The timing of worm exclusion in dogs repeatedly infected with the cestode Echinococcus multilocularis.

Experimental Echinococcus multilocularis infection and deworming was repeated three or five times in nine dogs at various re-infection schedules. The mean number of worms decreased more than 91% in dogs with repeated infection, compared to first infection controls (n= 6). The copro-antigen assay and the egg count in the faeces suggested that the worm burden gradually decreased each time the dogs were re-infected. To examine whether such worm exclusion was a non-specific response, five dogs were sequentially infected with the parasite four times and subsequently fed freely for 6 months. Even after the 6-month interval, the five dogs that were infected five times with the parasite were still able largely to exclude the adult worms. The results suggested that the ability of worm exclusion in dogs that developed a resistance did not become rapidly extinct. Observation of the condition of faeces and the excretion of hooks in the faeces of repeatedly infected dogs revealed that the exclusion of worms started at the first week after the re-infection, and it continued during the patent period. Serum antibodies specific to the parasite antigen increased gradually until the third infection and significantly decreased during the 6-month interval. There was little enhancement of serum antibodies after the fifth infection in most dogs, although no clear correlation was observed between the antibody response and the worm burden. These findings suggested the possibility of developing a vaccine.

EFFECT OF SEASONAL CHANGES ON TESTICULAR MORPHOLOGY AND THE EXPRESSION OF CIRCADIAN CLOCK GENES IN JAPANESE WOOD MICE (APODEMUS SPECIOSUS).

This study aimed to determine the seasonality of reproduction throughout the year in Japanese wood mice (Apodemus speciosus). The effect of seasonal changes on testicular morphology and the periodic expression of circadian clock genes in the hypothalamus and testes of male individuals was evaluated. We also examined the morphology of the testes and caudae epididymides of male mice. In addition, RT-PCR analysis was carried out with mRNA extracted from the hypothalamus and testes to evaluate the expression of the circadian clock genes Clock, Bmal1, Per1, and Cry1. The complete induction of testicular activity was detected from February to April and from August to October, with testes weight increasing with the completion of spermatogenesis (reproductive season). From May to early June and from November to early January, testicular weight declined, the seminiferous tubules reduced in size, spermatogenesis was arrested, and sperm were not produced (non-reproductive season). From mid- June to July and mid-January, the re-induction of testicular activity for spermatogenesis was observed in the seminiferous tubules (transitional season). Out of the four examined genes, Cry1 had the highest expression level in both the hypothalamus and testes throughout the year, followed by Bmal1, Per1, and Clock. The expression of Bmal1 was significantly lower in the hypothalamus and testes during the transitional season compared to the reproductive and non-reproductive seasons. Cry1 transcript levels were also significantly lower in the hypothalamus and testes during the transitional season compared to the reproductive season. In conclusion, the results indicating changes in testicular morphology revealed annual reproductive, non-reproductive, and transmission periods in Japanese wood mice. When an increase in testicular activity was observed indicating the onset of the reproductive season, the mean day length was approximately 11–13 h. The expression of the circadian clock genes Bmal1 and Cry1 in the hypothalamus and testes during the reproductive season was significantly higher than that of the same genes during the transitional season. Consequently, completion of spermatogenesis occurred in the seminiferous tubules of Japanese wood mice testes during the reproductive period.

DyP-type peroxidases comprise a novel heme peroxidase family.

Dye-decolorizing peroxidase (DyP) is produced by a basidiomycete (Thanatephorus cucumeris Dec 1) and is a member of a novel heme peroxidase family (DyP-type peroxidase family) that appears to be distinct from general peroxidases. Thus far, 80 putative members of this family have been registered in the PeroxiBase database (http://peroxibase.isbsib.ch/) and more than 400 homologous proteins have been detected via PSI-BLAST search. Although few studies have characterized the function and structure of these proteins, they appear to be bifunctional enzymes with hydrolase or oxygenase, as well as typical peroxidase activities. DyP-type peroxidase family suggests an ancient root compared with other general peroxidases because of their widespread distribution in the living world. In this review, firstly, an outline of the characteristics of DyP from T. cucumeris is presented and then interesting characteristics of the DyP-type peroxidase family are discussed.

