Bilateral movement training promotes axonal remodeling of the corticospinal tract and recovery of motor function following traumatic brain injury in mice.

Traumatic brain injury (TBI) results in severe motor function impairment, and subsequent recovery is often incomplete. Rehabilitative training is considered to promote restoration of the injured neural network, thus facilitating functional recovery. However, no studies have assessed the effect of such trainings in the context of neural rewiring. Here, we investigated the effects of two types of rehabilitative training on corticospinal tract (CST) plasticity and motor recovery in mice. We injured the unilateral motor cortex with contusion, which induced hemiparesis on the contralesional side. After the injury, mice performed either a single pellet-reaching task (simple repetitive training) or a rotarod task (bilateral movement training). Multiple behavioral tests were then used to assess forelimb motor function recovery: staircase, ladder walk, capellini handling, single pellet, and rotarod tests. The TBI+rotarod group performed most forelimb motor tasks (staircase, ladder walk, and capellini handling tests) better than the TBI-only group did. In contrast, the TBI+reaching group did not perform better except in the single pellet test. After the injury, the contralateral CST, labeled by biotinylated dextran amine, formed sprouting fibers into the denervated side of the cervical spinal cord. The number of these fibers was significantly higher in the TBI+rotarod group, whereas it did not increase in the TBI+reaching group. These results indicate that bilateral movement training effectively promotes axonal rewiring and motor function recovery, whereas the effect of simple repetitive training is limited.

Effect of dietary ascorbate on lipogenesis and lipolysis activities in black sea bream, Acanthopagrus schlegelii.

To assess the effect of dietary ascorbate on lipid metabolism, 1-year black sea bream (Acanthopagrus schlegelii) were reared on a casein-based purified diet and an ascorbate fortified diet (1,100 mg of L: -ascorbyl-2- monophosphate-Mg/kg diet). The fortified ascorbate was effectively incorporated into the fish body and elevated muscle carnitine content. Fortifications of dietary ascorbate depressed activities of glucose-6-phosphate dehydrogenase and NADP-isocitrate dehydrogenase as lipogenic enzymes in the hepatopancreas and intraperitoneal fat body. Starvation after feeding experiment activated carnitine palmitoyltransferase as a lipolysis enzyme in the hepatopancreas in both control and vitamin C(VC) groups, while the lipolysis activity was significantly higher in VC group. These results confirmed that dietary ascorbate depressed lipogenesis and activated lipolysis, i.e., influenced the lipid metabolism of black sea bream.

Dietary intake and urinary excretion of selenium in the Japanese adult population: the INTERMAP Study Japan.

OBJECTIVE: This study is to examine the relationship between dietary selenium intake and 24-h urinary selenium excretion in Japanese population samples participating in the INTERMAP Study. METHODS: Using highly standardized methods, we assessed individual dietary selenium intake from four 24-h dietary recalls and measured urinary selenium excretion in two timed 24-h urine collections in 1145 Japanese participants (574 men and 571 women) ages 40-59 years in four areas of Japan. RESULTS: The medians of dietary selenium intake were 177.5 microg/day in men and 139.8 microg/day in women; the medians of 24-h urinary selenium excretion were 127.9 microg/day in men and 109.4 microg/day in women, that is, urinary excretion was estimated to be 73% of dietary intake in men and 77% in women. Dietary selenium intake was significantly correlated with 24-h urinary selenium excretion (r=0.24 in men, r=0.18 in women; P<0.001). With dietary selenium intake and urinary selenium excretion expressed per kg of body weight, values were similar for men and women (dietary intake, 2.7 microg/kg body weight in men and 2.5 microg/kg body weight in women; urinary excretion, 2.0 microg/kg body weight in men and 2.0 microg/kg body weight in women). CONCLUSION: Dietary intake and 24-h urinary excretion of selenium are related in the Japanese adult population.

Effect of pectin enhancement on plasma quercetin and fecal flora in rutin-supplemented mice.

