Myelin lesion in the aspartoacylase (Aspa) knockout rat, an animal model for Canavan disease.

Canavan disease (CD) is a fatal hereditary neurological disorder caused by a mutation in the aspartoacylase (ASPA) gene and characterized by neurological signs and vacuolation in the central nervous system (CNS). The mutation inhibits the hydrolysis of N-acetyl-aspartate (NAA) resulting in accumulation of NAA in the CNS. A new Aspa-knockout rat was generated by transcription activator-like effector nuclease (TALEN) technology. Herein we describe the pathological and morphometrical findings in the brain and spinal cords of Aspa-knockout rats. Although Aspa-knockout rats did not show any neurological signs, vacuolation with swollen axons, hypomyelination, and activated swollen astrocytes were observed mainly in the brainstem reticular formation, ascending and descending motor neuron pathway, and in the olfactory tract. Morphometrical analysis revealed no obvious change in the number of neurons. These changes in the CNS are similar to human CD, suggesting that this animal model would be useful for further study of treatment and understanding the pathophysiology of human CD.

Segmental aplasia of a uterine horn in two RccHan:WIST rats.

Aplasia of the uterine horn caused by developmental defects has been reported in several species but has not been reported in RccHan:WIST rats. We encountered spontaneous segmental aplasia of a right uterine horn in two RccHan:WIST rats and detailed its pathological characteristics. The right uterine horn of both rats had similar gross and histological appearances. At necropsy, there was segmental loss of the tissues corresponding to normal right uterine horn, which consisted of a fibrous band connected to the uterine cervix. A cystic structure with clear and colorless fluid was observed in the cranial segment of the right uterine horn close to the right oviduct. The cystic structure was thought to be a partially developed tissue to the right uterine horn. The cystic structure seemed to be derived from the right uterine horn. Histologically, a single layer of cuboidal epithelium lined the luminal surface of the cystic structure, the endometrium was thin, and no uterine glands were observed. The fibrous band was composed of α-SMA positive smooth muscle cells, connective tissue, and blood vessels, but cytokeratin AE1/AE3 positive epithelium and uterine endometrium were absent. Based on these gross findings and histological features, segmental aplasia of a uterine horn was diagnosed. To our knowledge, these cases of segmental aplasia of a uterine horn are the first ones described in RccHan:WIST rats.

Ecrg4 peptide is the ligand of multiple scavenger receptors.

Esophageal cancer-related gene 4 (Ecrg4) encodes a hormone-like peptide that is believed to be involved in a variety of physiological phenomena, including tumour suppression. Recent progress in the study of Ecrg4 has shown that Ecrg4 is a proinflammatory factor and induces the expression of several cytokines and chemokines in macrophages/microglia. However, the detailed molecular mechanisms of Ecrg4 signalling, especially the Ecrg4 receptors, remain poorly understood. Here, using retrovirus-mediated expression cloning, we identified lectin-like oxidised low-density lipoprotein receptor-1 (LOX-1) as a membrane protein that binds amino acid residues 71-132 of Ecrg4 (Ecrg4(71-132)). Moreover, in addition to LOX-1, several scavenger receptors, such as Scarf1, Cd36 and Stabilin-1, facilitated the efficient internalisation of Ecrg4(71-132) into cells. A broad competitive inhibitor of scavenger receptors, polyinosinic acid, reduced both the binding of Ecrg4(71-132) and the activation of NF-κB in microglia. This activation was dependent on MyD88, an adaptor protein that recruits signalling proteins to Toll-like receptors (TLRs), with the consequent induction of various immune responses. These data suggest that multiple scavenger receptors recognise Ecrg4(71-132) and transduce its signals, together with TLRs, in microglia.

Candidate genes responsible for early key events of phenobarbital-promoted mouse hepatocellular tumorigenesis based on differentiation of regulating genes between wild type mice and humanized chimeric mice.

