Endogenous analgesic effect of pregabalin: A double-blind and randomized controlled trial.

BACKGROUND: Conditioned pain modulation (CPM) is widely used to measure endogenous analgesia, and a recent study indicated that drugs that act on endogenous analgesia are more effective in individuals with lower CPM. Recent animal studies have indicated that pregabalin activates endogenous analgesia by stimulating the descending pain inhibitory system. The present study examined whether the analgesic effect of pregabalin is greater in individuals with lower original endogenous analgesia using CPM. METHODS: Fifty-nine healthy subjects were randomly assigned to either a pregabalin group or a placebo group, and 50 of them completed the study. CPM was measured before and after pregabalin or placebo administration. The correlation of initial CPM to change in CPM was compared between the pregabalin and placebo groups. RESULTS: Initial CPM was significantly correlated with the change in CPM in the pregabalin group (r = -0.73, p < 0.0001) but not in the placebo group (p = 0.56) (difference in correlation coefficients between groups; p = 0.004). Furthermore, the initial CPM significantly affected the change in CPM in the pregabalin group but not in the placebo group (pregabalin group: adj R2  = 0.51, p < 0.001, y = -0.54x + 2.98; placebo group: p = 0.56, significant difference in regression slopes; p = 0.015). These results indicate that pregabalin has a higher endogenous analgesic effect in individuals with lower original endogenous analgesia. SIGNIFICANCE: The analgesic effect of pregabalin depends on the original endogenous analgesia status. Its effect on conditioned pain modulation (CPM) was stronger for subjects with lower original endogenous analgesia, suggesting that the mechanism of pregabalin involves the improvement of endogenous analgesia.

Hydrothermal impacts on trace element and isotope ocean biogeochemistry.

Hydrothermal activity occurs in all ocean basins, releasing high concentrations of key trace elements and isotopes (TEIs) into the oceans. Importantly, the calculated rate of entrainment of the entire ocean volume through turbulently mixing buoyant hydrothermal plumes is so vigorous as to be comparable to that of deep-ocean thermohaline circulation. Consequently, biogeochemical processes active within deep-ocean hydrothermal plumes have long been known to have the potential to impact global-scale biogeochemical cycles. More recently, new results from GEOTRACES have revealed that plumes rich in dissolved Fe, an important micronutrient that is limiting to productivity in some areas, are widespread above mid-ocean ridges and extend out into the deep-ocean interior. While Fe is only one element among the full suite of TEIs of interest to GEOTRACES, these preliminary results are important because they illustrate how inputs from seafloor venting might impact the global biogeochemical budgets of many other TEIs. To determine the global impact of seafloor venting, however, requires two key questions to be addressed: (i) What processes are active close to vent sites that regulate the initial high-temperature hydrothermal fluxes for the full suite of TEIs that are dispersed through non-buoyant hydrothermal plumes? (ii) How do those processes vary, globally, in response to changing geologic settings at the seafloor and/or the geochemistry of the overlying ocean water? In this paper, we review key findings from recent work in this realm, highlight a series of key hypotheses arising from that research and propose a series of new GEOTRACES modelling, section and process studies that could be implemented, nationally and internationally, to address these issues.This article is part of the themed issue 'Biological and climatic impacts of ocean trace element chemistry'.

Long-term effect of epidural injection with sustained-release lidocaine particles in a rat model of postoperative pain.

BACKGROUND: Single applications of sustained-release local anaesthetics may provide prolonged pain relief without requiring indwelling catheters, but have not yet been investigated for epidural postoperative pain management. We synthesized injectable sustained-release lidocaine particles (SRLPs) from biodegradable polymers and examined their effect in a rat model of postoperative pain. METHODS: Two types of polylactic acid particles, SRLP-10 and SRLP-25, containing 10% or 25% lidocaine, respectively, were generated and the lidocaine release was evaluated in vitro for 14 days. The SRLPs were then injected epidurally in the male Sprague-Dawley rats immediately before they received a hindpaw incision (the postoperative pain model), and hindpaw hypersensitivity was evaluated with the von Frey test. Motor paralysis and coordination were also assessed using a paralysis score and rota-rod test. Neurotoxicity and inflammation of the spinal cord, cauda equina, and tissue surrounding the injection site were histologically evaluated. RESULTS: In vitro, SRLP-10 and SRLP-25 released lidocaine over 7 and 3 days, respectively. The in vivo injection of SRLP-10 (80 mg) produced anti-hypersensitivity with no evidence of motor paralysis for 7 days after the paw incision, and SRLP-25 (60 mg) inhibited postoperative hypersensitivity for 7 days. Temporary motor paralysis (15 min) was observed after the injection of SRLP-25 (even with 40 mg). Foreign body reactions were observed around the SRLP injection site at 1 and 4 weeks after injection. No histopathological changes were observed at 1 or 4 weeks. CONCLUSIONS: The epidural injection of SRLPs produced prolonged anti-hypersensitivity in a rat model of postoperative pain with no major complications.

