Ingredients such as trehalose and hesperidin taken as supplements or foods reverse alterations in human T cells, reducing asbestos exposure-induced antitumor immunity.

Exposure of human immune cells to asbestos causes a reduction in antitumor immunity. The present study aimed to investigate the recovery of reduced antitumor immunity by several ingredients taken as supplements or foods, including trehalose (Treh) and glycosylated hesperidin (gHesp). Peripheral blood CD4+ cells were stimulated with IL­2, anti­CD3 and anti­CD28 antibodies for 3 days, followed by further stimulation with IL­2 for 7 days. Subsequently, cells were stimulated with IL­2 for an additional 28 days. During the 28 days, cells were cultured in the absence or presence of 50 µg/ml chrysotile asbestos fibers. In addition, cells were treated with 10 mM Treh or 10 µM gHesp. Following culture for 28 days, reverse transcription­quantitative PCR was performed to assess the expression levels of transcription factors, cytokines and specific genes, including matrix metalloproteinase­7 (MMP­7), nicotinamide nucleotide transhydrogenase (NNT) and C­X­C motif chemokine receptor 3, in unstimulated cells (fresh) and cells stimulated with PMA and ionomycin (stimuli). The results demonstrated that compared with the control group, chrysotile­exposure induced alterations in MMP­7, NNT and IL­17A expression levels were not observed in the 'Treh' and 'gHesp' groups in stimulated cells. The results suggested that Treh and gHesp may reverse asbestos exposure­induced reduced antitumor immunity in T helper cells. However, further investigation is required to confirm the efficacy of future trials involving the use of these compounds with high­risk human populations exposed to asbestos, such as workers involved in asbestos­handling activities.

Cholesteryl Pullulan Encapsulated TNF-α Nanoparticles Are an Effective Mucosal Vaccine Adjuvant against Influenza Virus.

We encapsulated tumor necrosis factor-α (TNF-α), a major proinflammatory cytokine, into cholesteryl pullulan (CHP) to prepare TNF/CHP nanoparticles. In this report, we describe the immune-enhancing capability of the nanoparticles to act as a vaccine adjuvant. TNF/CHP nanoparticles showed excellent storage stability and enhanced host immune responses to external immunogens. The nanoparticles were effective via the nasal route of administration for inducing systemic IgG1 as well as mucosal IgA. We applied the nanoparticles in a model experimental influenza virus infection to investigate their adjuvant ability. TNF/CHP nanoparticles combined with a conventional split vaccine protected mice via nasal administration against a lethal challenge of A/PR/8/34 (H1N1) influenza virus. Mechanistic studies showed that the nanoparticles enhanced antigen uptake by dendritic cells (DCs) and moderately induced the expression of inflammation-related genes in nasopharynx lymphoid tissue (NALT), leading to the activation of both B and T cells. Preliminary safety study revealed no severe toxicity to TNF/CHP nanoparticles. Slight-to-moderate influences in nasal mucosa were observed only in the repeated administration and they seemed to be reversible. Our data show that TNF/CHP nanoparticles effectively enhance both humoral and cellular immunity and could be a potential adjuvant for vaccines against infectious diseases, especially in the mucosa.

The cyanine Dye NK-4 improves scopolamine-induced memory impairments in mice.

The aim of this study is to evaluate the effects of NK-4, a kind of cyanine dye, on cholinergic memory deficits in mice. We examined whether NK-4 could reverse scopolamine-induced amnesia in mice since NK-4 displays a potent and selective inhibitory effect on acetylcholinesterase (AChE) in vitro. Intraperitoneal administration of NK-4 significantly reversed scopolamine-induced cognitive impairments in mice in the Y maze and the passive avoidance tests, and NK-4 also improved spatial learning ability in the Morris water maze test. Despite NK-4 displaying remarkable AChE inhibitory activity in vitro, we could not detect a significant reduction of AChE activity in brain homogenates of NK-4-treated mice. Although the mechanism through which NK-4 reverses cognitive impairments in scopolamine-treated mice remains unclear, these data suggest that NK-4 may have potential as a therapeutic agent for the treatment of dementia.

