Dog leukocyte antigen genotyping across class I and class II genes in beagle dogs as laboratory animals.

Dog leukocyte antigen (DLA) polymorphisms have been found to be associated with inter-individual variations in the risk, susceptibility, and severity of immune-related phenomena. While DLA class II genes have been extensively studied, less research has been performed on the polymorphisms of DLA class I genes, especially in beagle dogs commonly used as laboratory animals for safety evaluations in drug development. We genotyped four DLA class I genes and four DLA class II genes by locus-specific Sanger sequencing using 93 laboratory beagle dogs derived from two different strains: TOYO and Marshall. The results showed that, for DLA class I genes, 11, 4, 1, and 2 alleles, including a novel allele, were detected in DLA-88, DLA-12/88L, DLA-64, and DLA-79, while, for DLA class II genes, 1, 10, 6, and 7 alleles were detected in DLA-DRA, DLA-DRB1, DLA-DQA1, and DLA-DQB1, respectively. It was estimated that there were 14 DLA haplotypes, six of which had a frequency of ≥ 5%. Furthermore, when comparing the DLA diversity between TOYO and Marshall strains, the most common alleles and haplotypes differed between them. This is the first study to genotype all DLA loci and determine DLA haplotypes including all DLA class I and class II genes in dogs. Integrating information on the DLA diversity of laboratory beagle dogs should reinforce their benefit as an animal model for understanding various diseases associated with a specific DLA type.

Astrocytic Neuroimmunological Roles Interacting with Microglial Cells in Neurodegenerative Diseases.

Both astrocytic and microglial functions have been extensively investigated in healthy subjects and neurodegenerative diseases. For astrocytes, not only various sub-types were identified but phagocytic activity was also clarified recently and is making dramatic progress. In this review paper, we mostly focus on the functional role of astrocytes in the extracellular matrix and on interactions between reactive astrocytes and reactive microglia in normal states and in neurodegenerative diseases, because the authors feel it is necessary to elucidate the mechanisms among activated glial cells in the pathology of neurological diseases in order to pave the way for drug discovery. Finally, we will review cyclic phosphatidic acid (cPA), a naturally occurring phospholipid mediator that induces a variety of biological activities in the brain both in vivo and in vitro. We propose that cPA may serve as a novel therapeutic molecule for the treatment of brain injury and neuroinflammation.

Integrin Signaling in the Central Nervous System in Animals and Human Brain Diseases.

The integrin family is involved in various biological functions, including cell proliferation, differentiation and migration, and also in the pathogenesis of disease. Integrins are multifunctional receptors that exist as heterodimers composed of α and ß subunits and bind to various ligands, including extracellular matrix (ECM) proteins; they are found in many animals, not only vertebrates (e.g., mouse, rat, and teleost fish), but also invertebrates (e.g., planarian flatworm, fruit fly, nematodes, and cephalopods), which are used for research on genetics and social behaviors or as models for human diseases. In the present paper, we describe the results of a phylogenetic tree analysis of the integrin family among these species. We summarize integrin signaling in teleost fish, which serves as an excellent model for the study of regenerative systems and possesses the ability for replacing missing tissues, especially in the central nervous system, which has not been demonstrated in mammals. In addition, functions of astrocytes and reactive astrocytes, which contain neuroprotective subpopulations that act in concert with the ECM proteins tenascin C and osteopontin via integrin are also reviewed. Drug development research using integrin as a therapeutic target could result in breakthroughs for the treatment of neurodegenerative diseases and brain injury in mammals.

Metabolic Plasticity of Astrocytes and Aging of the Brain.

As part of the blood-brain-barrier, astrocytes are ideally positioned between cerebral vasculature and neuronal synapses to mediate nutrient uptake from the systemic circulation. In addition, astrocytes have a robust enzymatic capacity of glycolysis, glycogenesis and lipid metabolism, managing nutrient support in the brain parenchyma for neuronal consumption. Here, we review the plasticity of astrocyte energy metabolism under physiologic and pathologic conditions, highlighting age-dependent brain dysfunctions. In astrocytes, glycolysis and glycogenesis are regulated by noradrenaline and insulin, respectively, while mitochondrial ATP production and fatty acid oxidation are influenced by the thyroid hormone. These regulations are essential for maintaining normal brain activities, and impairments of these processes may lead to neurodegeneration and cognitive decline. Metabolic plasticity is also associated with (re)activation of astrocytes, a process associated with pathologic events. It is likely that the recently described neurodegenerative and neuroprotective subpopulations of reactive astrocytes metabolize distinct energy substrates, and that this preference is supposed to explain some of their impacts on pathologic processes. Importantly, physiologic and pathologic properties of astrocytic metabolic plasticity bear translational potential in defining new potential diagnostic biomarkers and novel therapeutic targets to mitigate neurodegeneration and age-related brain dysfunctions.