Few reports have considered the effects of dietary fiber on plasma quercetin and the intestinal flora. We investigated the effects of pectin on the plasma and fecal flora of mice fed a diet supplemented with the quercetin glycoside rutin. Male mice were randomly divided into 2 groups, which were fed a pectin-rutin (PR) or cellulose-rutin (CR) diet for 14 d. Plasma quercetin and isorhamnetin metabolites were measured by high-performance liquid chromatography. Feces were immediately processed with bacteriological procedures. The fecal flora was investigated. Plasma quercetin and isorhamnetin concentrations were significantly higher in the PR diet group, as was the plasma isorhamnetin/quercetin ratio. The composition of the intestinal flora differed between the 2 dietary groups. The total number of fecal bacteria was significantly larger in the PR group, in which most types of bacteria were more abundant, with the exceptions of bifidobacteria, fusiform-shaped bacteria, and staphylococci. The lower gut seemed to be the major absorption site for rutin. Pectin might thus enhance the bioavailability of quercetin from rutin by altering the metabolic activity of the intestinal flora and/or gut physiological function.

p53-dependent hyperthermic enhancement of tumour growth inhibition by X-ray or carbon-ion beam irradiation.

To elucidate p53-dependency on combined treatment with radiation and hyperthermia, growth inhibition and apoptosis were analysed using transplantable human tumour. Human head and neck squamous cell carcinoma (HNSCC) cells carrying different p53 genes were transplanted into the thigh of nude mice. When the mean diameter of tumour reached 5-6 mm, the tumours were exposed to X-rays (2 Gy) or Carbon-ion (C-) beams (1 Gy) followed by heating at 42 degrees C for 20 min. Tumour growth inhibition was evaluated by measuring the diameters of tumour. The induction of apoptosis and accumulation of apoptosis-related proteins were also analysed by immunohistochemical staining. Synergistic enhancement of tumour growth inhibition by hyperthermia was observed in wild-type p53 tumours treated with X-rays or C-beams but not in mutant p53 tumours. The incidence of apoptotic cells and activated-caspase-3-positive cells after combined treatment with them were significantly high in wild-type p53 tumours compared with that in mutant p53 tumours. The hyperthermic enhancement of tumour growth inhibition by X-ray- or C-beam-irradiation was p53-dependent, suggesting that it might be highly correlated with p53-dependent apoptosis.

Heat-induced growth inhibition and apoptosis in transplanted human head and neck squamous cell carcinomas with different status of p53.

To examine p53-dependency in hyperthermic cancer therapy, heat-induced growth inhibition and apoptosis in transplanted human head and neck squamous cell carcinoma (HNSCC) tumours were analysed with different status of p53 into nude mice. The tumour tissue from HNSCC cell line (SAS) transfected with mutant p53 gene (SAS/mp53) or control vector containing neo gene (SAS/neo) was transplanted into the subcutaneous tissue of the thigh of nude mice using a trocar. Hyperthermia was performed at 42 degrees C when the mean diameter of tumour was 5-6mm. The diameter of tumours was measured using vernier calipers and tumour weight (TW) and the relative tumour weight (RW) was calculated. Tumour regrowth delay was evaluated when the RW reached 5-fold against the control group. The accumulation of p53 and Bax proteins was examined by an immunohistochemical technique. Apoptotic cells in the sections were detected by staining of DNA ends using an immunohistochemical technique. SAS/mp53 tumours showed more heat-resistance than SAS/neo tumours. The p53-positively staining cells were observed in untreated SAS/mp53 tumours, but not in untreated SAS/neo tumours. After heat treatment, the accumulation of p53 and Bax proteins was observed in SAS/neo tumours, but little in SAS/mp53 tumours. The incidence of apoptotic cells induced by heat treatment was very low in SAS/mp53 tumours compared with SAS/neo tumours. In conclusion, the heat-induced growth inhibition of a transplanted HNSCC may be correlated with the induction of p53-dependent Bax-mediated apoptosis. Thus, p53 status appears to be one of the useful parameters for the predictive assays in hyperthermic cancer therapy.