Sequence analysis of the agaB gene encoding a new beta-agarase from Vibrio sp. strain JT0107.

An agarase gene (agaB) was cloned from genomic DNA of Vibrio sp. strain JT0107. Analysis of the 3200 nucleotide sequence just before the agarase 0107 gene (agaA) which existed in genomic DNA of Vibrio sp. strain JT0107 revealed a putative single open reading frame coding for 955 amino acids. Comparison of the deduced amino acid sequence of AgaB to that of agarase 0107 revealed the existence of partially highly homologous regions. A part of this gene was expressed in Escherichia coli to yield a protein with agarase activity. This is the first report of evidence by genetic analysis that at least two different kinds of agarases exist in strain JT0107.

Carbon-11-d-threo-methylphenidate binding to dopamine transporter in baboon brain.

UNLABELLED: The more active d-enantiomer of methylphenidate (dl-threo-methyl-2-phenyl-2-(2-piperidyl)acetate, Ritalin) was labeled with 11C(t1/2:20.4 min) to characterize its binding, examine its specificity for the dopamine transporter and evaluate it as a radio-tracer for the presynaptic dopaminergic neuron. METHODS: PET studies were carried out in the baboon. The pharmacokinetics of [11C]dl-threo-methylphenidate, [11C]l-threo-MP and with its racemate ([11C]dl-threo-methylphenidate, [11C]MP). Nonradioactive methylphenidate was used to assess the reversibility and saturability of the binding. GBR 12909, 3 beta-(4-iodophenyl)tropane-2-carboxylic acid methyl ester (beta-CIT), tomoxetine and citalopram were used to assess the binding specificity. RESULTS: The ratio between radioactivity in the striatum and that in the cerebellum (ST/CB) after injection of [11C]d-threo-MP was higher than that for [11C]MP and [11C]l-threo-MP (3.3 for d-, 2.2 for racemic and 1.1 for l- in the same baboon). Most of the striatal binding of [11C]d-threo-MP was displaceable by injection of nonradioactive MP. Pretreatment with nonradioactive MP (0.5 mg/kg), GBR12909 (1.5 mg/kg) and RTI-55 (0.3 mg/kg) markedly reduced striatal but not cerebellar uptake of [11C]d-threo-MP. In all cases, the ST/CB after pretreatment was reduced by about 60% compared to 43% for [11C]MP. The ratios of distribution volumes at steady-state for the ST/CB for the three separate studies in the same baboon were reduced by about 50%, as compared with 37% for [11C]MP. In contrast, pretreatment with tomoxetine (3.0 mg/kg) or citalopram (2.0 mg/kg) did not change [11C]d-threo-MP kinetics; the ST/CB after pretreatment was similar to that for the control. CONCLUSION: These results demonstrate the saturable, reversible and specific binding of [11C]d-threo-MP to the dopamine transporter on the baboon brain, suggesting that [11C]d-threo-MP will be a useful PET tracer for the presynaptic dopaminergic neuron in living human brain.

Binding of d-threo-[11C]methylphenidate to the dopamine transporter in vivo: insensitivity to synaptic dopamine.

The regional distribution of [11C]d-threo-methylphenidate in mouse brain was very similar to that of [3H]WIN 35,428 ((-)-2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane), and the two radioligands were displaced from striatum similarly after administration of the potent cocaine analog RTI-55 ((-)-2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane). However, while striatal [3H]WIN 35,428 increased between 5 and 30 min, striatal [11C]d-threo-methylphenidate halved. Thus [11C]d-threo-methylphenidate binds similarly to but more reversibly than [3H]WIN 35,428. The methyl ester of L-DOPA (L-3,4-dihydroxyphenylalanine; 200 mg/kg) plus benserazide plus clorgyline, which markedly elevates rat striatal extracellular dopamine (Wachtel and Abercrombie, 1994, J. Neurochem. 63, 108), decreased the mouse striatum-to-cerebellum ratio for [11C]d-threo-methylphenidate at 30 min by 13% (P < 0.05). In positron emission tomographic (PET) baboon studies [11C]d-threo-methylphenidate binding was insensitive to drugs expected to lower endogenous dopamine. These experiments suggest that normal synaptic dopamine does not compete for binding with [11C]d-threo-methylphenidate, and will not affect PET measures of dopamine transporter availability.