Neuroimmunological Implications of AQP4 in Astrocytes.

The brain has high-order functions and is composed of several kinds of cells, such as neurons and glial cells. It is becoming clear that many kinds of neurodegenerative diseases are more-or-less influenced by astrocytes, which are a type of glial cell. Aquaporin-4 (AQP4), a membrane-bound protein that regulates water permeability is a member of the aquaporin family of water channel proteins that is expressed in the endfeet of astrocytes in the central nervous system (CNS). Recently, AQP4 has been shown to function, not only as a water channel protein, but also as an adhesion molecule that is involved in cell migration and neuroexcitation, synaptic plasticity, and learning/memory through mechanisms involved in long-term potentiation or long-term depression. The most extensively examined role of AQP4 is its ability to act as a neuroimmunological inducer. Previously, we showed that AQP4 plays an important role in neuroimmunological functions in injured mouse brain in concert with the proinflammatory inducer osteopontin (OPN). The aim of this review is to summarize the functional implication of AQP4, focusing especially on its neuroimmunological roles. This review is a good opportunity to compile recent knowledge and could contribute to the therapeutic treatment of autoimmune diseases through strategies targeting AQP4. Finally, the author would like to hypothesize on AQP4's role in interaction between reactive astrocytes and reactive microglial cells, which might occur in neurodegenerative diseases. Furthermore, a therapeutic strategy for AQP4-related neurodegenerative diseases is proposed.

Aquaporin 4-dependent expression of glial fibrillary acidic protein and tenascin-C in activated astrocytes in stab wound mouse brain and in primary culture.

We previously reported that aquaporin 4 (AQP4) has a neuroimmunological function via astrocytes and microglial cells involving osteopontin. AQP4 is a water channel localized in the endofoot of astrocytes in the brain, and its expression is upregulated after a stab wound to the mouse brain or the injection of methylmercury in common marmosets. In this study, the correlation between the expression of AQP4 and the expression of glial fibrillary acidic protein (GFAP) or tenascin-C (TN-C) in reactive astrocytes was examined in primary cultures and brain tissues of AQP4-deficient mice (AQP4/KO). In the absence of a stab wound to the brain or of any stimulation of the cells, the expressions of both GFAP and TN-C were lower in astrocytes from AQP4/KO mice than in those from wild-type (WT) mice. High levels of GFAP and TN-C expression were observed in activated astrocytes after a stab wound to the brain in WT mice; however, the expressions of GFAP and TN-C were insignificant in AQP4/KO mice. Furthermore, lipopolysaccharide (LPS) stimulation activated primary culture of astrocytes and upregulated GFAP and TN-C expression in cells from WT mice, whereas the expressions of GFAP and TN-C were slightly upregulated in cells from AQP4/KO mice. Moreover, the stimulation of primary culture of astrocytes with LPS also upregulated inflammatory cytokines in cells from WT mice, whereas modest increases were observed in cells from AQP4/KO mice. These results suggest that AQP4 expression accelerates GFAP and TN-C expression in activated astrocytes induced by a stab wound in the mouse brain and LPS-stimulated primary culture of astrocytes.

Immunological function of aquaporin-4 in stab-wounded mouse brain in concert with a pro-inflammatory cytokine inducer, osteopontin.

