Biomedical subjects

Toshihiko Kadoya

Publications and source records attributed to Toshihiko Kadoya.

At least 19 recordsLinked to original sources

PubMed2006

A carbohydrate-binding protein, Galectin-1, promotes proliferation of adult neural stem cells.

In the subventricular zone of the adult mammalian forebrain, neural stem cells (NSCs) reside and proliferate to generate young neurons. We screened factors that promoted the proliferation of NSCs in vitro by a recently developed proteomics technique, the ProteinChip system. In this screen, we identified a soluble carbohydrate-binding protein, Galectin-1, as a candidate. We show herein that Galectin-1 is expressed in a subset of slowly dividing subventricular zone astrocytes, which includes the NSCs. Based on results from intraventricular infusion experiments and phenotypic analyses of knockout mice, we demonstrate that Galectin-1 is an endogenous factor that promotes the proliferation of NSCs in the adult brain.

Animals↗

PubMed2006

Optimization of elution salt concentration in stepwise elution of protein chromatography using linear gradient elution data. Reducing residual protein A by cation-exchange chromatography in monoclonal antibody purification.

Our simple method for optimization of the elution salt concentration in stepwise elution was applied to the actual protein separation system, which involves several difficulties such as detection of the target. As a model separation system, reducing residual protein A by cation-exchange chromatography in human monoclonal antibody (hMab) purification was chosen. We carried out linear gradient elution experiments and obtained the data for the peak salt concentration of hMab and residual protein A, respectively. An enzyme-linked immunosorbent assay was applied to the measurement of the residual protein A. From these data, we calculated the distribution coefficient of the hMab and the residual protein A as a function of salt concentration. The optimal salt concentration of stepwise elution to reduce the residual protein A from the hMab was determined based on the relationship between the distribution coefficient and the salt concentration. Using the optimized condition, we successfully performed the separation, resulting in high recovery of hMab and the elimination of residual protein A.

Antibodies, Monoclonal↗

PubMed2005

Rational methods for predicting human monoclonal antibodies retention in protein A affinity chromatography and cation exchange chromatography. Structure-based chromatography design for monoclonal antibodies.

Rational methods for predicting the chromatographic behavior of human monoclonal antibodies (hMabs) in protein A affinity chromatography and cation exchange chromatography from the amino acid sequences information were proposed. We investigated the relation between the structures of 28 hMabs and their chromatographic behavior in protein A affinity chromatography and cation exchange chromatography using linear gradient elution experiments. In protein A affinity chromatography, the elution pH of the hMabs was correlated with not only the structure of the Fc region (subclass), but also that of the variable region. The elution pH of hMabs that have LYLQMNSL sequences in between the CDR2 and CDR3 regions of the heavy chain became lower among the same subclass of hMabs. In cation exchange chromatography, the peak salt concentrations IR of hMabs that have the same sequences of variable regions (or that have a structural difference in their Fc region, which puts them into a subclass) were similar. The IR values of hMabs were well correlated with the equilibrium association constant Ke, and also with the surface positive charge distribution of the variable region of the heavy chain (corrected surface net positive charge (cN) of the VH region). Based on these findings, we developed rational methods for predicting the retention behavior, which were also tested with eight additional hMabs. By considering the information on the number of binding sites associated with protein adsorption as determined experimentally, and the surface positive charge distribution from the three-dimensional structure of Mab A, we hypothesized that hMabs is separated by cation exchange chromatography as the surface positive charge distribution of the VH region is recognized.

Amino Acid Sequence↗

PubMed2005

Oxidized galectin-1 advances the functional recovery after peripheral nerve injury.