Effects of NK-4 in a transgenic mouse model of Alzheimer's disease.

Beta-amyloid (Aß) peptides are considered to play a major role in the pathogenesis of Alzheimer's disease (AD) and molecules that can prevent pathways of Aß toxicity may be potential therapeutic agents for treatment of AD. We have previously reported that NK-4, a cyanine photosensitizing dye, displays neurotrophic and antioxidant activities. In this study, we report the effects of NK-4 on the toxicity of Aß and on cognitive function and Aß concentration in a transgenic mouse model of AD (Tg2576). In vitro, NK-4 effectively protected neuronal cells from toxicity induced by Aß. In addition, it displayed profound inhibitory activities on Aß fibril formation. In vivo, Tg2576 mice received an intraperitoneal injection at 100 or 500 µg/kg of NK-4 once a day, five times a week for 9 months. Administration of NK-4 to the mice attenuated impairment of recognition memory, associative memory, and learning ability, as assessed by a novel object recognition test, a passive avoidance test, and a water maze test, respectively. NK-4 decreased the brain Aß concentration while increasing the plasma amyloid level in a dose-dependent manner. NK-4 also improved memory impairments of ICR mice induced by direct intracerebroventricular administration of Aß. These lines of evidence suggest that NK-4 may affect multiple pathways of amyloid pathogenesis and could be useful for treatment of AD.

Neurotrophic effects of a cyanine dye via the PI3K-Akt pathway: attenuation of motor discoordination and neurodegeneration in an ataxic animal model.

BACKGROUND: Neurotrophic factors may be future therapeutic agents for neurodegenerative disease. In the screening of biologically active molecules for neurotrophic potency, we found that a photosensitizing cyanine dye, NK-4, had remarkable neurotrophic activities and was a potent radical scavenger. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we evaluated the effect of NK-4 on the protection of neurons against oxidative damage and investigated the associated intracellular signaling pathways. Subsequently, we evaluated the effect of NK-4 in an animal model of neurodegeneration. In vitro, NK-4 showed dose-dependent protection of PC12 cells from toxicity induced by oxidative stress caused by hydrogen peroxide (H(2)O(2)) or 6-hydroxydopamine (6-OHDA). Comparison of extracellular signal-regulated kinase signaling pathways between treatment with NK-4 and nerve growth factor (NGF) using K252a, an inhibitor of the NGF receptor TrkA, revealed that NK-4 activity occurs independently of NGF receptors. LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, blocked the protective effect of NK-4, and NK-4 caused activation of Akt/protein kinase B, a downstream effector of PI3K. These results suggest that the neuroprotective effects of NK-4 are mediated by the PI3K-Akt signaling pathway. NK-4 treatment also attenuated stress-induced activation of SAPK/JNK, which suggests that NK-4 activates a survival signaling pathway and inhibits stress-activated apoptotic pathways independently of the TrkA receptor in neuronal cells. In vivo, administration of NK-4 improved motor coordination in genetic ataxic hamsters, as assessed by rota-rod testing. Histological analysis showed that cerebellar atrophy was significantly attenuated by NK-4 treatment. Notably, the Purkinje cell count in the treated group was threefold higher than that in the vehicle group. CONCLUSIONS/SIGNIFICANCE: These results suggest that NK-4 is a potential agent for therapy for neurodegenerative disorders based on the activation of survival signaling pathways.

Therapeutic effect of PEGylated TNFR1-selective antagonistic mutant TNF in experimental autoimmune encephalomyelitis mice.