During injury to the central nervous system (CNS), astrocytes and microglia proliferate and migrate around the lesion sites. Recently, it has been reported that one of the water channels, aquaporin-4 (AQP4) is seemed to have a role in astroglial migration and glial scar formation caused by brain injury, although its molecular mechanism is largely unknown. In the present study, we examined the expression profiles in wild-type (WT) and AQP4-deficient (AQP4/KO) mice after a stab wound to the cerebral cortex. Three days after the stab wound, AQP4 expression was observed in activated microglia around the lesion site as well as in astrocytes. A microarray analysis revealed that 444 genes around the lesion site were upregulated 3 days after the wounding in WT mice. Surprisingly, most of these up-regulations were significantly attenuated in AQP4/KO mice. Real-time RT-PCR and immunofluorescence showed that osteopontin (OPN) expression around the lesion site was much lower in AQP4/KO mice than in WT mice. Moreover, the up-regulation of pro-inflammatory cytokines was significantly attenuated in AQP4/KO mice. Taken together, these results suggest that AQP4 plays an important role in immunological function in concert with OPN under pathological conditions in the CNS.

An awareness survey on childcare facilities and acoustic environment in Japan's residential areas.

A study conducted in Japan aimed to understand how childcare facilities should coexist with the local community. The researchers used a sound survey, demographic survey, and logistic regression analysis to study residents' noise awareness in various areas. They found that higher land prices led to lower approval of new childcare facilities. The study also revealed that those more sensitive to noise and less willing to participate in public events at childcare facilities were more significantly opposed to the establishment of new facilities.

An astrocyte-specific enhancer of the aquaporin-4 gene functions through a consensus sequence of POU transcription factors in concert with multiple upstream elements.

Aquaporin-4, a predominant water channel in the brain, is specifically expressed in astrocyte endfeet and plays a central role in water homeostasis, neuronal activity, and cell migration in the brain. It has two dominant isoforms called M1 and M23, whose mRNA is driven by distinct promoters located upstream of exons 0 and 1 of the aquaporin-4 gene, respectively. To identify cis-acting elements responsible for the astrocyte-specific transcription of M1 mRNA, the promoter activity of the 5'-flanking region upstream of exon 0 in primary cultured mouse astrocytes was examined by luciferase assay, and sequences, where nuclear factors bind, were identified by electrophoretic mobility shift assay. An astrocyte-specific activity enhancing transcription from the M1 promoter was observed within ∼2 kb from the transcriptional start sites of M1 mRNA. At least five elements clustered within the 286-bp region were found to function as a novel astrocyte-specific enhancer. Among the five elements, a consensus sequence of Pit-1/Oct/Unc-86 (POU) transcription factors was indispensable to the astrocyte-specific enhancer since disruption of the POU motif completely abolished the enhancer activity in astrocytes. However, the POU motif alone had little activity, indicating the requirement for cooperation with other upstream elements to exert full enhancer activity.

The neuroprotective function of 2-carba-cyclic phosphatidic acid: Implications for tenascin-C via astrocytes in traumatic brain injury.

We examined the mechanism how 2-carba-cyclic phosphatidic acid (2ccPA), a lipid mediator, regulates neuronal apoptosis in traumatic brain injury (TBI). First, we found 2ccPA suppressed neuronal apoptosis after the injury, and increased the immunoreactivity of tenascin-C (TN-C), an extracellular matrix protein by 2ccPA in the vicinity of the wound region. 2ccPA increased the mRNA expression levels of Tnc in primary cultured astrocytes, and the conditioned medium of 2ccPA-treated astrocytes suppressed the apoptosis of cortical neurons. The neuroprotective effect of TN-C was abolished by knockdown of TN-C. These results indicate that 2ccPA contributes to neuroprotection via TN-C from astrocytes in TBI.

Direct determination of bromine in plastics by electrothermal vaporization/inductively coupled plasma mass spectrometry using a tungsten boat furnace vaporizer and an exchangeable sample cuvette system.

A tungsten boat furnace vaporization inductively coupled plasma mass spectrometry (TBF/ICP-MS) method has been applied to the direct determination of bromine in plastic samples. In the pretreatment, the plastic sample is spread over a small sample cuvette made of tungsten by treating it with a strongly basic organic solution, e.g., octanol or diisobutyl ketone in the presence of potassium hydroxide. The cuvette is placed on a tungsten boat furnace, with which the electrothermal vaporizer is equipped. At the vaporization step, a widely spread thin layer of the sample facilitates its efficient evaporation and introduction into an ICP mass spectrometer. The most remarkable feature is that all the bromine species in plastic samples are decomposed to form a thermally stable inorganic salt during the pretreatment procedure. Therefore, the bromine content in plastic samples can be measured by a calibration curve method constructed with an aqueous standard solution of potassium bromate(V). The detection limit (3sigma) was estimated to be 0.77 pg of bromine, which corresponds to a concentration of 0.31 ng g(-1) of bromine in plastic samples when a sample amount taken of 2.5 mg is studied. The relative standard deviation was calculated to be 2.2%. Analytical results of some plastic samples, which contained both inorganic bromide salts and also organic bromine species, are given.