Oxidized galectin-1 has been shown to promote axonal regeneration from transected-nerve sites in an in vitro dorsal root ganglion (DRG) explant model as well as in in vivo peripheral nerve axotomy models. The present study provides evidence that oxidized galectin-1 advances the restoration of nerve function after peripheral nerve injury. The sciatic nerve of adult rats was transected and the distal nerve was frozen after being sutured into a proximal site with four epineurial stitches. An osmotic pump delivered oxidized galectin-1 peripherally to the surgical site. Functional recovery was assessed by measurement of the degree of toe spread of the hind paw for 3 months after the sciatic nerve lesion. The recovery curves of toe spread in the test group showed a statistically significant improvement of functional recovery after day 21 by the application of oxidized recombinant human galectin-1 (rhGAL-1/Ox) compared to the control group. This functional recovery was supported by histological analysis performed by light microscopic examination. The regenerating myelinated fibers at the site 21 mm distal to the nerve-transected site were quantitatively examined at 100 days after the operation. The frequency distribution of myelinated fiber diameters showed that exogenous rhGAL-1/Ox increased the number and diameter of regenerating myelinated fibers; the number of medium-sized (6-11 microm in diameter) fibers increased significantly (P<0.05). These results indicate that oxidized galectin-1 promotes the restoration of nerve function after peripheral nerve injury. Thus, rhGAL-1/Ox may be a factor for functional restoration of injured peripheral nerves.

Animals↗

PubMed2005

Neuroprotective effect of oxidized galectin-1 in a transgenic mouse model of amyotrophic lateral sclerosis.

Abnormal accumulation of neurofilaments in motor neurons is a characteristic pathological finding in amyotrophic lateral sclerosis (ALS). Recently, we revealed that galectin-1, whose oxidized form has axonal regeneration-enhancing activity, accumulates in the neurofilamentous lesions in ALS. To investigate whether oxidized galectin-1 has a beneficial effect on ALS, oxidized recombinant human galectin-1 (rhGAL-1/ox) or physiological saline was injected into the left gastrocnemius muscle of the transgenic mice over-expressing a mutant copper/zinc superoxide dismutase (SOD1) with a substitution of histidine to arginine at position 46 (H46R SOD1). The H46R SOD1 transgenic mice, which represented a new animal model of familial ALS, were subsequently assessed for their disease onset, life span, duration of illness, and motor function. Furthermore, the number of remaining large anterior horn cells of spinal cords was also compared between the two groups. The results showed that administration of rhGAL-1/ox to the mice delayed the onset of their disease and prolonged the life of the mice and the duration of their illness. Motor function, as evaluated by a Rotarod performance, was improved in rhGAL-1/ox-treated mice. Significantly more anterior horn neurons of the lumbar and cervical cords were preserved in the mice injected with rhGAL-1/ox than in those injected with physiological saline. The study suggests that rhGAL-1/ox administration could be a new therapeutic strategy for ALS.

Age of Onset↗

PubMed2005

Oxidized galectin-1 is an essential factor for peripheral nerve regeneration.

Although many factors have been implicated in the regenerative response of peripheral axons to nerve injury, the signals that prompt neurons to extend processes in peripheral nerves after axotomy are not well-understood. As shown in the first chapter, oxidized recombinant human galectin-1 (rhGAL-1/Ox), which lacks lectin activity, promotes initial axonal growth in an in vitro peripheral nerve regeneration model at low concentrations (pg/ml). At a similarly low concentration, rhGAL-1/Ox has also been shown to be effective in enhancing axonal regeneration using in vivo experiments. Moreover, the application of functional anti-rhGAL-1 antibody strongly inhibited axonal regeneration in vivo as well as in vitro. Since galectin-1 (GAL-1) is expressed in the regenerating sciatic nerves as well as in both sensory and motoneurons, these results indicate that GAL-1, which is secreted into the extracellular space, is subsequently oxidized and then may regulate initial repair after axotomy. This possibility was confirmed by Western blot analysis, which revealed that both reduced and oxidized forms of GAL-1 are present in culture media of DRG neurons and immortalized adult mouse Schwann cells (IMS32). Externalized GAL-1/Ox has been found to stimulate macrophages to secrete an axonal regeneration-promoting factor. From these results, we propose that axonal regeneration occurs in axotomized peripheral nerves as a result of cytosolic reduced GAL-1 being released from Schwann cells and injured axons, which then becomes oxidized in the extracellular space. GAL-1/Ox in the extracellular space stimulates macrophages to secrete a factor that promotes axonal growth and Schwann cell migration, thus enhancing peripheral nerve regeneration and functional recovery. These results suggest that rhGAL-1/Ox may be a novel factor for functional restoration of injured peripheral nerves.