Pinguecula and contact lenses.

PURPOSE: To assess the relationship between age and the incidence and severity (determined by a grading system) of pinguecula in contact lens (CL) wearers, and to compare the grade of pinguecula between CL wearers and non-wearers. METHODS: A total of 600 CL wearers (94 wore hard CLs (HCLs) and 506 wore soft CLs (SCLs)) aged 11-60 years and 579 non-wearers aged 10-60 years were enrolled. The age, gender, medical history, ocular history, and grade of pinguecula at two locations (nasal and temporal) were determined in all subjects. RESULTS: There was an age-related increase in the grade of pinguecula among both CL wearers and non-wearers. The grade of pinguecula at the temporal conjunctiva was higher in CL wearers than in non-wearers (P=0.01907), whereas it was higher in HCL wearers than SCL wearers at both the nasal and temporal conjunctiva (P<0.00001 and P<0.00001). CONCLUSIONS: This was the first assessment of the severity of pinguecula in a large consecutive series of CL wearers. Our results suggest that the use of CLs is an important risk factor for pinguecula.

Positive effect of balance training with visual feedback on standing balance abilities in people with incomplete spinal cord injury.

OBJECTIVES: (1) To evaluate the learning potential and performance improvements during standing balance training with visual feedback (VBT) in individuals with incomplete spinal cord injury (SCI) and (2) to determine whether standing static and dynamic stability during training-irrelevant tasks can be improved after the VBT. SETTING: National Rehabilitation Center for Persons with Disabilities, Tokorozawa, Japan. METHODS: Six participants with chronic motor and sensory incomplete SCI who were able to stand for at least 5 min without any form of assistive device performed the VBT, 3 days per week, for a total of 12 sessions. During the training, participants stood on a force platform and were instructed to shift their center of pressure in the indicated directions as represented by a cursor on a monitor. The performance and the rate of learning were monitored throughout the training period. Before and after the program, static and dynamic stability was assessed. RESULTS: All participants showed substantial improvements in the scores, which varied between 236±94 and 130±14% of the initial values for different exercises. The balance performance during training-irrelevant tasks was significantly improved: for example, the area inside the stability zone after the training reached 221±86% of the pre-training values. CONCLUSION: Postural control can be enhanced in individuals with incomplete SCI using VBT. All participants showed substantial improvements during standing in both game performance and training-irrelevant tasks after the VBT.

Involvement of tazarotene-induced gene 1 in proliferation and differentiation of human adipose tissue-derived mesenchymal stem cells.

OBJECTIVE: Mesenchymal stem cells (MSC) have both self-renewal and multilineage differentiation potential, and bone marrow-derived MSC have been applied for tissue regeneration and repair. Although adipose tissue-derived MSC (ASC) have emerged as an alternative cell source, little information is available regarding the biologic difference between ASC derived from visceral and subcutaneous fat. Therefore, we aimed to compare the proliferation and gene expression profile of cultured human visceral ASC (VASC) and subcutaneous ASC (SASC), and to identify a novel gene involved in proliferation and differentiation of ASC. MATERIALS AND METHODS: We performed microarray analysis of cultured VASC and SASC, and investigated the role of tazarotene-induced gene 1 (TIG1), a most differentially expressed gene, in the proliferation and differentiation of ASC. RESULTS: SASC proliferated faster than VASC for over 10 passages, and TIG1 expression was consistently up-regulated in VASC of humans, rats and mice. Overexpression of the TIG1 gene in human SASC inhibited cell proliferation, whereas knockdown of TIG1 expression by siRNA promoted cell proliferation. In addition, overexpression of the TIG1 gene in SASC enhanced their differentiation into adipocytes, and promoted up-regulation of peroxisome proliferators-activated receptor gamma and CCAAT/enhancer binding protein alpha. On the other hand, TIG1 overexpression in SASC inhibited their differentiation into osteocytes and the expression of osteocalcin. CONCLUSION: TIG1 plays an important role in regulating proliferation and differentiation of ASC.

