images using in vivo TPLSM. Immunohistochemical analysis of ZO-1 was performed to support the morphological findings of TPLSM. We established an organ-stabilizing system to evaluate the real-time imaging of visceral organs in actin–GFP transgenic mice using in vivo TPLSM. DSS-induced colitis showed irregularity of crypt architecture, disappearance of crypts, inflammatory cell infiltration and increased rolling of white blood cells along the vasculature. In addition, the intercellular distance of mucosal cells in the crypt and vascular endothelial cells in the intestinal wall was increased in the intestinal mucosa during DSS colitis. In DSS colitis, there was remarkable loss of mucosal and vascular endothelial ZO-1 expression, as could be seen by a decrease in ZO-1 staining. In conclusion, our observations suggested the possibility that our TPLSM imaging system can be used to clarify the pathophysiological changes in various diseases using longitudinal studies of microscopic changes in the same animal over long periods of time.

The small GTPase Rac regulates neuronal behavior, but whether it also functions in neural progenitor cells has not yet been explored. Here we report that Rac contributes to the regulation of nuclear migration in neocortical progenitor cells. Rac1 is expressed by progenitor cells in a unique spatiotemporal pattern. Cross-sectional immunohistochemical examination revealed intense Rac1 immunoreactivity at the ventricular surface. Similar staining patterns were obtained by immunofluorescence for a Rac-activator, Tiam1, and by reactions to detect the GTP-bound (active) form of Rac. En face inspection of the ventricular surface revealed that apical Rac1 localization was most frequent in M-phase cells, and the endfeet of cells in other cell cycle phases also showed apical Rac1 distribution at lower frequencies. To ask whether progenitor cell behavior prior to and during M phase is Rac-dependent, we monitored individual DiI-labeled progenitor cells live in the presence of a Rac inhibitor, NSC23766. We observed significantly retarded adventricular nuclear migration, as well as cytokinesis failures. Similar inhibitory effects were obtained by forced expression of a dominant-negative Rac1. These results suggest that Rac may play a role in interkinetic nuclear migration in the developing mouse brain.

Rab GTPases and their effectors play important roles in membrane trafficking between cellular compartments in eukaryotic cells. In the present study, we examined the roles of Rab11B and its effectors in insulin secretion in pancreatic beta-cells. In the mouse insulin-secreting cell line MIN6, Rab11 was co-localized with insulin-containing granules, and over-expression of the GTP- or the GDP-bound form of Rab11B significantly inhibited regulated secretion, indicating involvement of Rab11B in regulated insulin secretion. To determine the downstream signal of Rab11-mediated insulin secretion, we examined the effects of various Rab11-interacting proteins on insulin secretion, and found that Rip11 is involved in cAMP-potentiated insulin secretion but not in glucose-induced insulin secretion. Analyses by immunocytochemistry and subcellular fractionation revealed Rip11 to be co-localized with insulin granules. The inhibitory effect of the Rip11 mutant was not altered in MIN6 cells lacking Epac2, which mediates protein kinase A (PKA)-independent potentiation of insulin secretion, compared with wild-type MIN6 cells. In addition, Rip11 was found to be phosphorylated by PKA in MIN6 cells. The present study shows that both Rab11 and its effector Rip11 participate in insulin granule exocytosis and that Rip11, as a substrate of PKA, regulates the potentiation of exocytosis by cAMP in pancreatic beta-cells.