Animals↗

PubMed2005

Regulation of the neuronal fate by DeltaFosB and its downstream target, galectin-1.

In mammals, the regulation of the cell fate to either proliferate, differentiate, arrest cell growth, or initiate programmed cell death is the most fundamental mechanism for maintaining normal cell function and tissue homeostasis. Under multiple signaling pathways, Jun and Fos family proteins are known to play important roles as components of an AP-1 (activator protein-1) complex, to regulate the transcription of various genes involved in cell proliferation, differentiation and programmed cell death. DeltaFosB, one of the AP-1 subunits encoded by alternatively spliced fosB mRNA, triggers one round of proliferation in quiescent rat embryo cell lines, followed by a different cell fate such as morphological alteration or delayed cell death. As one of the downstream targets of the DeltaFosB in rat3Y1 cell line, we identified rat galectin-1 and its novel variant, galectin-1beta, and demonstrated that the expression of galectin-1 is required for the proliferative activation of quiescent rat1A cells by DeltaFosB, thus indicating that galectin-1 is one of functional targets of DeltaFosB. The expression of DeltaFosB is highly inducible in the adult brain in response to various insults such as ischemic reperfusion injury, seizure induced by electric stimulation or cocaine administration. On the other hand, galectin-1 has also been shown to be involved in the regeneration of damaged axons in the peripheral nerve, as well as in neurite outgrowth or synaptic connectivity in the olfactory system during development. We herein propose that DeltaFosB together with galectin-1, may therefore mediate neuroprotection and neurogenesis in response to brain damage.

Animals↗

PubMed2005

Structural and functional studies of galectin-1: a novel axonal regeneration-promoting activity for oxidized galectin-1.

Recently, we discovered oxidized galectin-1 as a factor that regulates initial axonal growth in the peripheral nerve after axotomy. Galectin-1 is a member of the galectins, a family of animal lectins ranging from Caenorhabditis elegans to humans, which is defined by their affinity for beta-galactosides and by significant sequence similarity in the carbohydrate-binding site. Galectin-1 is a homodimer with a subunit molecular mass of 14.5 kDa, which contains six cysteine residues per subunit. The cysteine residues should be in a free state in order to maintain a molecular structure that is capable of showing lectin activity. However, our structural analysis revealed that the axonal regeneration-promoting factor exists as an oxidized form of galectin-1, containing three intramolecular disulfide bonds. The oxidized galectin-1 exhibited marked peripheral nerve regeneration-promoting activity, although it showed no lectin activity. It was also revealed that oxidized galectin-1 exists as a monomer in a physiological solution. Galectin-1 seems to have a variety of biological functions. These functions could vary according to the time at which a biological function is taking place, as well as the site in which a biological function is taking place. In addition, these functions could vary according to the structure of galectin-1 by which a particular biological function is taking place. Disulfide bond formation alters the structure of galectin-1, so as to confer the novel ability to promote axonal regeneration. Oxidized galectin-1 likely acts as an autocrine or paracrine factor to promote axonal regeneration, functioning more like a cytokine than as a lectin.

Amino Acid Sequence↗

PubMed2004

Oxidized galectin-1 stimulates macrophages to promote axonal regeneration in peripheral nerves after axotomy.

Various neurotrophic factors that promote axonal regeneration have been investigated in vivo, but the signals that prompt neurons to send out processes in peripheral nerves after axotomy are not well understood. Previously, we have shown oxidized galectin-1 (GAL-1/Ox) promotes initial axonal growth after axotomy in peripheral nerves. However, the mechanism by which GAL-1/Ox promotes axonal regeneration remains unclear and is the subject of the present study. To identify possible target cells of GAL-1/Ox, a fluorescently labeled recombinant human GAL-1/Ox (rhGAL-1/Ox) was incubated with DRG neurons, Schwann cells, and intraperitoneal macrophages from adult rats. Only the cell surfaces of intraperitoneal macrophages bound the rhGAL-1/Ox, suggesting that these cells possess a receptor for GAL-1/Ox. Experiments examining tyrosine phosphorylation revealed that rhGAL-1/Ox stimulated changes in signal transduction pathways in these macrophages. These changes caused macrophages to secrete an axonal growth-promoting factor. This was demonstrated when conditioned media of macrophages stimulated with rhGAL-1/Ox in 48 hr culture strongly enhanced axonal regeneration from transected-nerve sites of DRG explants. Furthermore, activated macrophage-conditioned media also improved Schwann cell migration from the transected-nerve sites. From these results, we propose that axonal regeneration occurs in axotomized peripheral nerves as a result of cytosolic reduced galectin-1 being released from Schwann cells and injured axons, which then becomes oxidized in the extracellular space. Oxidized galectin-1 then stimulates macrophages to secrete a factor that promotes axonal growth and Schwann cell migration, thus enhancing peripheral nerve regeneration.