Comprehensive analysis of cardiac function, blood biomarkers and histopathology for milrinone-induced cardiotoxicity in cynomolgus monkeys.

The objective of this study was to elucidate the underlying cardiotoxic mechanism of milrinone, a cAMP phosphodiesterase 3 inhibitor, by evaluating cardiac functions, blood biomarkers including cardiac troponin I (cTnI), microRNAs (miR-1, miR-133a and miR-499a) and various endogenous metabolites, and histopathology in conscious cynomolgus monkeys. Milrinone at doses of 0, 3 and 30 mg/kg were orally administered to monkeys (n = 3-4/group), and the endpoints were evaluated 1 to 24 h post-dosing. Milrinone caused myocardial injuries characterized by myocardial degeneration/necrosis, cell infiltration and hemorrhage 24 h after drug administration. Cardiac functional analysis revealed that milrinone dose-dependently increased the maximum upstroke velocity of the left ventricular pressure and heart rate, and decreased the QA interval and systemic blood pressure 1-4 h post-dosing, being associated with pharmacological action of the drug. In the blood biomarker analysis, only plasma cTnI was dose-dependently increased 4-7 h after drug administration, suggesting that cTnI is the most sensitive biomarker for early detection of milrinone-induced myocardial injuries. In the metabolomics analysis, high dose of milrinone induced transient changes in lipid metabolism, amino acid utilization and oxidative stress, together with the pharmacological action of increased cAMP and lipolysis 1 h post-dosing before the myocardial injuries were manifested by increased cTnI levels. Taken together, milrinone showed acute positive inotropic and multiple metabolic changes including excessive pharmacological actions, resulting in myocardial injuries. Furthermore, a comprehensive analysis of cardiac functions, blood biomarkers and histopathology can provide more appropriate information for overall assessment of preclinical cardiovascular safety.

Cloning and expression of cDNA for interleukin 4 from the MSKR inbred strain of Mus musculus molossinus.

The 5'-upsteam region of the genomic gene and cDNA encoding interleukin 4 (IL-4) has been isolated and sequenced from the inbred mouse strain MSKR derived from a Japanese wild mouse using polymerase chain reaction (PCR) methodology. The cDNA sequence of IL-4 of MSKR was found to contain several nucleotide alterations, which result in amino acid substitutions, in comparison with that of inbred mouse BALB/c. In MSKR, IL-4 was expressed at high levels in thymus and spleen as revealed by reverse transcriptase PCR method and Northern hybridization. The tissue-specific expression profile was quite similar to that of laboratory mice.

Astrocytic endfeet re-cover blood vessels after removal by laser ablation.

The astrocyte, one of the glial cells, plays many functional roles. These include provision of nutrients from blood vessels to neurons, supply of neurotransmitters and support of blood-brain barrier (BBB) integrity. Astrocytes are known to support the integrity of BBB through maintenance of the tight junction between endothelial cells of blood vessels. However, evidence of its direct contribution to BBB is lacking owing to technical limitations. In this study, astrocytic endfeet covering blood vessels were removed by the laser ablation method with two photon laser scanning microscopy in in vivo mouse brain, and the re-covering of blood vessels with the astrocytic endfeet was observed in about half of the cases. Blood vessels kept their integrity without astrocytic endfoot covers: leakage of plasma marker dyes, Evans Blue or dextran-conjugated fluorescein, was not observed from stripped blood vessels, while ablation of vascular walls induced extravasation of Evans Blue. These results suggest that the astrocytic endfeet covering blood vessels do not contribute to the immediate BBB barrier.

The crucial protective role of glutathione against tienilic acid hepatotoxicity in rats.