Comparison of 1-week and 2-week triple therapy with omeprazole, amoxicillin, and clarithromycin in peptic ulcer patients with Helicobacter pylori infection: results of a randomized controlled trial.

This study was a comparison of 1-week and 2-week triple therapies with omeprazole, amoxicillin, and clarithromycin (OAC) in patients with peptic ulcer disease and Helicobacter pylori infection. A total of 147 peptic ulcer patients with H. pylori infection assessed by histology and culture were randomly treated with omeprazole 20mg bid + amoxicillin 1000mg bid + clarithromycin 400mg bid for either 1 week (OAC1w) or 2 weeks (OAC2w). Both groups then received omeprazole 20mg daily for 2 weeks followed by ranitidine 300mg daily for 4 weeks. Eradication of H. pylori was assessed by histology, culture, and the 13C-urea breath test (13C-UBT) at least 6 weeks after cessation of antimicrobial therapy. Intention-to-treat eradication rates were 78.2% (95%CI 69%-87%) with OAC1w and 88.4% (95%CI 81%-96%) with OAC2w. Per-protocol eradication rates were 86.0% (95%CI 78%-94%) with OAC1w, 97.0% (95%CI 93%-100%) with OAC2w. There was no significant difference in the eradication rates between OAC1w and OAC2w. Side effects were mild and self-limiting in both groups. In conclusion, both 1- and 2-week triple therapy with OAC are well tolerated and provide good eradication rates in peptic ulcer patients in Japan. The eradication rate of the 2-week regimen was higher than that of the 1-week regimen, but the difference was not statistically significant. Further studies including long-term economic considerations are required to determine the optimal duration of treatment.

Adrenalectomy potentiates the morphine--but not cocaine-induced place preference in rats.

The conditioned place preference paradigm is commonly used to study the reinforcing properties of various drugs. In the present study, the effect of adrenalectomy (ADX) on the morphine-induced place preference was examined in rats. Morphine produced a significant preference for the drug-associated place in sham-operated (sham) and ADX rats. In sham rats, only the highest dose of morphine (8 mg/kg, i.p.) produced a significant preference, while in ADX rats, lower doses of morphine (1 and 2 mg/kg, i.p.) produced a significant preference for the drug-associated place. Furthermore, the morphine-induced place preference was blocked by the dopamine D1 antagonist SCH23390 in both sham and ADX rats. On the other hand, the cocaine-induced place preference was not affected by ADX. In the present study, we found that ADX potentiates the reinforcing effect induced by morphine, but not that induced by cocaine, which suggests that the enhancement by ADX may be due to a change in opioid receptors, morphine metabolism and/or some other cause, but not a change in dopamine receptors.

Role of beta-adrenoceptors in the expression of morphine withdrawal signs.

The effects of intracerebroventricular (i.c.v.) pretreatment with the noradrenergic neurotoxin DSP-4 and beta 1- and beta 2-adrenoceptor antagonists on the expression of morphine withdrawal signs were investigated in mice. Mice were chronically treated with morphine (8-45 mg/kg, s.c.). Several withdrawal signs were observed following naloxone challenge in morphine-dependent mice which had been pretreated with vehicle. Treatment with DSP-4 before the naloxone challenge suppressed the expression of morphine withdrawal signs, including jumping and "wet dog" shakes. Similarly, pretreatment with the beta 1-antagonist atenolol significantly reduced the incidence of naloxone-precipitated jumping and "wet dog" shakes. However, pretreatment with the beta 2-antagonist ICI118,551 suppressed the expression of "wet dog" shakes, but not that of jumping. These findings suggest that the central noradrenergic system may mediate the expression of withdrawal signs. The blocking effects of beta-antagonists indicate that naloxone-precipitated jumping may be mediated predominantly by beta 1-adrenoceptors, while naloxone-precipitated "wet dog" shakes may be mediated by both beta 1- and beta 2-adrenoceptors.