Lysine decarboxylase of Vibrio parahaemolyticus: kinetics of transcription and role in acid resistance.

AIM: The aim of this study was to investigate the detailed mechanisms of acid resistance in Vibrio parahaemolyticus. METHODS AND RESULTS: All 11 strains of V. parahaemolyticus survived lethal acidic conditions following acid adaptation, and accumulation of cadaverine was detected. The addition of lysine improved survival, suggesting that lysine decarboxylase plays a role in the adaptive acid tolerance response. Two open reading frames (ORF) in V. parahaemolyticus, which are separated by a noncoding region, were found to be highly homologous to bacterial lysine decarboxylase (cadA) and lysine/cadaverine antiporter (cadB) genes. Transcriptional analyses of this operon revealed acid induction and enhanced induction by external lysine. The relative expression ratio of each transcript was found to follow the trend of cadA mRNA > cadB mRNA > cadBA bi-cistronic mRNA. A mutated strain, with a disrupted cadA gene, showed attenuated acid survival. CONCLUSIONS: We identified the lysine decarboxylase gene operon of V. parahaemolyticus. Expression of this operon was induced under acidic conditions. The cadA-mutated strain constructed in this study showed weaker tolerance to acidic conditions than the wild-type strain. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrio parahaemolyticus utilizes the lysine decarboxylation pathway for survival in acidic conditions.

Bone defects in latent TGF-beta binding protein (Ltbp)-3 null mice; a role for Ltbp in TGF-beta presentation.

The latent transforming growth factor (TGF)-beta binding proteins (LTBP)-1, -3 and -4 bind the latent form of the multipotent cytokine TGF-beta. To examine the function of the LTBPs, we made a null mutation of Ltbp-3 by gene targeting. The homozygous mutant animals developed cranio-facial malformations by 12 days. By three months, there was a pronounced rounding of the cranial vault, extension of the mandible beyond the maxilla, and kyphosis. The mutant animals developed osteosclerosis of the long bones and vertebrae as well as osteoarthritis between 6 and 9 months of age. These latter phenotypic changes were similar to those described for mice that have impaired TGF-beta signaling. Thus, we suggest that Ltbp-3 plays an important role in regulating TGF-beta bioavailability as the phenotype of the Ltbp-3 null mouse appears to result from decreased TGF-beta signaling. Histological examination of the skulls from null animals revealed no effects on calvarial suture closure. However, the synchondroses in the skull base were obliterated within 2 weeks of birth. This is in contrast to the wild-type synchondroses, which remain unossified throughout the life of the animal and enable growth of the skull base through endochondral ossification. Histological changes in mutant basooccipital-basosphenoid synchondrosis were observed 1.5 days after birth. Compared with wild-type or heterozygous littermates, the basooccipital-basosphenoid synchondrosis of Ltbp-3 null mice contained increased numbers of hypertrophic chondrocytes. The expression of bone sialoprotein-1 (a marker for osteoblasts) was observed in cells surrounding the synchondrosis at postnatal day 1.5 indicating ectopic ossification. The expression of Indian hedgehog (Ihh) (a marker for chondrocytes committed to hypertrophic differentiation) was found through the basooccipital-basosphenoid synchondrosis, whereas the expression of parathyroid hormone related protein (PTHrP), which inhibits chondrocyte differentiation, appeared to be diminished in Ltbp-3 null mice. This suggests that Ltbp-3 may control chondrocyte differentiation by regulating TGF-beta availability. TGF-beta may regulate PTHrP expression either downstream of Ihh or independently of Ihh signaling.

Apolipoprotein J/clusterin is induced in vascular smooth muscle cells after vascular injury.