To investigate the hepatotoxic potential of tienilic acid in vivo, we administered a single oral dose of tienilic acid to Sprague-Dawley rats and performed general clinicopathological examinations and hepatic gene expression analysis using Affymetrix microarrays. No change in the serum transaminases was noted at up to 1000 mg/kg, although slight elevation of the serum bile acid and bilirubin, and very mild hepatotoxic changes in morphology were observed. In contrast to the marginal clinicopathological changes, marked upregulation of the genes involved in glutathione biosynthesis [glutathione synthetase and glutamate-cysteine ligase (Gcl)], oxidative stress response [heme oxygenase-1 and NAD(P)H dehydrogenase quinone 1] and phase II drug metabolism (glutathione S-transferase and UDP glycosyltransferase 1A6) were noted after 3 or 6 h post-dosing. The hepatic reduced glutathione level decreased at 3-6 h, and then increased at 24 or 48 h, indicating that the upregulation of NF-E2-related factor 2 (Nrf2)-regulated gene and the late increase in hepatic glutathione are protective responses against the oxidative and/or electrophilic stresses caused by tienilic acid. In a subsequent experiment, tienilic acid in combination with l-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of Gcl caused marked elevation of serum alanine aminotransferase (ALT) with extensive centrilobular hepatocyte necrosis, whereas BSO alone showed no hepatotoxicity. The elevation of ALT by this combination was observed at the same dose levels of tienilic acid as the upregulation of the Nrf2-regulated genes by tienilic acid alone. In conclusion, these results suggest that the impairment of glutathione biosynthesis may play a critical role in the development of tienilic acid hepatotoxicity through extensive oxidative and/or electrophilic stresses.

Selective ablation of an astroglial subset by toxic gene expression driven by tenascin promoter.

OBJECTIVE: In vitro culture, one of the astroglia-derived extracellular matrix proteins, tenascin, expressed highly in fibrous astrocytes, whereas it expressed only low levels in protoplasmic astrocytes. We devised a method of selectively altering the population of astroglial subsets in primary culture of astrocytes derived from embryonic mouse brains using toxic gene expression driven by the tenascin promoter. METHODS: We have identified that the segment of 512-bases of 5'-flanking plus 243-bases leader sequences of the mouse tenascin gene contains maximum promoter activity in primary culture of astrocytes by deletion analysis of 5'-upstream region. This promoter element was used to specifically express the herpes simplex virus thymidine kinase (HSV-TK) gene in tenascin-positive astrocytes. RESULTS: This strategy allowed us to selectively decrease tenascin-positive astrocytes at the optimal concentration of ganciclovir, which is cytotoxic in HSV-TK-expressing cells. DISCUSSION: This approach should be useful for examining the role of the tenascin-negative astroglial subset in the development and regeneration of the central nervous system.

Involvement of cytochrome P450-mediated metabolism in tienilic acid hepatotoxicity in rats.

Tienilic acid is reported to be converted into electrophilic metabolites by cytochrome P450 (CYP) in vitro. In vivo, however, the metabolites have not been detected and their effect on liver function is unknown. We previously demonstrated that tienilic acid decreased the GSH level and upregulated genes responsive to oxidative/electrophilic stresses, such as heme oxygenase-1 (Ho-1), glutamate-cysteine ligase modifier subunit (Gclm) and NAD(P)H dehydrogenase quinone 1 (Nqo1), in rat liver, as well as inducing hepatotoxicity by co-treatment with the glutathione biosynthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO). In this study, for the first time, we identified a glutathione-tienilic acid adduct, a stable conjugate of putative electrophilic metabolites with glutathione (GSH), in the bile of rats given a single oral dose of tienilic acid (300mg/kg). Furthermore, a tienilic acid-induced decrease in the GSH level and upregulation of Ho-1, Gclm and Nqo1 were completely blocked by pretreatment with the CYP inhibitor 1-aminobenzotriazole (ABT, 66mg/kg, i.p.). The increase in the serum ALT level and hepatocyte necrosis resulting from the combined dosing of BSO and tienilic acid was prevented by ABT, despite a low hepatic GSH level. These findings suggest that the electrophilic metabolites of tienilic acid produced by CYP induce electrophilic/oxidative stresses in the rat liver and this contributes to the hepatotoxicity of tienilic acid under impaired GSH biosynthesis.

Alteration of inflammatory cytokine production in the injured central nervous system of tenascin-deficient mice.