Multiple sclerosis (MS) is an inflammatory demyelinating disease, the pathogenesis of which is related to elevated serum levels of tumor necrosis factor-α (TNF). Although anti-TNF therapy has been tested as a potential treatment for MS, no remission of symptoms was observed. Recent reports indicated that the TNFR1 signal was responsible for the pathogenesis of murine experimental autoimmune encephalomyelitis (EAE), while the TNFR2 signal was responsible for recovery of the pathogenesis of EAE. Therefore, selective blocking of TNFR1 appears to be a promising strategy for the treatment of MS. In this regard, we previously succeeded in developing a novel TNFR1-selective antagonistic TNF mutant (R1antTNF) by using phage display technology. Here, we have examined the therapeutic potential of R1antTNF using EAE mice. Treatment with PEGylated R1antTNF (PEG-R1antTNF) significantly improved the clinical score and cerebral demyelination at the onset of EAE. Considerable suppression of Th1 and Th17-type response was also observed in spleen and lymph node cells of mice given PEG-R1antTNF. Moreover, the administration of PEG-R1antTNF suppressed the infiltration of inflammatory cells containing Th1 and Th17 cells into the spinal cord. These results suggest that selective blocking of TNFR1 by PEG-R1antTNF could be an effective therapeutic strategy against MS.

Cyanine dyes attenuate cerebral ischemia and reperfusion injury in rats.

Some photosensitizing cyanine dyes act on the immune system to enhance the phagocytic capacity of macrophages. In this study, we examined whether these dyes have neurotrophin-like activities and neuroprotective effects in vitro and in vivo. By screening more than 250 cyanine dyes, we found that NK-4 and NK-150, which belong to a group of pentamethine trinuclear cyanine dyes, significantly potentiated nerve growth factor (NGF)-primed neurite outgrowth of PC12HS cells in nanomolar to micromolar concentrations. Both NK-4 and NK-150 showed a remarkable hydroxyl radical-scavenging activity using an in vitro electron spin resonance (ESR)-based technique. They also effectively scavenged peroxy radicals, and in addition, NK-4 acted on superoxides to a similar extent as ascorbate. In vivo, NK-4 and NK-150 prevented cerebral ischemic injury induced by 2 h middle cerebral artery occlusion (MCAO) and 24 h reperfusion in rats. Dyes were intravenously administrated twice 1 h after the occlusion and immediately after the start of reperfusion. NK-4 and NK-150 (100 µg/kg) reduced cerebral infarct volumes by 57.0% and 46.0%, respectively. Those dyes also decreased brain swelling in the ischemic semispheres. As a result, administration of NK-4 and NK-150 provided substantial improvements in MCAO-induced neurological deficits in a dose-dependent manner. These results suggest that NK-4 and NK-150 effectively prevented ischemia-induced brain injury through their potent neurotrophin-like activity as well as antioxidative activity.

The treatment of established murine collagen-induced arthritis with a TNFR1-selective antagonistic mutant TNF.

Blocking the binding of TNF-alpha to TNF receptor subtype-1 (TNFR1) is an important strategy for the treatment of rheumatoid arthritis (RA). We recently succeeded in developing a TNFR1-selective antagonistic TNF mutant, R1antTNF. Here, we report the anti-inflammatory effects of R1antTNF in a murine collagen-induced arthritis model. To improve the in vivo stability of R1antTNF, we first engineered PEG (polyethylene glycol)-modified R1antTNF (PEG-R1antTNF). In prophylactic protocols, PEG-R1antTNF clearly improved the incidence, and the clinical score of arthritis due to its long plasma half-life. Although, the effect of PEG-R1antTNF on the incidence and production of IL1-beta was less than that of the existing TNF-blocking drug Etanercept, its effect on severity was almost as marked as Etanercept. Interestingly, in therapeutic protocols, PEG-R1antTNF showed greater therapeutic effect than Etanercept. These data suggest that the anti-inflammatory effects of PEG-R1antTNF depend on the stage of arthritis. Recently, there has been much concern over the reactivation of viral infection caused by TNF blockade. Unlike Etanercept, PEG-R1antTNF did not reactivate viral infection. Together, these results indicate that selective inhibition of TNF/TNFR1 could be effective in treating RA and that PEG-R1antTNF could serve as a promising anti-inflammatory drug for this purpose.