BACKGROUND: Understanding the precise molecular mechanisms underlying the phenomenon of restenosis after PTCA may help us to develop a new strategy for the treatment of restenosis after PTCA. The purpose of this study was to identify the genes involved in vascular restenosis. METHODS AND RESULTS: Applying a differential hybridization method to a model of the balloon-injured rabbit aorta, we identified 6 cDNA clones that were upregulated after injury. Northern blot showed that 5 genes, but not apolipoprotein J (apoJ)/clusterin, were constitutively expressed in noninjured aorta and upregulated after balloon injury. ApoJ mRNA was not detectable in noninjured aorta (control), began to be expressed at 6 hours after injury, showed a peak level at 24 hours (a 48-fold increase), gradually declined, and returned to the control level at 24 weeks. Western blot and immunohistochemistry demonstrated no expression of apoJ protein in noninjured aorta, an expression of apoJ at 2 days after balloon injury, and a peak level (a 55-fold increase) at 2 to 8 weeks. The expression of apoJ protein continued until 24 weeks after injury. In situ hybridization revealed that apoJ mRNA was expressed in smooth muscle cells (SMCs) of media at 2 days after injury and in SMCs of media and neointima at 2 weeks. To analyze the function of apoJ, stably transfected rabbit SMCs were created. The expression of apoJ stimulated proliferation and migration of SMCs. CONCLUSIONS: ApoJ is dramatically induced in media and neointima after vascular injury, suggesting that apoJ contributes to restenosis after angioplasty.

[Colony and phage-plaque direct sequencings by dye-terminator methods].

Direct sequencing using lambda phage DNA and E. coli colonies with plasmid DNA is a very powerful technique. Almost all of the reported direct sequencing methods involve either radioactive sequencing or fluorescent dye-primer sequencing. We present a direct colony sequencing strategy that uses a dye terminator (BigDye terminator kit) together with dye primer sequencing. We found that single-colony sequencing with the terminator yielded about 500 base pairs of sequence information. Signal strength was not improved when the number of cycles increased to 40. The colony used for the sequencing was estimated to contain about 5.6 x 10(7) cells. In addition, although a single plaque consisted of 2 x 10(6) cells, the pfu was not high enough to read with single-cycle sequencing, and only about 300 base pairs of sequence information were obtained from a single plaque using two cycle-sequencing reactions (re-cycle sequencing). The optimal amounts of the template were 500 ng of purified lambda DNA and 1 x 10(7) pfu of the lambda phage suspension, but with BigDye terminator it was possible to detect as little as 50 ng of purified lambda DNA and 2 x 10(6) pfu for lambda phage suspensions. Thus, colony direct sequencing and plaque direct sequencing are estimated to be very useful for rapid and high-throughout screening of genomic and cDNA libraries.

Direct neurotoxicity of tetracaine on growth cones and neurites of growing neurons in vitro.

BACKGROUND: Local anesthetics have direct neurotoxicity on neurons. However, precise morphologic changes induced by the direct application of local anesthetics to neurons have not yet been fully understood. Also, despite the fact that local anesthetics are sometimes applied to the sites where peripheral nerves may be regenerating after injury, the effects of local anesthetics on growing or regenerating neurons have never been studied. METHODS: Three different neuronal tissues (dorsal root ganglion, retinal ganglion cell layer, and sympathetic ganglion chain) were isolated from an age-matched chick embryo and cultured for 20 h. Effects of tetracaine were examined microscopically and by a quantitative morphologic assay, growth cone collapse assay. RESULTS: Tetracaine induced growth cone collapse and neurite destruction. Three neuronal tissues showed significantly different dose-response, both at 60 min and at 24 h after the application of tetracaine (P < 0.01). The ED50 values (mean +/- SD) at 60 min were 1.53+/-1.05 mM in dorsal root ganglion, 0.15+/-0.05 mM in retinal, and 0.06+/-0.02 mM in sympathetic ganglion chain cultures. The ED50 values at 24 h were 0.43+/-0.15 mM in dorsal root ganglion, 0.07+/-0.03 mM in retinal, and 0.02+/-0.01 mM in sympathetic ganglion chain cultures. Concentration of nerve growth factor in the culture media did not influence the ED50 values. The growth cone collapsing effect was partially reversible in dorsal root ganglion and retinal neurons. However, in the sympathetic ganglion culture, no reversibility was observed after exposure to 1 mM tetracaine for 10 or for 60 min. Bupivacaine had similar neurotoxicity to the three types of growing neurons. (The ED50 values at 60 min were 2.32+/-0.50 mM in dorsal root ganglion, 0.96+/-0.16 mM in retinal, and 0.18+/-0.05 mM in sympathetic ganglion chain cultures. The ED50 values at 24 h were 0.34+/-0.09 mM in dorsal root ganglion, 0.21+/-0.06 mM in retinal, and 0.45+/-0.10 mM in sympathetic ganglion chain cultures.) CONCLUSIONS: Short-term exposure to tetracaine produced irreversible changes in growing neurons. Growth cones were quickly affected, and neurites degenerated subsequently. Sensitivity varied with neuronal type and was not influenced by the concentration of nerve growth factor. Because a similar phenomenon was observed after exposure to bupivacaine, the toxicity to growing neurons may not be unique to tetracaine.

