Magnetic Polystyrene Nanoparticles Prepared by Emulsion Solvent-Evaporation for the Chemiluminescent Immunoassay.

In clinical diagnosis, magnetic polystyrene nanoparticles (MPS NPs) are commonly applied to, e.g., the chemiluminescent immunoassay (CLEIA). However, the conventional preparation method of MPS NPs requires a long duration of heating to form polymer particles, which is inefficient. In this study, we prepared MPS NPs by emulsion solvent-evaporation without heating. We evaluated the effect of the solvent in the water and organic phases on the magnetic particle content. MPS NPs prepared by 4% (v/v) MeOH aqueous solution and adding stearic acid (SA) (4MeSA-MPS NPs) exhibited the highest magnetic particle content. Furthermore, CLEIA analysis indicates that the C-reactive protein detection limit is 80 pg/mL. Thus, 4MeSA-MPS NPs are promising for clinical diagnoses.

Solubilized eggshell membrane supplies a type III collagen-rich elastic dermal papilla.

Signs of aging in facial skin correlate with lifespan and chronic disease; however, the health of aging skin has not been extensively studied. In healthy young skin, the dermis forms a type III collagen-rich dermal papilla, where capillary vessels supply oxygen and nutrients to basal epidermal cells. Chicken eggshell membranes (ESMs) have been used as traditional medicines to promote skin wound healing in Asian countries for many years. Previously, we designed an experimental system in which human dermal fibroblasts (HDFs) were cultured on a dish with a solubilized ESM (S-ESM) bound to an artificial phosphorylcholine polymer; we found that genes that promoted the health of the papillary dermis, such as those encoding type III collagen, were induced in the S-ESM environment. The present study found that a gel with a ratio of 20% type III/80% type I collagen, similar to that of the baby skin, resulted in a higher elasticity than 100% type I collagen (p < 0.05) and that HDFs in the gel showed high mitochondrial activity. Thus, we decided to perform further evaluations to identify the effects of S-ESM on gene expression in the skin of hairless mice and found a significant increase of type III collagen in S-ESM. Picrosirius Red staining showed that type III collagen significantly increased in the papillary dermis after S-ESM treatment. Moreover, S-ESM application significantly improved human arm elasticity and reduced facial wrinkles. ESMs may have applications in extending lifespan by reducing the loss of tissue elasticity through the increase of type III collagen.

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.

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.

Increased interferon alpha receptor 2 mRNA levels is associated with renal cell carcinoma metastasis.

BACKGROUND: Interferon-alpha (IFN-alpha) is one of the central agents in immunotherapy for renal cell carcinoma (RCC) and binds to the IFN-alpha receptor (IFNAR). We investigated the role of IFNAR in RCC. METHODS: We quantified IFNAR mRNA expression in paired tumor and non-tumor samples from the surgical specimens of 103 consecutive patients with RCC using a real-time reverse transcription polymerase chain reaction (RT-PCR), and IFNAR2 protein using Western blotting. RESULTS: The absolute level of IFNAR1 and IFNAR2 mRNAs in tumor and non-tumor tissues did not correlate with the malignant and metastatic profiles. The relative yields of the PCR product from the tumor tissue to that from the corresponding non-tumor tissue (T/N) for the expression of IFNAR mRNAs were calculated. While the T/N ratio of IFNAR1 did not correlate with any factor, a high T/N ratio of IFNAR2 correlated with poor differentiation (P < 0.05), local invasion (P < 0.001), and metastasis (P < 0.0001). By multivariate analysis, a high T/N ratio of IFNAR2 predicted a shortened overall survival in all cases (P < 0.05) and a shorter disease-free survival in those without metastasis (M0; 68 cases, P < 0.05). Impressively, patients with a poorer response to IFN-alpha treatment had a higher IFNAR2 T/N ratio than those who had a good response (P < 0.05). IFNAR2c protein expression was higher in the primary tumors in patients with metastases (M1; 35 cases) compared to those without ( P < 0.0001). CONCLUSION: IFNAR2 is associated with the progression of RCC.

Role of p53 in the inhibitory effects of interferon-alpha subtypes on proliferation of hepatocellular carcinoma cells.