Phenobarbital (PB) is a nongenotoxic hepatocellular carcinogen in rodents. PB induces hepatocellular tumors by activating the constitutive androstane receptor (CAR). Some previous research has suggested the possible involvement of epigenetic regulation in PB-promoted hepatocellular tumorigenesis, but the details of its molecular mechanism are not fully understood. In the present study, comprehensive analyses of DNA methylation, hydroxymethylation and gene expression using microarrays were performed in mouse hepatocellular adenomas induced by a single 90 mg kg-1 intraperitoneal injection dose of diethylnitrosamine (DEN) followed by 500 ppm PB in the diet for 27 weeks. DNA modification and expression of hundreds of genes are coordinately altered in PB-induced mouse hepatocellular adenomas. Of these, gene network analysis showed alterations of CAR signaling and tumor development-related genes. Pathway enrichment analysis revealed that differentially methylated or hydroxymethylated genes belong mainly to pathways involved in development, immune response and cancer cells in contrast to differentially expressed genes belonging primarily to the cell cycle. Furthermore, overlap was evaluated between the genes with altered expression levels with 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) alterations in mouse hepatocellular adenoma induced by DEN/PB and the genes with altered expression levels in the liver of CD-1 mice or humanized chimeric mice treated with PB for 7 days. With the integration of transcriptomic and epigenetic approaches, we detected candidate genes responsible for early key events of PB-promoted mouse hepatocellular tumorigenesis. Interestingly, these genes did not overlap with genes altered by the PB treatment of humanized chimeric mice, thus suggesting a species difference between the effects of PB in mouse and human hepatocytes.

Alteration of microRNA expressions in the pons and medulla in rats after 3,3'-iminodipropionitrile administration.

Although 3,3'-iminodipropionitrile (IDPN) is widely used as a neurotoxicant to cause axonopathy due to accumulation of neurofilaments in several rodent models, its mechanism of neurotoxicity has not been fully understood. In particular, no information regarding microRNA (miRNA) alteration associated with IDPN is available. This study was conducted to reveal miRNA alteration related to IDPN-induced neurotoxicity. Rats were administered IDPN (20, 50, or 125 mg/kg/day) orally for 3, 7, and 14 days. Histopathological features were investigated using immunohistochemistry for neurofilaments and glial cells, and miRNA alterations were analyzed by microarray and reverse transcription polymerase chain reaction. Nervous symptoms such as ataxic gait and head bobbing were observed from Day 9 at 125 mg/kg. Axonal swelling due to accumulation of neurofilaments was observed especially in the pons, medulla, and spinal cord on Day 7 at 125 mg/kg and on Day 14 at 50 and 125 mg/kg. Furthermore, significant upregulation of miR-547* was observed in the pons and medulla in treated animals only on Day 14 at 125 mg/kg. This is the first report indicating that miR-547* is associated with IDPN-induced neurotoxicity, especially in an advanced stage of axonopathy.

Ecrg4 contributes to the anti-glioma immunosurveillance through type-I interferon signaling.

Esophageal cancer-related gene 4 (Ecrg4), a hormone-like peptide, is thought to be a tumor suppressor, however, little is known about the mechanism of how Ecrg4 suppresses tumorigenesis. Here, we show that the ecrg4 null glioma-initiating cell (GIC) line, which was generated from neural stem cells of ecrg4 knockout (KO) mice, effectively formed tumors in the brains of immunocompetent mice, whereas the transplanted ecrg4 wild type-GIC line GIC(+/+) was frequently eliminated. This was caused by host immune system including adaptive T cell responses, since depletion of CD4+, CD8+, or NK cells by specific antibodies in vivo recovered tumorigenicity of GIC(+/+). We demonstrate that Ecrg4 fragments, amino acid residues 71-132 and 133-148, which are produced by the proteolitic cleavage, induced the expression of pro-inflammatory cytokines in microglia in vitro. Moreover, blockades of type-I interferon (IFN) signaling in vivo, either depleting IFN-α/ß receptor 1 or using stat1 KO mice, abrogated the Ecrg4-dependent antitumor activity. Together, our findings indicate a major antitumor function of Ecrg4 in enhancing host immunity via type-I IFN signaling, and suggest its potential as a clinical candidate for cancer immunotherapy.

Claudin-1 Binder Enhances Epidermal Permeability in a Human Keratinocyte Model.

Tight junctions (TJs) are complex biochemical structures that seal the intercellular space and prevent the free movement of solutes across epithelial cell sheets. Modulating the TJ seal is a promising option for increasing the transdermal absorption of drugs. Within TJs, the binding of the claudin (CLDN) family of tetratransmembrane proteins through cis- and trans-interactions is an integral part of seal formation. Because epidermal TJs contain CLDN-1 and CLDN-4, a binder for these CLDNs may be a useful modulator of the permeability of the epidermal barrier. Here, we investigated whether m19, which can bind to CLDN-1/-4 (also CLDN-2/-5), modulates the integrity of epidermal TJs and the permeability of cell sheets to solutes. Treatment of normal human epidermal keratinocytes (NHEKs) with the CLDN binder reduced the integrity of TJs. A CLDN-1-specific binder (a monoclonal antibody, clone 7A5) also weakened the TJ seal in NHEKs. Although m19 attenuated the TJ barrier in human intestinal epithelial cells (Caco-2), 7A5 did not. Treatment of NHEKs with 7A5 enhanced permeation of a paracellular permeation marker. These findings indicate that CLDN-1 is a potential target for modulating the permeability of the epidermis, and that our CLDN-1 binder is a promising candidate molecule for development as a dermal absorption enhancer.