Novel protein engineering strategy for creating highly receptor-selective mutant TNFs.

Tumor necrosis factor (TNF) plays important roles in host defense and in preventing tumor formation by acting via its receptors, TNFR1 and TNFR2, functions of which are less understood. To this end, we have been isolating TNF receptor-selective mutants using phage display technique. However, generation of a phage library with large repertoire (>10(8)) is impeded by the limited transformation efficiency of Escherichia coli. Therefore, it is currently difficult to create a mutant library containing amino acid substitutions in more than seven residues. To overcome this problem, here we have used two different TNF mutant libraries, each containing random substitutions at six selected amino acid residues, and utilized a gene shuffling method to construct a randomized mutant library containing substitutions at 12 different amino acid residues of TNF. Consequently, using this library, we identified TNF mutants with greater receptor-selectivity and enhanced receptor-specific bioactivity than the existing mutants.

Fast binding kinetics and conserved 3D structure underlie the antagonistic activity of mutant TNF: useful information for designing artificial proteo-antagonists.

Tumour necrosis factor (TNF) is an important cytokine that induces an inflammatory response predominantly through the TNF receptor-1 (TNFR1). A crucial strategy for the treatment of many autoimmune diseases, therefore, is to block the binding of TNF to TNFR1. We previously identified a TNFR1-selective antagonistic mutant TNF (R1antTNF) from a phage library containing six randomized amino acid residues at the receptor-binding site (amino acids 84-89). Two R1antTNFs, R1antTNF-T2 (A84S, V85T, S86T, Y87H, Q88N and T89Q) and R1antTNF-T8 (A84T, V85P, S86A, Y87I, Q88N and T89R), were successfully isolated from this library. Here, we analysed R1antTNF-T8 using surface plasmon resonance spectroscopy and X-ray crystallography to determine the mechanism underlying the antagonistic activity of R1antTNF. The kinetic association/dissociation parameters of R1antTNF-T8 were higher than those of wild-type TNF, indicating more rapid bond dissociation. X-ray crystallographic analysis suggested that the binding mode of the T89R mutation changed from a hydrophobic to an electrostatic interaction, which may be responsible for the antagonistic behaviour of R1antTNF. Knowledge of these structure-function relationships will facilitate the design of novel TNF inhibitors based on the cytokine structure.

Creation of interferon-alpha8 mutants with amino acid substitutions against interferon-alpha receptor-2 binding sites using phage display system and evaluation of their biologic properties.

In this study, we describe the creation of three interferon-alpha (IFN-alpha)8 mutants with markedly higher antiviral and antiproliferative activities in comparison with those of the wild-type (wt)IFN-alpha8, wtIFN-alpha2, and IFN-con1 using a phage display system. Sequence analysis showed that three out of the six hot-spot amino acid residues of wtIFN-alpha8 known to be important for the interaction with the IFN-alpha receptor-2 (IFNAR-2)-binding sites were substituted to other amino acids and the others remained. Although affinity analysis revealed that the dissociation constant (K(D)) of IFN-alpha8 mutants was almost the same with that of wtIFN-alpha8, furthermore, the rates of association (k(a)) and dissociation (k(d)) were relatively lower. These results suggest that changes in the surface electronic charge of amino acid residues lead to changes in binding affinity and kinetics (prolonged dissociation time) toward the IFNAR-2, resulting in the modification of the biological activity. Moreover, our results demonstrate that the molecular engineering of the IFN-alpha8 provides important insight into action of IFN and also it would be useful in the development of therapeutically prominent IFN preparations than those used in clinical practice.