Effects of a lipopolysaccharide from Pantoea agglomerans on the cocaine-induced place preference.

A lipopolysaccharide from Pantoea agglomerans (LPSp) was purified, and its effect on the cocaine-induced place preference was examined in rats. Cocaine (4 mg/kg, i.p.) produced a significant place preference. Administration of LPSp (5-1000 micrograms/kg, i.p.) alone resulted in neither preference nor aversion for either the drug- or saline-associated place. However, pretreatment with LPSp (500 and 1000 micrograms/kg, i.p.) abolished the place preference that had been induced by cocaine. Furthermore, treatment with LPSp (500 micrograms/kg, i.p.) abolished cocaine (20 mg/kg, i.p.)-induced locomotor enhancement in mice. These results suggest that while LPSp itself may possess neither reinforcing nor locomotor enhancing effects, it blocks both the reinforcing and the locomotor enhancing effects of cocaine. Therefore, LPSp might be useful in pharmacotherapy for prevention of recurrent cocaine abuse.

Mapping catechol-O-methyltransferase in vivo: initial studies with [18F]Ro41-0960.

Ro41-0960 is a potent, fluorine containing COMT inhibitor which has be en reported to cross the blood brain barrier and to inhibit COMT in the brain. It is structurally similar to Ro40-7592 which is currently undergoing clinical trials in Parkinson's disease. Positron emission tomographic (PET) studies in baboon using F-18 labeled Ro41-0960 demonstrated a negligible uptake in the brain both at tracer doses and with the addition of unlabeled drug (1.5 mg/kg) at all times through a 90 min experimental interval. The brain to plasma ratios of F-18 averaged about 0.025. Region of interest analysis of the brain tissue area suggests that most of F-18 in the brain was due to the blood in the brain and not the brain tissue itself. However, high uptake was observed in the kidneys and in other organs which are known to have high COMT activity. Studies in mice showed that at 30 min after injection of tracer, F-18 in kidneys was largely as unchanged [18F]Ro41-0960 and that it could be displaced with unlabeled Ro41-0960. The fact that the average brain to blood ratio for mice (n=12) was 0.04, and that similar HPLC metabolite patterns were observed for brain and blood, provides consistent evidence that nearly all the F-18 in the brain represents F-18 in the cerebral blood vessels. These studies raise the question of whether the central pharmacological effects of Ro41-0960 are due to its presence in the brain. They also provide the first example of a positron emitter labeled radiotracer for COMT, and provide initial encouraging evidence that [18F]Ro41-0960 may be used to examine COMT in peripheral organs in vivo.

Heat balance of rats acclimated to diurnal 2-hour feeding.

Heat production (M) and heat loss (H) of ad lib fed rats and of rats fed for 2-hr daily for 2 months (2-hr fed rats) were simultaneously measured by direct and indirect calorimetry over a period of 48 hours. The ad lib fed rats showed a clear nocturnal increase in M and H, which consisted of several discrete increases associated with discrete rises in feeding activity. In each increase, however, M and H were somewhat out of phase with each other. During feeding, a positive shift of heat storage occurred, which was repaid by H being higher than M thereafter. No such increases of M and H were observed in either the fasted ad lib fed rats or the 2-hr fed rats. The 2-hr fed rats showed a very large increase in M and H following the meal, which obscured the nocturnal increase of metabolic rate. Without food, this diurnal increase was greatly reduced but not to the minimum level, and two definite peaks of M and H associated with increased food exploratory activity, one in the day and the other at night, were obtained. There was a sharp fall in RQ 1-2 hr prior to the diurnal feeding. These observations suggest that, besides diet-induced heat production, feeding can be an additional time cue for increasing energy metabolism, but a more basic biological clock mechanism synchronizing the day-night cycle drives circadian rhythms of physical activity and biochemical processes related to energy expenditure in rats.