Atopic dermatitis management: comparing the treatment patterns of dermatologists in Japan, U.S.A. and U.K.

BACKGROUND: The incidence of atopic dermatitis (AD) is increasing worldwide. No large-scale study has previously compared the therapeutic management of this condition in different countries. OBJECTIVES: The purpose of this study was to determine the treatment preferences of dermatologists in Japan, the U.S.A. and the U.K., and investigate their relationship with certain factors pertaining to the physician and his practice. METHODS: A questionnaire was sent to all registered members of dermatological societies in Japan, the U.S.A. and the U.K. Responses were collated and statistical analysis performed using chi2, Mantel-Haenszel and Breslow heterogeneity tests. RESULTS: Three thousand six hundred and eighty-eight completed surveys were returned. U.S.A. and U.K. physicians were significantly more aggressive in prescribing systemic medications, such as steroids, antibiotics and immunosuppressants, compared with those in Japan. Japanese dermatologists also utilized topical steroids significantly less. The use of alternative remedies was highest in Japan. All three countries had a relatively high degree of optimism for topical immunosuppressants, but less so for other emerging therapies. CONCLUSIONS: Both similarities and differences in the therapy of AD exist in the three countries studied. Factors related to the physician, patient population and culture may influence these observations.

Dose- and time-dependence of radiation-induced nitric oxide formation in mice as quantified with electron paramagnetic resonance.

In vivo nitric oxide (NO) formation was quantified in mice after exposure to high-dose whole-body X-ray irradiation. NO produced and accumulated in the livers of irradiated mice was determined using NO trapping method with iron-dithiocarbamate complex combined with electron paramagnetic resonance (EPR) spectroscopy. When mice were irradiated with 50 Gy X-ray, NO formation peaked in approximately 3 h after the irradiation was terminated. Dose-dependence study indicated that NO formation measured 5 h after irradiation was leveled off at the dose higher than 50 Gy. Administration of NO synthase inhibitor, N(G)-monomethyl L-arginine (L-NMMA) shortly after irradiation completely abolished the NO signal, indicating that radiation-induced NO is produced through L-arginine-dependent NO synthase pathways. These results suggest that irradiation of X-ray initiates inflammation processes, resulting in delayed NO synthase expression and NO formation.

Cisplatin-based chemotherapy for postoperative recurrence in non-small cell lung cancer patients: relation of the in vitro chemosensitive test to clinical response.

The usefulness of the in vitro chemosensitivity test, the collagen gel droplet embedded culture drug- sensitivity test (CD-DST, Int J Oncol 11: 449, 1997), in cisplatin-based combined chemotherapy for postoperative recurrent tumors in non-small cell lung cancer (NSCLC) patients was retrospectively analyzed. CD-DST data for cisplatin (or carboplatin), etoposide, 5-fluorouracil, mitomycin C, and vindesine were obtained in 311 surgically resected primary lesions. Of them, 25 patients were practically treated with first-line cisplatin- or carboplatin-based chemotherapy for postoperative initial recurrence. Nine (36%) of them responded to the combined chemotherapy for recurrent lesions, including one with complete remission, whereas 16 did not, with no change in 5 and progression in 11. Seven (70%) of 10 patients receiving combined chemotherapy using two or three in vitro sensitive chemoagents showed good responses, whereas there was no responder among the patients receiving chemotherapy including no in vitro sensitive chemoagents. In particular, of 11 patients showing good sensitivity to cisplatin or carboplatin on CD-DST, 8 (73%) responded to chemotherapy, whereas only one (7%) of 14 patients showing cisplatin- or carboplatin-resistance on CD-DST was a responder. Thus, CD-DST results for the chemoagents, especially cisplatin or carboplatin, correlated with chemotherapeutic response, indicating that the CD-DST analysis of surgically resected primary NSCLC tumors is a practically useful indicator of the clinical effect of first-line cisplatin-based combined chemotherapy for postoperative recurrence.