Structure-function relationship of tumor necrosis factor (TNF) and its receptor interaction based on 3D structural analysis of a fully active TNFR1-selective TNF mutant.

Tumor necrosis factor (TNF) is an important cytokine that suppresses carcinogenesis and excludes infectious pathogens to maintain homeostasis. TNF activates its two receptors [TNF receptor (TNFR) 1 and TNFR2], but the contribution of each receptor to various host defense functions and immunologic surveillance is not yet clear. Here, we used phage display techniques to generate receptor-selective TNF mutants that activate only one TNFR. These TNF mutants will be useful in the functional analysis of TNFR. Six amino acids in the receptor binding interface (near TNF residues 30, 80, and 140) were randomly mutated by polymerase chain reaction. Two phage libraries comprising over 5 million TNF mutants were constructed. By selecting the mutants without affinity for TNFR1 or TNFR2, we successfully isolated 4 TNFR2-selective candidates and 16 TNFR1-selective candidates, respectively. The TNFR1-selective candidates were highly mutated near residue 30, whereas TNFR2-selective candidates were highly mutated near residue 140, although both had conserved sequences near residues 140 and 30, respectively. This finding suggested that the phage display technique was suitable for identifying important regions for the TNF interaction with TNFR1 and TNFR2. Purified clone R1-6, a TNFR1-selective candidate, remained fully bioactive and had full affinity for TNFR1 without activating TNFR2, indicating the usefulness of the R1-6 TNF mutant in analyzing TNFR1 receptor function. To further elucidate the receptor selectivity of R1-6, we examined the structure of R1-6 by X-ray crystallography. The results suggested that R31A and R32G mutations strongly influenced electrostatic interaction with TNFR2, and that L29K mutation contributed to the binding of R1-6 to TNFR1. This phage display technique can be used to efficiently construct functional mutants for analysis of the TNF structure-function relationship, which might facilitate in silico drug design based on receptor selectivity.

Suppressive effects of a cyanine dye against herpes simplex virus (HSV)-1 infection.

In this study, we demonstrate that a cyanine dye, lumin, significantly suppressed cytopathic effect by herpes simplex virus (HSV)-1 toward human amnionic FL cell and also it reduced replication of HSV-1 in a dose-dependent manner. In addition, lumin additively augmented the antiviral effect of interferon (IFN)-alpha. Furthermore, fluorescence microscopic study showed that lumin (not IFN-alpha) itself remarkably induced alkalinization of intracellular organelle, suggesting the inhibition of virus invasion into the cells. These results suggest that lumin exerts an antiviral action against HSV-1 with the independent pathways of IFN-alpha and also it would become a therapeutically effective drug in clinical practice.

The therapeutic effect of TNFR1-selective antagonistic mutant TNF-alpha in murine hepatitis models.

Tumor necrosis factor-alpha (TNF-alpha) is critically involved in a wide variety of inflammatory pathologies, such as hepatitis, via the TNF receptor-1 (TNFR1). To develop TNFR1-targeted anti-inflammatory drugs, we have already succeeded in creating a TNFR1-selective antagonistic mutant TNF-alpha (R1antTNF) and shown that R1antTNF efficiently inhibits TNF-alpha/TNFR1-mediated biological activity in vitro. In this study, we examined the therapeutic effect of R1antTNF in acute hepatitis using two independent experimental models, induced by carbon tetrachloride (CCl(4)) or concanavalin A (ConA). In a CCl(4)-induced model, treatment with R1antTNF significantly inhibited elevation in the serum level of ALT (alanine aminotransferase), a marker for liver damage. In a ConA-induced T-cell-mediated hepatitis model, R1antTNF also inhibited the production of serum immune activated markers such as IL-2 and IL-6. These R1antTNF-mediated therapeutic effects were as good as or better than those obtained using conventional anti-TNF-alpha antibody therapy. Our results suggest that R1antTNF may be a clinically useful TNF-alpha antagonist in hepatitis.