Effect of addition of water-soluble polysaccharides on bacterial cellulose production in a 50-L airlift reactor.

Bacterial cellulose (BC) production was carried out in a batch cultivation of Acetobacter xylinum in a 50-L internal loop airlift reactor by addition of water-soluble polysaccharides into the medium. When 0.1% (w/w) agar was added, BC production reached 8.7 g/L compared with 6.3 g/L in the control, and duration of the cultivation period to reach the maximum concentration of BC was almost half of that without addition of polysaccharides. During cultivation, BC was formed into pellets whose size was smaller when the productivity of BC was higher, indicating that increase in the relative viscosity by addition of polysaccharides hindered formation of large clumps of BC and increase in the volumetric oxygen transfer coefficient at high flow rate led to increase in BC productivity.

cDNA cloning and genetic analysis of a novel decolorizing enzyme, peroxidase gene dyp from Geotrichum candidum Dec 1.

A novel decolorizing peroxidase gene (dyp) was cloned from a cDNA library of a newly isolated strain of fungus Geotrichum candidum Dec 1. The open reading frame of 1494 nucleotides which corresponds to dyp predicts a primary translation product of 498 amino acids, M(r) 53,306. The deduced amino acid sequence of DyP does not contain the typical conserved motif which is characteristic of heme-containing peroxidases in the plant peroxidase superfamily. Comparison of the deduced amino acid sequence of DyP with that of a peroxidase from Polyporaceae sp. suggests that these proteins share highly homologous regions.

Efficient production of a heterologous peroxidase, DyP from Geotrichum candidum Dec 1, on solid-state culture of Aspergillus oryzae RD005.

The productivity of a peroxidase (DyP) originating from Geotrichum candidum Dec 1 was enhanced in the solid-state culture using Aspergillus oryzae RD005. When the humidity, water content, and temperature were adjusted to 60%, 50% and 27 degrees C, respectively, the productivity of DyP reached 5.3 g per kilogram wheat bran, which was used as the solid medium. The yield of 5.3 g per kg wheat bran corresponded to the yield of a 56 kg submerged culture. The productivity per gram carbon of the medium in the solid-state culture was 4.1-fold that in the submerged culture.

Effect of molasses on the production and activity of dye-decolorizing peroxidase from Geotrichum candidum Dec1.

The production of dye-decolorizing peroxidase (DyP) was investigated by cultivating Geotrichum candidum Dec1 using molasses as a carbon source. Molasses at concentrations greater than 10 g.l(-1) was found to increase the decolorization activity of the culture broth toward dye, reactive blue 5 mainly because the amount of enzyme produced was enhanced. However, complete inhibition of DyP activity by molasses was observed at the concentration of 20 g.l(-1), indicating that the inhibitory effect of molasses on the culture broth activity to decolorize the dye was involved. When the culture broth was diluted 25 times, the dye-decolorizing activity was 7 times as much as that of non-diluted culture broth. The molasses fractions separated by gel chromatography (300-400 ml and 400-500 ml fractions) completely inhibited the purified DyP. On the basis of these results, we propose a scheme to control both positive and negative effects of molasses on the dye decolorization process.

Removal of a high load of ammonia gas by a marine bacterium, Vibrio alginolyticus.