Cellular uptake and in vivo distribution of polyhistidine peptides.

Cell-penetrating peptides (CPPs) are arginine/lysine-rich sequences, and they are effectively internalized into cells. In this process, positive charge is crucial. In the present study, we found polyhistidine peptides (PHPs), as the novel CPP, which are efficiently internalized into cells in a positive charge-independent manner. Interestingly, cellular uptake of the PHPs increased as the chain length increased, reaching a maximum uptake at H16 (HHHHHHHHHHHHHHHH-NH2). This H16 peptide showed up to 14.6-fold higher cell-penetrating capacity against HT1080 human fibrosarcoma cells relative to a major CPP, the octa-arginine (RRRRRRRR-NH2) peptide. Cellular uptake of the H16 peptide is mainly due to macropinocytosis and most of the H16 peptide localizes in the lysosome and Golgi apparatus. However, a cytoplasmic pro-apoptotic domain (KLAKLAKKLAKLAK-NH2) conjugated to the H16 peptide showed cytotoxic effects. This indicates that a proportion of the H16 peptide escapes from the macropinosome to the cytoplasm. In a protein transduction study, green fluorescence protein fused to the H16 peptide (GFP-H16) was purified by Ni-NTA chromatography, detected using an anti-His-tag antibody and internalized into cells. This serial process reveals that H16 functions as a His-tag and protein transduction domain. Furthermore, in vivo distribution analysis showed that the H16 peptide accumulates immediately in tumor tissue and is retained up to 132h following injection into the tumor (HT1080 human fibrosarcoma)-bearing mice. This is the first observation of a His-polymer being internalize into cells efficiently. The findings suggest that PHPs are novel CPPs. In particular, the H16 peptide represents a promising drug delivery carrier candidate in medical and biotechnological fields.

A Spontaneous Oligodendroglioma in the Lumbar Portion of the Spinal Cord in a Young BrlHan:WIST@Jcl (GALAS) Rat.

Oligodendroglioma is a rare tumor originating from oligodendrocytes found mainly in the cerebrum in aged rats. Only a few reports have shown spontaneous occurrence of this tumor in the spinal cord, and no report has mentioned its occurrence in young rats. We encountered a case of spontaneous oligodendroglioma in the lumbar portion of the spinal cord in a young (9 weeks old) female BrlHan:WIST@Jcl (GALAS) rat. Here we report the detailed histopathological and immunohistochemical characteristics of this case. No clinical signs, no gross lesions at necropsy, and no specific changes in hematology or blood biochemistry were observed. The tumor was located in the dorsal funiculus in the lumbar portion of the spinal cord and widely spread to the dorsal root nerve. The neoplastic cells showed a sheet-like growth pattern and had small round nuclei, clear cytoplasm and distinct cell borders that resulted in a honeycomb pattern. No mitotic figures or other histological lesions were observed. The neoplastic cells were positively stained for Olig2 and PCNA. The present case was considered to be a low-grade oligodendroglioma based on the histological and immunohistochemical features. To our knowledge, our case is considered to be extremely rare and the first report in a young rat.

Cerebellar ataxia suspected to be caused by Oxytropis glabra poisoning in western Mongolian goats.

In the last five years in western Mongolia, a neurological disorder and resultant economic loss have developed in goats, sheep, cattle and horses: association of the disease with ingestion of Oxytropis glabra, a toxic plant, was suggested. Affected goats showed neurological signs, including ataxia, incoordination, hind limb paresis, fine head tremor and nystagmus. Three goats, one with moderate clinical signs and the other two with severe clinical signs, were necropsied and examined to describe and characterize the histologic, immunohistochemical and ultrastructural lesions. Although no gross pathological changes were observed in a variety of organs including the central nervous system of these goats, microscopic examination of the cerebellum demonstrated degenerative changes in all these goats, such as vacuolar changes and loss of Purkinje cells, torpedo formation in the granular layer, increased number of spheroids in the cerebellar medulla, and loss of axons and myelin sheaths of Purkinje cells. The chemical analysis of the dried plant detected 0.02-0.05% (dry weight basis) of swainsonine. This is the first report describing the clinical and pathological findings in Mongolian goats suspected to be affected by O. glabra poisoning.