Detection of freeze-injured Escherichia coli O157:H7 cells from foods by resuscitation prior to selective enrichment.

We tried to detect Escherichia coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 using an enrichment method with modified EC broth supplemented with novobiocin (mEC + n). When the samples were cultured for enrichment immediately after inoculation of freeze-injured cells, E. coli O157:H7 was not detected in 13 out of 18 samples. However, allowing the food samples to stand for 3 h at room temperature prior to enrichment in mEC + n remarkably improved recovery of E. coli O157:H7 except for some acidic foods. E. coli O157:H7 was detected in the acidic foods by introducing a resuscitation step of 3-h of incubation in a non-selective broth at room temperature prior to enrichment with mEC + n.

Identification of a common receptor for three types of rat cytokine-induced neutrophil chemoattractants (CINCs).

Rat cytokine-induced neutrophil chemoattractant-1 (CINC-1), CINC-2 and CINC-3/macrophage inflammatory protein-2 (MIP-2), members of the CXC chemokine family, are potent chemotactic factors for neutrophils. In order to identify the receptor for CINCs, rat CXC chemokine receptor 2 (CXCR2) was cloned and expressed in HEK293 cells. CINC-1, CINC-2 and CINC-3 induced calcium mobilizations dose-dependently in CXCR2-transfected cells, whereas formyl-methionyl-leucyl-phenylalanine (FMLP) did not. CINC-3 induced enhancement of cytoplasmic calcium concentration more potently than CINC-1 and CINC-2, and desensitized calcium transients induced by CINC-1 and CINC-2, which were essentially identical to those observed in rat neutrophils. In addition, anti-CXCR2 serum inhibited neutrophil chemotactic activities of three types of CINCs almost completely. The mutant CINC-3, whose amino-terminal amino acid sequence (SELR) was replaced to AAR, lost chemotactic activity of its own but inhibited that of CINC-1 and CINC-2 potently, and that of CINC-3 weakly. The results indicate that rat CXCR2 on neutrophils is the unique receptor for all three types of CINCs, and CINC-1/-2 and CINC-3 exert different biological activities through the common receptor.

Selective enrichment with a resuscitation step for isolation of freeze-injured Escherichia coli O157:H7 from foods.

We studied injury of Escherichia coli O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured E. coli O157:H7 cells from foods. Food samples inoculated with E. coli O157:H7 were stored for 16 weeks at -20 degrees C in a freezer. Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Large populations of E. coli O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples. In an experiment to detect E. coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modified E. coli broth without bile salts at 25 degrees C for 2 h and then selectively enriched at 42 degrees C for 18 h by adding bile salts and novobiocin. Our enrichment method was further evaluated by isolating E. coli O157:H7 from frozen foods inoculated with the organism prior to freezing. Two hours of resuscitation at 25 degrees C in nonselective broth improved recovery of E. coli O157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating E. coli O157:H7 from frozen foods contaminated with injured E. coli O157:H7 cells.

Molecular cloning and expression of two distinct human chondroitin 4-O-sulfotransferases that belong to the HNK-1 sulfotransferase gene family.