While interferon-alpha (IFN-alpha) subtypes share a common specific receptor composed of two subunits, interferon-alpha receptor (IFNAR)-1 and IFNAR-2, their subtype activities are exhibited via several intracellular signaling pathways and thus subsequently show different biological effects. Anti-proliferative effects of single treatment with IFN-alpha subtypes or 5-fluorouracil (FU), and of combined treatment with each IFN-alpha subtype and 5-FU were examined on three hepatocellular carcinoma cell lines, HepG2, HLE and PLC/PRF/5. HepG2 and PLC/PRF/5 cells were susceptible to the combination treatment, but HLE cells were not. Proliferation of PLC/PRF/5 cells was also inhibited by the IFN-alpha subtypes singly. In addition, apoptosis was observed in HepG2 cells upon treatment with 5-FU alone and with the combination treatment, and in PLC/PRF/5 cells after single treatment with the IFN-alpha subtypes and after the combination treatment. IFN-alpha subtypes induced cell cycle arrest in the G2/M phase in HepG2 and PLC/PRF/5. Analyses by Western blotting and immunoprecipitation revealed increased p53 phosphorylation in HepG2 and PLC/PRF/5 cells but not in HLE cells after combined treatment. Single treatment with IFN-alpha subtypes promoted p53 activation only in PLC/PRF/5 cells. These results propose that IFN-alpha subtypes induce cells to undergo apoptosis through p53 activation directly and indirectly, in collaboration with 5-FU, further suggesting the presence of distinct signal pathways for IFN-alpha-induced apoptosis.

Growth inhibitory effects of interferon-alpha subtypes vary according to human liver cancer cell lines.

BACKGROUND: Interferon (IFN)-alpha preparations used in the treatment of viral and neoplastic disease consist of single or multiple IFN-alpha subtypes that may possess different biological activity, but there are no data on liver cancer cells. METHODS: Antiproliferative effects and the mechanisms of growth inhibition of five IFN-alpha subtypes (alpha1, alpha2, alpha5, alpha8 and alpha10) were examined in vitro using 13 human liver cancer cell lines. RESULTS: The antiproliferative effect of each IFN-alpha subtype was different in each cell line. The 50% growth inhibitory concentration (IC50) on an antiviral unit basis showed that alpha5 presented the most potent antiproliferative effects in 11 of the 13 cell lines, and alpha8 in two cell lines. On average, the antiproliferative effects were strong in descending order from alpha5, alpha8, alpha10, alpha2 to alpha1. On weight basis, the most potent antiproliferative effect was shown by alpha8 in nine of the 13 cell lines, alpha5 in four cell lines, and the potency of the effects on average in descending order was alpha8, alpha5, alpha10, alpha2 and alpha1. No significant difference was observed between natural and recombinant alpha2. The mechanism of growth inhibition of each subtype in HAK-1B and KMCH-1 cell lines were apoptosis and S-phase arrest, and their induction levels were related to a certain degree to the antiproliferative effects. CONCLUSIONS: Our findings show that the antiproliferative effect of each IFN-alpha subtype varies according to the cell line, but that the cells are relatively or absolutely responsive to alpha5 and alpha8 subtypes.

Acceptor specificity of trehalose phosphorylase from Thermoanaerobacter brockii: production of novel nonreducing trisaccharide, 6-O-alpha-D-galactopyranosyl trehalose.

We investigated the acceptor specificity of a thermostable trehalose phosphorylase from Thermoanaerobacter brockii ATCC 35047 (TbTP) was examined using beta-D-glucose-1-phosphate (beta-G1P) as a glucosyl donor and oligosaccharides as the acceptor. Oligosaccharides with a reducing-end glucose residue as the C-6 substituent (e.g., isomaltose, gentiobiose, melibiose, isomaltotriose, and isopanose) were found to be successful acceptors. The transfer products of isomaltose, gentiobiose, and melibiose were isolated and characterized as 6-O-alpha-D-glucopyranosyl trehalose (alpha-GlcTre), 6-O-beta-D-glucopyranosyl trehalose (beta-GlcTre), and 6-O-alpha-D-galactopyranosyl trehalose (alpha-GalTre), respectively. To produce alpha-GalTre, a novel nonreducing trisaccharide, the reaction conditions of alpha-GalTre were examined using trehalose as a glucosyl donor. As a result, the yield of alpha-GalTre reached 40.5%.

Up-regulation of an extracellular superoxide dismutase-like activity in hibernating hamsters subjected to oxidative stress in mid- to late arousal from torpor.