A rat model of postthoracotomy pain: behavioural and spinal cord NK-1 receptor assessment.

PURPOSE: To develop a new rat model of postthoracotomy pain for investigating its mechanisms and clarifying neurochemical changes. METHODS: Male Wistar rats were randomly assigned to three groups that underwent either fourth and fifth intercostal nerve ligation, cutting of the fourth and fifth ribs, or a sham operation in which only pleura was cut. For behavioural response assessment during the following month, pinch and touch were used as mechanical stimuli, and acetone was used as a cold thermal stimulus. In addition, (125)I-substance P autoradiography was used to determine neurokinin (NK) receptor density in spinal cord laminae I and II at one to six weeks after surgery. RESULTS: In rats with nerve ligation, hypersensitivity to noxious and non-noxious stimuli continued throughout the month. The "mirror phenomenon" was observed. The lowest threshold was obtained in the dorsomedial portion of the T4 dermatome on the side of surgery. In rats with rib cutting, a lowered threshold to noxious and non-noxious stimuli was observed for two weeks. In rats with sham operations, hypersensitivity was seen only at postoperative day one. NK-1 receptor density on the side of operation increased significantly in rats with nerve ligation from day seven to 28. Receptor density was highest on day 14 (22.97 +/- 1.04 fmol x mg(-1) tissue vs. control, 16.22 +/- 0.43), representing a 50% receptor excess on the side of ligation compared to the contralateral side. CONCLUSION: Intercostal nerve damage induces long-term postthoracotomy pain and an increase of spinal NK-1 receptors in rats. This model may be useful for investigation of postthoracotomy pain.

[The trends of Vibrio parahaemolyticus foodborne outbreaks in Tokyo: 1989-2000].

The total number of foodborne outbreaks due to Vibrio parahaemolyticus in Tokyo during the last 12 years between 1989 and 2000 were 710. The number of outbreaks in a year was 55 in 1989, 75 in 1990, and there was a gradual decrease to 24 outbreaks in 1993 which was the smallest number during those 12 years. After 1994, the number of outbreaks increased dramatically year by year until 1998 (107 outbreaks). Then they had decreased slightly to 74 in 1999, 65 in 2000. The monthly incidence of V. parahaemolyticus foodborne outbreaks showed a peak in August (44.2%) each year. In the last 12 years, 88.7% of V. parahaemolyticus foodborne outbreaks occurred during the 3 months between July and September, while 99.9% occurred between June and October. The most prevalent serotype of V. parahaemolyticus also changed, the most prevalent was O4:K4 in 1989, O4:K8 in both 1990 and 1991, O1:K56 in 1992, and O4:K8 from 1993 through 1995. Serotype O3:K6 became the most prevalent in 1996 and has remained so to date. In addition, the new serotype O4:K68 had also appeared in 1998. The number of outbreaks due to serotype O4:K68 followed that of O3:K6. Thus, the trends of V. parahaemolyticus foodborne outbreaks during the last 12 years in Tokyo showed various characteristics and dramatic changes in causal organisms.

[Detection of visceral ischemia with doplex scanning method of mesenteric arteries: in patients with acute type B aortic dissection].

Between March, 1997 and January, 1999, 11 patients with acute type B aortic dissection underwent doplex scanning evaluation of mesenteric arteries for the early detection of visceral ischemia. Peak systolic velocity (PSV) of the celiac artery (CeA) and superior mesenteric artery (SMA) was measured on their admission. Mean PSV of CeA in the non-ischemic group (8 patients) and in the ischemic group (3 patients) was 1.66 +/- 0.34 m/sec and 3.60 +/- 0.49 m/sec (p = 0.0481), respectively. Mean PSV of the SMA in the non-ischemic group and in the ischemic group was 1.93 +/- 0.52 m/sec and 3.33 +/- 0.37 m/sec (p = 0.00768), respectively. All patients with PSV of the mesenteric arteries above 3.00 m/sec presented visceral ischemia that required emergency operation. If PSV of the mesenteric arteries exceeds 3.00 m/sec, urgent surgical repair should be considered.