Establishment of antihuman IFN-alpha8-specific monoclonal antibodies and their application in the enzyme-linked immunosorbent assay (ELISA).

In the present study, we describe the generation of a series of anti-interferon-alpha8 (IFN-alpha8)-specific monoclonal antibodies (mAbs), their characterization, and the establishment of a sandwich enzyme-linked immunosorbent assay (ELISA) system for human IFN-alpha8. The sandwich ELISA system is highly sensitive to human natural IFN-alpha8 (nIFN-alpha8), with a minimum detection limit of 50 pg/mL, which did not cross-react with the other IFN preparations and several cytokines tested. Using this ELISA system, pharmacokinetic properties of an IFN-alpha preparation administered in mice were examined. We found that IFN-alpha8 has higher vascular permeability and stability than IFN-alpha2 in the circulation. These results suggest that this ELISA would be very useful for determination of IFN-alpha8 protein concentrations in various experimental samples and also of pharmacokinetic properties of IFN-alpha preparations in human.

Creation and X-ray structure analysis of the tumor necrosis factor receptor-1-selective mutant of a tumor necrosis factor-alpha antagonist.

Tumor necrosis factor-alpha (TNF) induces inflammatory response predominantly through the TNF receptor-1 (TNFR1). Thus, blocking the binding of TNF to TNFR1 is an important strategy for the treatment of many inflammatory diseases, such as hepatitis and rheumatoid arthritis. In this study, we identified a TNFR1-selective antagonistic mutant TNF from a phage library displaying structural human TNF variants in which each one of the six amino acid residues at the receptor-binding site (amino acids at positions 84-89) was replaced with other amino acids. Consequently, a TNFR1-selective antagonistic mutant TNF (R1antTNF), containing mutations A84S, V85T, S86T, Y87H, Q88N, and T89Q, was isolated from the library. The R1antTNF did not activate TNFR1-mediated responses, although its affinity for the TNFR1 was almost similar to that of the human wild-type TNF (wtTNF). Additionally, the R1antTNF neutralized the TNFR1-mediated bioactivity of wtTNF without influencing its TNFR2-mediated bioactivity and inhibited hepatic injury in an experimental hepatitis model. To understand the mechanism underlying the antagonistic activity of R1antTNF, we analyzed this mutant using the surface plasmon resonance spectroscopy and x-ray crystallography. Kinetic association/dissociation parameters of the R1antTNF were higher than those of the wtTNF, indicating very fast bond dissociation. Furthermore, x-ray crystallographic analysis of R1antTNF suggested that the mutation Y87H changed the binding mode from the hydrophobic to the electrostatic interaction, which may be one of the reasons why R1antTNF behaved as an antagonist. Our studies demonstrate the feasibility of generating TNF receptor subtype-specific antagonist by extensive substitution of amino acids of the wild-type ligand protein.

The combination of IFN-alpha2 and IFN-alpha8 exhibits synergistic antiproliferative activity on renal cell carcinoma (RCC) cell lines through increased binding affinity for IFNAR-2.

Although there are at least 13 interferon-alpha (IFN-alpha) subtypes in humans, interactions between the subtypes remain unknown. To understand IFN-alpha interactions, we examined the antiproliferative activities and the receptor binding affinities of different combinations of IFN-alpha2 and IFN-alpha8 using six renal cell carcinoma (RCC) cell lines. Although IFN-alpha8 was the more potent subtype, synergistic and antagonistic antiproliferative effects were also observed in certain combinations of IFN-alpha2 and IFN-alpha8. To analyze the interactions between IFN-alpha2 and IFN-alpha8, the receptor-binding kinetics of different combinations of IFN-alpha2 and IFN- alpha8 to the IFN-alpha receptors, IFNAR-1 or IFNAR-2, were measured using a surface plasmon resonance-based biosensor. Unexpectedly, the receptor binding kinetics to IFNAR-2 but not to IFNAR-1 were mutually related to antiproliferative activity and increase in the binding speed (K(a)) for IFNAR-2. Moreover, we observed the increased fluorescence intensity (FI) of biotin-labeled IFN-alpha8 to IFNAR-2 by receptor binding inhibition assay with unlabeled IFN-alpha2 but not the other combinations. These findings indicate that the binding manner of IFN-alpha8 for IFNAR-2 is different from that of IFN-alpha2, suggesting that binding of IFN-alpha8 rather than binding of IFN-alpha2 to IFNAR-2 leads to activation and subsequent antiproliferative activity despite the same antiviral activity in RCC.