Using an expression cloning strategy, the cDNA encoding the human HNK-1 sulfotransferase (HNK-1ST) has been cloned. During this cloning we found that HNK-1ST and other Golgi-associated sulfotransferases cloned before share homologous sequences including the RDP motif (Ong, E., Yeh, J.-C., Ding, Y., Hindsgaul, O., and Fukuda, M. (1998) J. Biol. Chem. 223, 5190-5195). Using this conserved sequence in HNK-1ST as a probe, we identified two expressed sequence tags in EST data base which have 31.6 and 30.7% identity with HNK-1ST at the amino acid levels. Expression of these two full-length cDNAs failed to form HNK-1 glycan nor to add sulfate to CD34 or NCAM. Surprisingly, proteins expressed by these cDNAs transferred sulfate to the C-4 position of N-acetylgalactosamine in chondroitin and desulfated dermatan sulfate, thus we named these two enzymes, chondroitin 4-O-sulfotransferase 1 and -2 (C4ST-1 and C4ST-2). Both C4ST-1 and C4ST-2, however, did not form 4, 6-di-O-sulfated N-acetylgalactosamine when chondroitin sulfate C was used as an acceptor. Moreover, analysis of (35)S-labeled dermatan sulfate formed by C4ST-1 indicate that sulfation preferentially took place in GlcA-->GalNAc unit than in IdoA-->GalNAc unit, suggesting that 4-O-sulfation at N-acetylgalactosamine may precede epimerization of glucuronic acid to iduronic acid during dermatan sulfate biosynthesis. Northern analysis demonstrated that the transcript for C4ST-1 is predominantly expressed in peripheral leukocytes and hematopoietic tissues while the C4ST-2 transcript is more widely expressed in various tissues. These results indicate C4ST-1 and C4ST-2 play complementary roles in chondroitin and dermatan sulfate synthesis in different tissues.

Selective and continuous degradation of carbazole contained in petroleum oil by resting cells of Sphingomonas sp. CDH-7.

Microbial degradation of carbazole (CA), a model of hard-removal heterocyclic nitrogen compounds contained in petroleum oil, was examined using Sphingomonas sp. CDH-7 isolated from a soil sample by screening for CA-assimilating microorganisms. CDH-7 used CA as a sole source of carbon and nitrogen, and metabolized CA to ammonia via anthranilic acid as an intermediate product. When CDH-7 was cultivated in the medium containing CA at the concentration of 500 mg/l (2.99 mM), CA was completely degraded within 50 h. By the reaction with the resting cells of CDH-7, 500 mg/l of CA was completely degraded within 4 h, with 1.64 mM of ammonia accumulated in the reaction mixture. When CA was added at the concentration of 100 mg/l (0.599 mM) periodically to the reaction mixture ten times, 925 mg/l (5.54 mM) of CA was degraded within 48 h by the resting cells, and 4.50 mM of ammonia was accumulated in the reaction mixture with a 75.1% molar conversion yield based on total CA added. The resting cells could almost completely degrade CA in a two-liquid-phase system which consists of water and organic solvent, even in the presence of 20% (v/v) isooctane, n-hexane, cyclohexane, and kerosene as a model petroleum oil. In the presence of an organic solvent system such as 20% (v/v) pxylene, toluene, and heptanol, however, CA degradation yields decreased.

Age-related changes in the response to thermal noxious heat and reduction of C-fibers by neonatal treatment with capsaicin.

Developmental changes of the response to nociceptive heat were examined in mice treated with capsaicin (50 mg/kg) on postnatal days 2-15. Tests of hot-plate (55 degrees C) and irradiation by infrared (IR test) were carried out after 10 days of capsaicin administration up to 120 days at intervals of 10 or 20 days. The time until forepaw (hot-plate) and hindpaw (IR test) withdrawal was assessed as the response latencies to suprathreshold and thermal threshold, respectively. Moreover, the numbers of unmyelinated C-fibers and myelinated fibers in the L4 dorsal roots of the animals treated on postnatal days 2 and 5 were counted on electron micrograph montages. Despite the marked reduction of C-fibers (60% mean decrease) in the 4 dorsal roots of the animals treated on postnatal day 2, thresholds were normal compared with those of controls. However, the animals treated with capsaicin on postnatal day 5 showed an apparent delay of thermal threshold latency only in the IR test, although the mean reduction of C-fibers was very likely the same as that of the animals pretreated on day 2. The reduction of C-fibers in mice treated on postnatal days 10 and 15 was lower than the animals treated on days 2 or 5, but their threshold latencies were significantly increased (hypoalgesia). A possible implication of these findings is discussed on the basis of the development of inhibitory systems in the intraspinal and supraspinal dorsal horn and sprouting from the surviving primary afferent neurons in the superficial dorsal horn.