Torpor-arousal cycles, one of the inherent features in hibernators, are associated with a rapid increase in body temperature and respiration, and it would lead to elevation of reactive oxygen species (ROS) generation. However, hibernators apparently tolerate this oxidative stress. We have observed in Syrian hamsters (Mesocricetus auratus) a maximal temperature shift and respiratory rate in mid- to late arousal (16-33 degrees C rectal temperature) from torpor. To examine plasma antioxidant status during arousal, we studied total superoxide radical-scavenging activity in plasma by electron spin resonance. The superoxide radical-scavenging activity reached a maximum at 32 degrees C, coincident with a peak in plasma uric acid levels, a ROS generation indicator. The up-regulated activity at 32 degrees C was attributable to the peak of the activity eluted at 260-kDa on gel-filtration chromatography, but was not to small antioxidant molecules such as ascorbate and alpha-tocopherol. The activity eluted at 260-kDa increased 3-fold at 32 degrees C compared with that of the torpid state, and was not detected either at 6 h after the onset of arousal or in the euthermic state. Moreover, the activity exhibited extracellular SOD-like properties: its induction in plasma by heparin injection and its affinity for heparin. Our results suggest that the 260-kDa extracellular SOD-like activity plays a role in the tolerance for the oxidative stress during arousal from torpor.

The effect of AgK114 on wound healing.

AgK114 is a newly isolated membrane-associated protein which is expressed on keratinocytes. Its expression is restricted to dermal sheath cells near sebaceous glands in normal skin. However, it is transiently induced by UV exposure or injury stimulation (Tatefuji T. et al., Biol. Pharm. Bull., 27, 1742-1749, 2004). Thus, the expression pattern of AgK114 suggested its potential role in wound healing response. We report here that expression of AgK114 is induced in the initial 24 h at the edge keratinocytes during keratinocyte migration, followed by disappearance once epithelialization is completed in the murine excisional wound model. We also demonstrate that exogenous recombinant mouse AgK114FL promoted wound healing process. Mouse AgK114FL up-regulated pro-matrix-metalloproteinase-9, vascular endothelial growth factor, transforming growth factor-beta, IL-6, and IL-1beta production in the early stage of wound tissue. Moreover, mouse AgK114FL induced the matrix-metalloproteinase-9 activity of wound fibroblasts prepared from impaired skin in the presence of proinflammatory cytokines. These results suggest that the AgK114 participates in the wound response during the healing process, and promotes wound repair.

Interferon-related mental deterioration after craniotomy for removal of metastatic renal cancer.

Interferon-related severe adverse events on the central nervous system are relatively rare, because interferon-alpha (INF-alpha) can not cross an intact blood-brain barrier. We experienced remarkable mental deterioration caused by INF-alpha administration in a 43-year-old man with renal cell carcinoma after surgical removal of a metastatic brain tumor. We detected a high concentration of INF-alpha in a cerebrospinal fluid sample, which was comparable to that in the serum at 24 h after the administration of INF-alpha; 5x10(6) IU i.m., suggesting that the blood-brain barrier was damaged somehow by the craniotomy. The mental deterioration improved shortly after discontinuation of the INF-alpha administration.

Purification, characterization, and gene cloning of a novel maltosyltransferase from an Arthrobacter globiformis strain that produces an alternating alpha-1,4- and alpha-1,6-cyclic tetrasaccharide from starch.

A glycosyltransferase, involved in the synthesis of cyclic maltosylmaltose [CMM; cyclo-{-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->}] from starch, was purified to homogeneity from the culture supernatant of Arthrobacter globiformis M6. The CMM-forming enzyme had a molecular mass of 71.7 kDa and a pI of 3.6. The enzyme was most active at pH 6.0 and 50 degrees C and was stable from pH 5.0 to 9.0 and up to 30 degrees C. The addition of 1 mM Ca2+ enhanced the thermal stability of the enzyme up to 45 degrees C. The enzyme acted on maltooligosaccharides that have degrees of polymerization of > or =3, amylose, and soluble starch to produce CMM but failed to act on cyclomaltodextrins, pullulan, and dextran. The mechanism for the synthesis of CMM from maltotetraose was determined as follows: (i) maltotetraose + maltotetraose --> 6(4)-O-alpha-maltosyl-maltotetraose + maltose and (ii) 6(4)-O-alpha-maltosyl-maltotetraose --> CMM + maltose. Thus, the CMM-forming enzyme was found to be a novel maltosyltransferase (6MT) catalyzing both intermolecular and intramolecular alpha-1,6-maltosyl transfer reactions. The gene for 6MT, designated cmmA, was isolated from a genomic library of A. globiformis M6. The cmmA gene consisted of 1,872 bp encoding a signal peptide of 40 amino acids and a mature protein of 583 amino acids with a calculated molecular mass of 64,637. The deduced amino acid sequence showed similarities to alpha-amylase and cyclomaltodextrin glucanotransferase. The four conserved regions common in the alpha-amylase family enzymes were also found in 6MT, indicating that 6MT should be assigned to this family.