Animals↗

PubMed2004

Synthesis, localization and externalization of galectin-1 in mature dorsal root ganglion neurons and Schwann cells.

We recently confirmed that oxidized galectin-1 is a novel factor enhancing axonal growth in peripheral nerves after axotomy, but the process of extracellular release and oxidization of endogenous galectin-1 in the injured nervous tissue remains unknown. In the present study, we examined the distribution of galectin-1 in adult rat dorsal root ganglia (DRG) in vivo and in vitro. By RT-PCR analysis and in situ hybridization histochemistry, galectin-1 mRNA was detected in both DRG neurons and non-neuronal cells. Immunohistochemical analyses revealed that galectin-1 was distributed diffusely throughout the cytoplasm in smaller diameter neurons and Schwann cells in DRG sections. In contrast, the immunoreactivity for galectin-1 was detected in almost all DRG neurons from an early stage in culture (3 h after seeding) and was restricted to the surface and/or extracellular region of neurons and Schwann cells at later stages in culture. In a manner similar to the primary cultured cells, we also observed the surface and extracellular expression of this molecule in immortalized adult mouse Schwann cells (IMS32). Western blot analysis has revealed that both reduced and oxidized forms of galectin-1 were detected in culture media of DRG neurons and IMS32. These findings suggest that galectin-1 is externalized from DRG neurons and Schwann cells upon axonal injury. Some of the molecules in the extracellular milieu may be converted to the oxidized form, which lacks lectin activity but could act on neural tissue as a cytokine.

Animals↗

PubMed2003

Galectin-1 is involved in the potentiation of neuropathic pain in the dorsal horn.

Galectin-1 is one of the endogenous-galactoside-binding lectins, suggested to be involved in a variety of functions, such as neurite outgrowth, synaptic connectivity, cell proliferation and apoptosis. This protein is expressed in the dorsal root ganglion (DRG) and the spinal cord in the developing and adult rats, especially intensely in small DRG neurons. In the present study, we examined whether galectin-1 is colocalized with TrkA or c-Ret mRNA in small DRG neurons and the effect of axotomy on the expression of galectin-1 in the spinal cord. About 20% of the DRG neurons showed intense galectin-1-immunoreactivity (IR). Of the intensely galectin-1-IR DRG neurons, 93.9% displayed c-Ret mRNA positive signals. On the other hand, only 6.8% displayed TrkA mRNA positive signals. Galectin-1-IR was increased in the dorsal horn at 1 to 2 weeks after axotomy. Intrathecal administration of anti-recombinant human galectin-1 antibody (anti-rhGAL-1 Ab) partially but significantly attenuated the upregulation of substance P receptor (SPR) in the spinal dorsal horn and the mechanical hypersensitivity induced by the peripheral nerve injury. These data suggest that endogenous galectin-1 may potentiate neuropathic pain after the peripheral nerve injury at least partly by increasing SPR in the dorsal horn.

Animals↗

PubMed2003

An N-terminal fragment of ProSAAS (a granin-like neuroendocrine peptide precursor) is associated with tau inclusions in Pick's disease.