Covalent split protein fragment-DNA hybrids generated through N-terminus-specific modification of proteins by oligonucleotides.

Semisynthetic protein-DNA hybrid molecules have recently attracted much attention as valuable tools for bioanalytical chemistry and nanobiotechnology. Here we describe a synthetic method for conjugating oligonucleotides to the N-terminus of recombinant proteins. Our strategy involves the conversion of amine-terminated oligonucleotides to thioester-functionalized oligonucleotides by using a bifunctional reagent bearing an N-hydroxysuccinimide ester and benzyl thioester group, followed by native chemical ligation with proteins containing an N-terminal cysteine. We applied this technique to construct split luciferase fragment-DNA hybrid systems in which the catalytic activity of split luciferase is restored by the re-assembly of each fragment through a specific DNA-protein or DNA-DNA interaction. Split protein fragment-DNA hybrids will offer new opportunities to explore the potential of protein-DNA conjugates for various applications.

Site-specific conjugation of oligonucleotides to the C-terminus of recombinant protein by expressed protein ligation.

We developed a convenient method for synthesizing homogeneous DNA-protein conjugates. The method is based on expressed protein ligation of intein-fusion proteins and oligonucleotides derivatized with a cysteine. A range of cysteinyl oligonucleotides were synthesized by using a new reagent 1 and were successfully applied to expressed protein ligation to attach the oligonucleotides specifically at the C-terminus of a recombinant protein.

Rectified photocurrent in a protein based molecular photo-diode consisting of a cytochrome b562-green fluorescent protein chimera self-assembled monolayer.

We fabricated a self-assembled monolayer (SAM) of a chimeric protein created as a novel model protein for an artificial light-harvesting complex (LHC) composed of two proteins, cytochrome b(562) (cytb(562)) mutated for SAM fabrication (cytb(562), N22C, G82C) and enhanced green fluorescent protein (EGFP). The SAM formation of chimeric protein on a single-crystalline Au(111) substrate was confirmed by atomic force microscopy (AFM) measurement. The rectified photocurrent of the chimeric protein SAM on a gold substrate was detected by light-illumination scanning tunneling microscopy (LI-STM) co-operated with a lock-in technique. The photocurrent generation of the chimeric protein SAM was wavelength-specific to the light-illumination (488 nm), which indicated that the EGFP part of the chimera plays a role as a sensitizer in the photo-induced electron transfer process.

Rational design of a protein-based molecular device consisting of blue fluorescent protein and zinc protoporphyrin IX incorporated into a cytochrome b562 scaffold.

To make a single molecular photo-device, it is essential to control the exact orientation of two types of proteins. We made a chimeric protein in which cytochrome b562 was linked to the N-terminus of enhanced green fluorescent protein, cytb562-EGFP. Within cytb562-EGFP, the excitation energy of EGFP was transferred to the cytochrome b562 cofactor fixed proximally to EGFP. Cytb562-EGFP was engineered so that iron protoporphyrin IX was substituted by zinc protoporphyrin IX to make it a suitable cofactor for photo-induced electron transfer. The photosensitizer pigment was optimized and the EGFP was replaced by a blue fluorescent mutant that gave 15% higher energy transfer efficiency.

S-peptide as a potent peptidyl linker for protein cross-linking by microbial transglutaminase from Streptomyces mobaraensis.

We have found that ribonuclease S-peptide can work as a novel peptidyl substrate in protein cross-linking reactions catalyzed by microbial transglutaminase (MTG) from Streptomyces mobaraensis. Enhanced green fluorescent protein tethered to S-peptide at its N-terminus (S-tag-EGFP) appeared to be efficiently cross-linked by MTG. As wild-type EGFP was not susceptible to cross-linking, the S-peptide moiety is likely to be responsible for the cross-linking. A site-directed mutation study assigned Gln15 in the S-peptide sequence as the sole acyl donor. Mass spectrometric analysis showed that two Lys residues (Lys5 and Lys11) in the S-peptide sequence functioned as acyl acceptors. We also succeeded in direct monitoring of the cross-linking process by virtue of fluorescence resonance energy transfer (FRET) between S-tag-EGFP and its blue fluorescent color variant (S-tag-EBFP). The protein cross-linking was tunable by either engineering S-peptide sequence or capping the S-peptide moiety with S-protein, the partner protein of S-peptide for the formation of ribonuclease A. The latter indicates that S-protein can be used as a specific inhibitor of S-peptide-directed protein cross-linking by MTG. The controllable protein cross-linking of S-peptide as a potent substrate of MTG will shed new light on biomolecule conjugation.