Divergent activities of interferon-alpha subtypes against intracellular hepatitis C virus replication.

BACKGROUNDS: Interferon (IFN)-alpha is represented by several structurally related subtypes that show different antiviral and anti-tumor effects. Here, we analyzed differential effects of IFN-alpha subtypes on intracellular hepatitis C virus (HCV) replication using HCV subgenomic replicon system as a model. METHODS: Huh7 and HeLa cells supporting expression of HCV replicon were treated with various concentrations of five recombinant human IFN-alpha subtypes 1, 2, 5, 8, and 10, and with IFN-alpha con1. The effects of IFNs on various cell-signaling pathways were assayed by using ISRE-, GAS-, AP1-, NF-kappa B-, CRE-, and SRE-luciferase reporter plasmids. RESULTS: Each IFN-alpha subtype suppressed HCV replication in a dose-dependent manner. Among them, IFN-alpha8 was the most effective, while IFN-alpha1 was the least effective with 50% inhibitory concentrations of 0.123IU/ml versus 0.375IU/ml, respectively. These differential effects against HCV replication did not correlate with levels of the IFN-responsive ISRE or GAS reporter activities, nor they did activate the other reporters, AP1, NF-kappa B, CRE and SRE. CONCLUSION: There were divergent effects of IFN-alpha subtypes against HCV replication that may be through JAK-STAT-independent pathways. Exploring further mechanisms of action may elucidate IFN-mediated cellular antiviral mechanisms.

Distribution of a novel protein AgK114 expression in the normal tissues of adult mice: dual expression of AgK114 and growth hormone in anterior pituitary cells.

The novel antigen K114 (AgK114) has been previously identified in normal hamster skin, and its expression has been up-regulated accompanying tissue damages of the skin, although there is no information on its biological functions. To determine the physiological role of AgK114, we prepared anti-mouse AgK114 monoclonal antibody and studied its tissue distribution in healthy adult mice by immunocytochemistry. A widespread and unique expression of AgK114 peptide was found in the selected organs of various systems (hair follicle cells and sebaceous gland of skin, ciliated epithelial cells of trachea and bronchial tube, striated portion of submandibular gland, distal convoluted tubule cells of kidney, ciliated epithelial cells of oviduct, medulla of adrenal gland and anterior lobe of pituitary gland). Interestingly, dual expression of AgK114 peptide and growth hormone in somatotrophs was found in anterior lobe of pituitary gland by double immunocytochemistry. AgK114 peptide was expressed widely in many regionally well-defined cellular systems in various peripheral tissues, suggesting that AgK114 peptide may have some roles of physiological functions in these organs. The data from our current study have provided a rationale for further studies of functional roles of AgK114 peptide in a variety of organs or tissues under physiological conditions.

Enhancement of thermostability of kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047 by random mutagenesis.

Random mutation by error-prone PCR was introduced into kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047. One thermostable mutant enzyme, D513N, was isolated. The D513N mutant enzyme showed an optimum temperature of 67.5-70 degrees C (the wild type, 65 degrees C), and thermostability up to 67.5 degrees C (the wild type, up to 60 degrees C). The half-lives of D513N were estimated to be 135 h at 60 degrees C, 110 min at 70 degrees C and 6 min at 75 degrees C, respectively. They were about 1.6-fold, 7-fold and 6-fold longer than those of the wild-type enzyme, respectively.

Inhibition of chlamydia trachomatis growth by human interferon-alpha: mechanisms and synergistic effect with interferon-gamma and tumor necrosis factor-alpha.

We have evaluated the effect of natural human interferon (IFN)-alpha on the growth of chlamydia trachomatis in human epithelial cells in vitro and revealed that IFN-alpha has reduced both growth and infectivity of C. trachomatis. The effect of IFN-alpha was reversed by the addition of exogenous L-tryptophan and iron to the culture medium, suggesting that antichlamydial effect of IFN-alpha was caused by depletion of intracellular tryptophan and iron, both of which are essential for chlamydial growth. When IFN-alpha was combined with another antichlamydial cytokines, IFN-gamma and tumor necrosis factor (TNF)-alpha, the effect was synergistically enhanced. Therefore, IFN-alpha would act coordinately with other cytokines such as IFN-gamma and TNF-alpha, and play an important role in host defense against infection and in the establishment of persistent chlamydial infection of host, in which the organism remains viable, but in a culture-negative state.

Effects of interferon-alpha subtypes on the TH1/TH2 balance in peripheral blood mononuclear cells from patients with hepatitis virus infection-associated liver disorders.