Suppressed Nna1 gene expression in the brain of ataxic Syrian hamsters.

Ataxic Syrian hamsters with an autosomal recessive trait were analyzed. Homozygotes showed moderate ataxia beginning at seven to eight weeks of age. They were fertile and lived more than two years. The affected hamsters exhibited an adult-onset degeneration of cerebellar Purkinje neurons, followed by a slow, mild reduction in the density of granule cells. Northern hybridization demonstrated that expression of Nna1, the gene responsible for the Purkinje cell degeneration (pcd) phenotype, was almost negligible in the brain of homozygous hamsters. These results strongly suggest that pcd-type mutation is involved in the ataxic phenotype of mutant hamsters.

Regulation of Candida albicans morphogenesis by tumor necrosis factor-alpha and potential for treatment of oral candidiasis.

BACKGROUND: Endogenous tumor necrosis factor-alpha (TNF-alpha) has a beneficial effect as an activation mediator of host defense against infection by the fungus Candida albicans (C. albicans). However, it is unclear whether exogenous TNF-alpha has a beneficial or detrimental effect against Candida. MATERIALS AND METHODS: The direct effect of TNF-alpha on CO2-induced morphological transformation of C. albicans blastoconidia was examined in vitro and the effect of TNF-alpha was determined in a mouse model of oral candidiasis. RESULTS: TNF-alpha suppressed hyphal formation from C. albicans blastoconidia directly and dose-dependently, whereas it did not affect the fungal budding rate at concentrations ranging from 0.01 to 10 microg/ml. In vivo, the oral administration of TNF-alpha significantly reduced the C. albicans CFU in tongue tissues of treated mice. Histopathologically, there was a decrease in the number and size of C. albicans fungi in the tongue tissues. CONCLUSION: Since orally administered TNF-alpha suppressed fungal burden in the tongue tissue without significant detrimental effects, TNF-alpha has potential as a therapeutic agent against Candida.

Purification, identification, characterization, and cDNA cloning of a high molecular weight extracellular superoxide dismutase of hamster that transiently increases in plasma during arousal from hibernation.

We previously studied antioxidant profiles in the plasma of hibernating Syrian hamsters and found a transient increase of a superoxide radical-scavenging activity during the arousal phase. In this report, we purified and identified the high molecular weight superoxide dismutase (SOD)-like factor from the plasma of arousing hamsters. The cyanide-sensitive 240 kDa SOD-like factor showed a significant homology to mammalian extracellular SOD (EC-SOD) reported, although the molecular mass of EC-SOD was 135 kDa. The cDNA cloning revealed that the 240 kDa SOD-like factor was identical to the hamster ortholog of EC-SOD. It consisted of 245 amino acid residues including a signal sequence of 20 amino acid residues. Five cysteine residues that would participate in inner- and inter-subunit bonds were well conserved among species. Interestingly, there were four potential N-glycosylation sites in hamster EC-SOD, whereas there is only one site in other species. The amino acid sequence analysis indicated that three of the four sites were modified. These results suggest that the anomalistically high molecular weight of hamster EC-SOD is ascribed, at least in part, to the addition of extra sugar chains. Furthermore, results obtained here also propose the involvement of EC-SOD in the antioxidative defense of hibernating hamsters.