The deposition of aggregated tau in cytoplasmic inclusions is one of the common neuropathological features in various dementing neurodegenerative disorders. At present, it remains unclear whether tau inclusions exert neurotoxicity or they are simply the consequence of neurodegeneration. In our approach for the analysis of the composition of tau inclusions, we detected the intense binding of anti-diacylglycerol kinase-zeta (DGK-zeta) antibodies to Pick bodies (PBs), which represent tau inclusions in Pick's disease. The polyclonal antibodies were found to cross-react with a 21-kDa protein, but not with tau or ubiquitin, on Western blots of normal human brain extracts. Analysis of the 21-kDa protein by two-dimensional-gel electrophoresis and mass-spectrometry revealed that the protein is an N-terminal fragment of proSAAS (a human granin-like neuroendocrine peptide precursor). Our results suggest that sequestration of the N-terminal fragment of proSAAS in intracellular PBs may cause a functional disturbance of neurons in Pick's disease.

Amino Acid Sequence↗

PubMed2003

Proteomic analysis of hematopoietic stem cell-like fractions in leukemic disorders.

DNA microarray analysis has been applied to identify molecular markers of human hematological malignancies. However, the relatively low correlation between the abundance of a given mRNA and that of the encoded protein makes it important to characterize the protein profile directly, or 'proteome,' of malignant cells in addition to the 'transcriptome.' To identify proteins specifically expressed in leukemias, here we isolated AC133(+) hematopoietic stem cell-like fractions from the bone marrow of 13 individuals with various leukemic disorders, and compared their protein profiles by two-dimensional electrophoresis. A total of 11 differentially expressed protein spots corresponding to 10 independent proteins were detected, and peptide fingerprinting combined with mass spectrometry of these proteins revealed them to include NuMA (nuclear protein that associates with the mitotic apparatus), heat shock proteins, and redox regulators. The abundance of NuMA in the leukemic blasts was significantly related to the presence of complex karyotype anomalies. Conditional expression of NuMA in a mouse myeloid cell line resulted in the induction of aneuploidy, cell cycle arrest in G(2)-M phases, and apoptosis. These results demonstrate the potential of proteome analysis with background-matched cell fractions obtained from fresh clinical specimens to provide insight into the mechanism of human leukemogenesis.

Adult↗

PubMed2003

Decreased galectin-1 immunoreactivity of the skin in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving motor neurons. In addition to motor neuron signs and symptoms, a lack of bedsores has been considered a feature of ALS. Recently, we revealed that galectin-1 is a component of the axonal spheroid, which is an early pathological change of the spinal cord in ALS. To investigate whether galectin-1 is associated with skin changes in ALS, we performed an immunohistochemical investigation using anti-galectin-1 antibodies. The present study revealed that galectin-1 immunoreactivity is reduced in the skin of patients with ALS, suggesting that cutaneous galectin-1 is involved in the pathological process of ALS.

Aged↗

PubMed2003

CrkL directs ASAP1 to peripheral focal adhesions.

Searching for proteins in platelets that can interact with the N-terminal SH3 domain of CrkL (using a combination of a pull-down assay followed by mass spectrometry), we have found that human platelets express an ADP-ribosylation factor (Arf)-specific GTPase-activating protein (GAP), ASAP1, as a CrkL-binding protein. In spreading platelets, most endogenous ASAP1 is localized at peripheral focal adhesions. To determine the physiologic significance of the CrkL-ASAP1 association, we overexpressed CrkL, ASAP1, or both in combination in COS7 cells. Unlike endogenous ASAP1 in platelets, overexpressed ASAP1 showed diffuse cytoplasmic distribution. However, when co-expressed with wild-type CrkL, both endogenous and expressed ASAP1 accumulated at CrkL-induced focal adhesions. An SH2-mutated CrkL, which cannot localize at focal adhesions, failed to recruit ASAP1 into focal adhesions. Thus, CrkL appears to be a lynchpin between ASAP1 and peripheral focal adhesions.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Oxidized galectin-1 stimulates the migration of Schwann cells from both proximal and distal stumps of transected nerves and promotes axonal regeneration after peripheral nerve injury.