Although tenascin-C (TN) is highly up-regulated during the proliferation of reactive astrocytes, little is known about the function of TN at injury sites in the central nervous system (CNS). Here, the function of TN-expressing astrocytes in the injured brain was investigated by analyzing TN-deficient mice with stab-wound injuries of the cerebral cortex. Glial fibrillary acid protein expression after injury was down-regulated earlier in TN-deficient mice than in wild-type (WT) mice. To evaluate immune responses in the injured CNS in the absence of TN, inflammatory cytokine production was examined after unilateral stab injuries of the cerebral cortex in TN-deficient and WT mice. The expression of interleukin (IL)-1beta, tumor necrosis factor-a and IL-6 was higher in TN-deficient mice, whereas levels of IL-4 and granulocyte colony-stimulating factor were lower in TN-deficient mice than WT mice. Our findings suggest that TN helps to regulate production of inflammatory cytokines in the injured brain.

Novel Distinctive Roles of Docking Proteins in Short-term Synaptic Plasticity of Frog Neuromuscular Transmission Revealed by Botulinum Neurotoxins.

Short-term synaptic plasticity (SSP) is a basic mechanism for temporal processing of neural information in synaptic transmission. Facilitation, the fastest component of SSP, has been extensively investigated with regard to Ca2+ signaling and other relevant substances. However, systematic analyses on the slower components of SSP, originated by Magleby and Zengel, have remained stagnant for decades, as few chemicals directly modifying these slower components have been identified. In combination with refined experimental protocols designed to study the stimulation frequency-dependence of SSP and botulinum neurotoxins A and C (BoNT-A and BoNT-C), we investigated SSP of frog neuromuscular transmission to clarify the roles of synaptosomal-associated protein of 25 kDa (SNAP-25) and syntaxin, SNARE proteins exclusively participating in vesicular events including docking, priming and exocytosis. We found that BoNT-A treatment eliminated slow potentiation, and BoNT-C poisoning abolished intermediate augmentation, two components of SSP. Fast facilitation was maintained after double poisoning with BoNT-A and -C, but the postsynaptic response became biphasic. A novel depression, termed repression, emerged by double poisoning. Repression was different from depletion because it developed even at a low-frequency stimulation of 1 Hz. We conclude that SNAP-25 and syntaxin not only play roles as cooperative exocytotic machinery, but also have roles in SSP.

Osteopontin is indispensable for activation of astrocytes in injured mouse brain and primary culture.

OBJECTIVES: Osteopontin (OPN) is an inflammatory cytokine inducer involved in cell proliferation and migration in inflammatory diseases or tumors. To investigate the function of OPN in astrocyte activation during brain injury, we compared OPN-deficient (OPN/KO) with wild-type (WT) mouse brains after stab wound injury and primary culture of astrocytes. METHODS: Primary cultures of astrocytes were prepared from either WT or OPN/KO postnatal mouse brains. Activation efficiency of astrocytes in primary culture was accessed using Western blotting by examining the protein levels of glial fibrillary acidic protein (GFAP) and tenascin-C (TN-C), which are markers for reactive astrocytes, following lipopolysaccharide (LPS) stimulation. Furthermore, the stab wound injury on the cerebral cortex as a brain traumatic injury model was used, and activation of astrocytes and microglial cells was investigated using immunofluorescent analysis on fixed brain sections. RESULTS: Primary cultures of astrocytes prepared from WT or OPN/KO postnatal mouse brains showed that only 25% of normal shaped astrocytes in a flask were produced in OPN/KO mice. The expression levels of both GFAP and TN-C were downregulated in the primary culture of astrocytes from OPN/KO mice compared with that from WT mice. By the immunofluorescent analysis on the injured brain sections, glial activation was attenuated in OPN/KO mice compared with WT mice. DISCUSSION: Our data suggest that OPN is essential for proper astrocytic generation in vitro culture prepared from mouse cerebral cortex. OPN is indispensable for astrocyte activation in the mouse brain injury model and in LPS stimulated primary culture. ABBREVIATIONS: AQP4: aquaporin 4; BBB: blood brain barrier; BrdU: bromo-deoxy uridine; CNS: central nervous system; GFAP: glial fibllirary acidic protein; IgG: immunoglobulin G; LPS: lipopolysaccharide; OPN: osteopontin; OPN/KO: osteopontin-deficient; TN-C: tenascin-C.