[Endoventricular circular patch plasty (dor operation) for postinfarction left ventricular aneurysm and ischemic cardiomyopathy].

The determination of purse-string suture line is one of the most important point in endoventricular circular patch plasty (Dor operation) for postinfarction left ventricular aneurysm (LVAN), especially for ischemic cardiomyopathy (ICM). We suggest following three points to decide appropriate suture line. First, the purse-string suture on the basal side should be placed on the 1-2 cm level under diagonal branch. Secondly, lateral wall should not be over excluded to maintain left ventricular function. And the third, akinetic or dyskinetic lesion of apex and septal wall should be excluded as much as possible. Nine cases of five LVAN and four ICM were underwent Dor operation in our institute from Dec. 1999 to Jan. 2000. All patients were weaned from cardiopulmonary bypass easily except one patient, who was operated under IABP support, because of his preoperative severe heart failure. All patients recovered well without any serious complications and postoperative left ventricular graphies were satisfactory. Left ventricular ejection fraction and stroke volume index were increased from 34 +/- 17 to 55 +/- 16% and from 38 +/- 7 to 47 +/- 6 ml/m2, end-diastric and systric volume index decreased from 141 +/- 37 to 88 +/- 19 ml/m2 and from 96 +/- 41 to 41 +/- 23 ml/m2 respectively. The Dor procedure adopted our idea led to satisfactory result in hemodynamic and also in morphologic study.

Effects of 5-HT2 and 5-HT3 receptors on the modulation of nociceptive transmission in rat spinal cord according to the formalin test.

We used the formalin test to clarify the 5-hydroxytryptamine (5-HT) receptor subtypes involved in the modulation of spinal nociceptive transmission in rats. Intrathecal administration of a 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)-tetraline (8-OH-DPAT; 1, 10, and 30 microg), or a 5-HT1B receptor agonist, 1, 4-dihydro-3-(1, 2, 3, 6-tetrahydro-4-pyridinyl)-5H-pyrrol (3, 2-b) pyridin-5-one (CP 93129; 1 and 10 microg), produced no significant change in the number of flinches. A 5-HT(2) receptor agonist, (+/-)-2, 5-dimethoxy-4-iodoamphetamine (DOI; 10, 30, and 100 microg), and a 5-HT3 receptor agonist, 2-methyl-5-HT (100 and 300 microg), produced dose-dependent decreases in the number of flinches in phases 1 (1 to 6 min) and 2 (10 to 61 min) of the test. The antinociceptive effects of DOI and 2-methyl-5-HT were antagonized by intrathecal pretreatment with a 5-HT2 receptor antagonist, ketanserin, and a 5-HT3 receptor antagonist, 3-tropanyl-3, 5-dichlorobenzoate (MDL-72222), respectively. These results suggest that 5-HT2 and 5-HT3 receptors in the spinal cord mediate antinociception to chemical stimuli.

A novel cryoprotective protein (CRP) with high activity from the ice-nucleating bacterium, Pantoea agglomerans IFO12686.

The ice-nucleating bacterium, Pantoea agglomerans IFO12686, induces the cryoprotective protein (CRP) by cold acclimation at 12 degrees C. The CRP was purified to apparent homogeneity by various chromatographies. We found that the purified CRP was a monomer of approximately 29,000 according to gel filtration chromatography and SDS-PAGE, and was a heat-stable protein. The CRP could protect freeze-labile enzymes, lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH) and isocitrate dehydrogenase (iCDH), against freezing-thawing denaturation. The activity of the CRP was about 3.5 x 10(4) times more effective than bovine serum albumin (BSA) and 2 x 10(6) times than COR26 from the ice-nucleating bacterium Pseudomonas fluorescens KUIN-1. We confirmed that the CRP was a novel protein, as judged by the a different molecule mass from the already-known cryoprotectants, and has an extremely high cryoprotective activity.