Oxidized galectin-1 has recently been identified as a key factor that plays important roles in initial axonal growth in injured peripheral nerves. The aim of this study was to investigate the effects of oxidized galectin-1 on regeneration of rat spinal nerves using acellular autografts (containing no viable cells) and allografts (containing no cell membranes) with special attention to the relationship between axonal regeneration and Schwann cell migration. Immunohistochemically, endogenous galectin-1 was expressed in dorsal root ganglion (DRG) neurons, spinal cord motoneurons, and axons and Schwann cells in normal sciatic nerves. Administration of oxidized recombinant human galectin-1 (rh-gal-lox, 5 ng/ml) in autograft model promoted axonal regeneration from motoneurons as well as from DRG neurons; this was confirmed by a fluorogold tracer study (p < 0.05). Anti-rh-gal-1 antibody (30 microg/ml) strongly inhibited axonal regrowth (p < 0.05). Pretreatment of allografts with rh-gal-lox stimulated the migration of Schwann cells not only from proximal stumps but also from distal stumps into the grafts, resulting in accelerated axonal regeneration (p < 0.05). Moreover, Schwann cell migration preceded the axonal growth in the presence of exogenous rh-gal-lox in the grafts. These results strongly suggest that local administration of exogenous rh-gal-lox promotes the migration of Schwann cells followed by axonal regeneration from both motor and sensory neurons, resulting in acceleration of neuronal repair. This technique may also be of value in the repair of human nerves.

Animals↗

PubMed2002

T lymphocytes constitutively produce an interferonlike cytokine limitin characterized as a heat- and acid-stable and heparin-binding glycoprotein.

Several reports have described "multifunctional" eukaryotic mRNAs producing more than one protein through alternative translational initiation at multiple AUG codons. There are 2 such codons in the 5' region of our recently cloned limitin gene where 2 open reading frames overlap by 34 nucleotides. The deduced protein translated from the first ATG contains 33 amino acids, lacks a signal peptide, and has no obvious effects on the transfected 293T cells. We found that the second ATG is more effective as a translational initiation site than the first ATG and yields a secreted protein of 182 amino acids with the same activity as products made with full-length limitin cDNA. Immunohistochemical and reverse transcription-polymerase chain reaction analysis revealed that the longer limitin protein is produced by mature T lymphocytes in spleen and thymus as well as by bronchial epithelial and salivary duct cells in healthy mice. Properties of recombinant limitin were determined, revealing it to be a serologically distinct, heat- and acid-stable, heparin-binding glycoprotein with the potential for dimerization. Although the longer limitin protein is structurally and characteristically related to type I interferons, its production is uniquely regulated by translation as well as transcription.

Acids↗

PubMed2002

Galectin-1 plays essential roles in adult mammalian nervous tissues. Roles of oxidized galectin-1.

Previous data have suggested that galectin-1 is expressed widely in nervous tissues at embryonic stages but becomes restricted mainly to peripheral nervous tissues with maturation. Though the expression is intense in adult mammalian peripheral neurons, there had been no report about functions of galectin-1 there. Recently we discovered a factor that enhanced peripheral axonal regeneration. The factor was identified as oxidized galectin-1 with three intramolecular disulfide bonds and showed no lectin activity. Oxidized recombinant human galectin-1 (rhGAL-1/Ox) showed the same nerve growth promoting activity at very low concentrations (pg/ml). rhGAL-1/Ox at similarly low concentration was also effective in in vivo experiments of axonal regeneration. Moreover, the application of functional anti-rhGAL-1 antibody strongly inhibited the axonal regeneration in vivo as well as in vitro. Since galectin-1 is expressed in the regenerating sciatic nerves as well as in both sensory neurons and motor neurons, these results suggest that galectin-1 is secreted into the extracellular space to be oxidized, and then, in its oxidized form, to regulate initial repair after axotomy. The administration of oxidized galectin-1 effectively promoted functional recovery after sciatic nerve injury in vivo. Oxidized galectin-1, hence, appears to play an important role in promoting axonal regeneration, working as a kind of cytokine, not as a lectin. Recent reports indicated additional roles of cytosolic galectin-1 in neural diseases, such as ALS. Furthermore galectin-1 has been proved to be a downstream target of DeltaFosB. In hippocampus of rat brain, expression of DeltaFosB is induced immediately after ischemia-reperfusion, suggesting that galectin-1 may also play important roles in central nervous system after injury.

Animals↗