Double-blind test on the efficacy of methylcobalamin on sleep-wake rhythm disorders.

The therapeutic effect of methylcobalamin (Met-12) on sleep-wake rhythm disorders was examined in a double-blind test. In the test group which was given a large dosage, a higher percentage of improvement was found compared to the control group with a small dosage, although the difference was not significant. The test group inconsistently showed significant improvement in both the sleep-wake cycle parameters and in clinical symptoms. The tendency was for the results to show a beneficial effect of Met-12 on rhythm disorders. However, because the percentage of improvement was low and significant improvement was inconsistent, Met-12 might be considered to have a low therapeutic potency and possible use as a booster for other treatment methods of the disorders.

Collaborative evaluation of detection methods for Escherichia coli O157:H7 from radish sprouts and ground beef.

For the evaluation of plating and immunological methods applicable to the detection of Escherichia coli O157:H7 from ground beef and radish sprouts, a collaborative study was conducted. It focused on a comparison of the efficiency of the plating and immunological methods using various plating agars and immuno-kits in combination with enrichment in modified E. coli broth supplemented with novobiocin (mEC + n), and using immunomagnetic separation. The plating media tested were sorbitol MacConkey agar (SMAC), SMAC supplemented with cefixime (0.05 mg/l) and potassium tellurite (2.5 mg/l) (CT-SMAC), and agars containing beta-glucuronidase substrates such as BCM O157 and CHROMagar O157. The immuno-kits used were Now E. coli, Path-Stick O157, VIP, EHEC-Tek ELISA System and Rapiblot E. coli O157. The 20 participating laboratories attempted to detect E. coli O157:H7 in 25 g chilled and frozen samples of ground beef uninoculated and inoculated with E. coli O157:H7 at levels of 138.9 and 23.9 cfu/25 g, and in 25 g chilled and frozen samples of radish sprouts uninoculated and inoculated at levels of 20.4 and 1.7 cfu/25 g. E. coli O157:H7 was recovered well from ground beef by all of the methods except direct plating with SMAC. For radish sprouts, the IMS-plating methods with CT-SMAC, BCM O157 and CHROMagar O157 were most efficient at detecting E. coli O157:H7 in more than 90% of the chilled samples inoculated at the level of 20.4 cfu/25 g. All the methods were less sensitive when applied to similar levels of E. coli O157:H7 in radish sprouts (20.4 cfu/25 g) compared with ground beef (23.9 cfu/25 g) especially if the sprouts were frozen. The sensitivity of the immuno-kits appeared to be similar to the IMS-plating methods, but the specificity was lower. Based on the results, we recommend the IMS-plating method using CT-SMAC and agars containing beta-glucuronidase substrate in combination with static enrichment incubation in mEC + n at 42 degrees C.

Reynosin from Sassurea lappa as inhibitor on CINC-1 induction in LPS-stimulated NRK-52E cells.

An inhibitor on CINC-1 (cytokine-induced neutrophil chemoattractant-1) induction in LPS-stimulated rat kidney epithelioid NRK-52E cells was purified from the roots of Sassurea lappa Clarke, a herbal medicine used in Korean traditional prescriptions for gastric intestinal diseases by a variety of column chromatographic procedures. The inhibitor was identified as reynosin, a sesquiterpene lactone isolated and characterized previously from Ambrosia confertiflora DC., and Magnolia grandiflora L. Reynosin exhibited a dose-dependent inhibition on CINC-1 induction in LPS-stimulated NRK-52E cells, where 50% of inhibitory effect was shown at the concentration of about 1 microM.

Effects of Japanese black tea on atherosclerotic disorders.

The atherogenic index was found to be significantly better in rats fed a high-cholesterol diet supplemented with black tea extract than in the ones not given the extract. It was also evident that black tea inhibited the proliferation of smooth muscle cells involved in the development and progression of atherosclerosis, and suppressed the production of oxidized low-density lipoprotein, a cause of lipid accumulation. It thus seems likely that black tea has an antiatherosclerotic action.