Interferon-alpha (IFN-alpha) has recently been shown to modulate in vitro T helper (Th) 1-driven responses in the peripheral blood mononuclear cells (PBMC) of patients with hepatitis B virus or C virus infection. In this study, we examined the in vitro effects of IFN-alpha subtypes (IFN-alpha1, -alpha2, -alpha5, -alpha8, and -alpha10) on the Th1/Th2 balance in PBMC obtained from patients with hepatitis virus infection-associated liver disorders and chronic hepatitis (CH), in comparison with the effect on healthy control volunteer PBMC. The Th1-type cell percentages and Th1/Th2 ratios were significantly higher in the PBMC of patients when compared with controls both before and after cultivation in vitro, with the IFN-alpha subtypes. The IFNalpha-5 induced an increase in the Th2-type cell percentages in both control and patient PBMC, resulting in that IFN-alpha5 lowered the Th1/Th2 ratio in patients with CH. Furthermore, statistical analysis revealed that IFN-alpha8 significantly promoted an increase in the Th1/Th2 ratios of PBMC from patients with CH and liver cirrhosis (LC) but not that of PBMC from patients with LC-hepatocellular carcinoma (HCC) and HCC. These findings imply that hepatitis virus infection and its disease status modify the effects of IFN-alpha subtypes on Th1 and Th2 immune balance in patients. Our findings should help to elucidate the mechanisms underlying successful IFN therapy for hepatitis virus infection and prevention of hepatocellular carcinogenesis.

Enzymatic synthesis of a novel cyclic pentasaccharide consisting of alpha-D-glucopyranose with 6-alpha-glucosyltransferase and 3-alpha-isomaltosyltransferase.

A novel cyclic pentasaccharide (CPS) and a branched cyclic pentasaccharide (6G-CPS) consisting of d-glucopyranose were synthesized with 6-alpha-glucosyltransferase (6GT) and 3-alpha-isomaltosyltransferase (IMT) from Bacillus globisporus N75. The structure of CPS was cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The other, 6G-CPS, had the structure cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-[alpha-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The formation of CPS was presumed to occur after the following four successive reactions: a 6-glucosyltransfer reaction with 6GT, a 4-glucosyltransfer reaction with 6GT, a 3-isomaltosyltransfer reaction with IMT, and a cyclization reaction with IMT.

An enzymatically produced novel cyclic tetrasaccharide, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->} (cyclic maltosyl-(1-->6)-maltose), from starch.

A bacterial strain M6, isolated from soil and identified as Arthrobacter globiformis, produced a novel nonreducing oligosaccharide. The nonreducing oligosaccharide was produced from starch using a culture supernatant of the strain as enzyme preparation. The oligosaccharide was purified as a crystal preparation after alkaline treatment and deionization of the reaction mixture. The structure of the oligosaccharide was determined by methylation analysis, mass spectrometry, and (1)H and (13)C NMR spectroscopy, and it was demonstrated that the oligosaccharide had a cyclic structure consisting of four glucose residues joined by alternate alpha-(1-->4)- and alpha-(1-->6)-linkages. The cyclic tetrasaccharide, cyclo-{-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->}, was found to be a novel oligosaccharide, and was tentatively called cyclic maltosyl-maltose (CMM). CMM was not hydrolyzed by various amylases, such as alpha-amylase, beta-amylase, glucoamylase, isoamylase, pullulanase, maltogenic alpha-amylase, and alpha-glucosidase, but hydrolyzed by isomalto-dextranase to give rise to isomaltose. This is the first report of the cyclic tetrasaccharide, which has alternate alpha-(1-->4)- and alpha-(1-->6)-glucosidic linkages.

A novel phenomenon predicting the entry into a state of hibernation in Syrian hamsters (Mesocricetus auratus).

When Syrian hamsters (Mesocricetus auratus) are bred in a cold and short-day environment, most animals go into hibernation after a certain period of time. However, to date it has not been possible to predict which hamster will enter hibernation. In this study, we subcutaneously implanted thermo-loggers in hamsters bred in the cold environment, and recorded the subcutaneous temperature at short intervals until they went into hibernation. A time series analysis of temperature disclosed that a fall of 0.4 to 0.8 degrees C in subcutaneous temperature was seen 5 to 16 days before entering hibernation, and this phenomenon continued for three days or more. No hamster went into the hibernation without displaying this signal. Although the mechanism by which this phenomenon takes place is not clear, it is a sign from the body, which is useful for indicating if a hamster will enter hibernation shortly.