A newly isolated marine bacterium, Vibrio alginolyticus was used to remove a high load of ammonia gas. By a stepwise increase in ammonia supply over the concentration range of 120-2000 ppm (v/v), complete removal of ammonia was observed from the start of the experiment in a suspended culture of the bacterium in basal medium containing 3% NaCl. When cells were inoculated onto an inorganic packing material in a biofilter, and a high load of ammonia was introduced continuously under nonsterile conditions, the average percentage of gas removed exceeded 85% for a 61-d operation. The maximum removal capacity and the complete removal capacity were 22.8 g-N/kg-dry packing material/d and 18.6 g-N/kg-dry packing material/d, respectively, which were about four times larger than those obtained in nitrifying sludge inoculated onto the same packing material. During this operation, the nonsterile air supply had no adverse effect on the removability of ammonia by V. alginolyticus.

Seasonal changes in heat balance of dogs acclimatized to outdoor climate.

Heat production (M), dry heat loss (R+C), evaporative heat loss (E) and rectal temperature (Tre) were measured in a direct calorimeter in female mongrel dogs acclimatized to outdoor climate at Kanazawa (latitude; 36 degrees 35" N), Japan. M and total dry and evaporative heat losses (HL) were minimum at calorimeter wall temperatures (TW) of 26-29 degrees C in summer and 22-26 degrees C in winter (thermoneutral temperature; TNT). The seasonal shift of the lower critical temperature was confirmed. At TW below TNT, the values of M and HL were significantly higher in summer. At TW above TNT, these values increased. A TNT and above, M and HL were significantly higher in winter. (R+C) decreased linearly with increasing TW in both seasons. AT TW below 26 degrees C, (R+C) were significantly higher in summer. At TW above 26 degrees C, E increased greatly. The values of E were significantly higher in winter at TW 29-32 degrees C. Tre remained nearly constant at TNT and below, and increased at TW above TNT in both seasons. Mean body surface temperature (Tsf) decreased with decreasing TW. Body thermal conductance (K) was minimum at TW below 26 degrees C in summer and at TW below 22 degrees C in winter. At TW above these temperatures, K increased significantly. Whole body insulation (I) was significantly higher in winter, particularly at TW 18 degrees C. These results suggest that the dogs reared outdoors in winter acclimatized to cold in two ways; by increasing the insulating effect of the fur coat and by elevating resting heat production.

Effects of tracheal bypass breathing on heat balance in rabbits.

The effects of obstruction of the air passage through the nose on hypothalamic temperature (Thy) and heat balance were studied in 37 conscious rabbits using a direct calorimeter. The obstruction of the air passage through the nose was made by forcing the animal to breathe through a tracheal bypass cannula at a calorimeter wall temperature (TW) of either 17, 22, 26, or 28 degrees C. By bypass breathing, Thy and sensible heat loss (HL dry) were increased without a significant change in colonic temperature in a warm environment. At TW 28 degrees C, Thy in bypass breathing was 39.52 +/- 0.13 degrees C while that in nose breathing was 39.30 +/- 0.11 degrees C (p less than 0.05). HL dry in bypass breathing was 29.3 +/- 0.8 W . m-2 while that in nose breathing was 24.8 +/- 0.6 W . m-2 (p < 0.01). However, evaporative heat loss (Hl wet) was not changed by bypass breathing, although the respiratory rate was increased slightly. These results indicate that the obstruction of air passing through the nose increases hypothalamic temperature and elicits a thermolytic response only through the increase in sensible heat loss in the TW 28 degrees C environment. Heat production (M) was not suppressed by bypass breathing at TW 28 degrees C, at which the value of M was already minimum (42.0 +/- 3.0 W . m-2). In the 17-26 degrees C range of TW, heat balance was roughly obtained in either nose or bypass breathing. At TW 28 degrees C, however, M was slightly higher than HL, and a positive shift of heat storage was assumed particularly in nose breathing.

Metabolic and cardiovascular changes during physical restrains in rats.

When conscious rats were physically restrained, their body temperature, heart rate and heat production increased within minutes. The rate of increase in heat production was less than that in heart rate, particularly in warm environments. Reduction of oxygen pulse, consequently either stroke volume or (A-V) O2 difference, was suggested. Stroke volume cardiac output and its fractional distribution to most of the calorigenic organs measured by the reference sample method using 85Sr-labelled microspheres decreased significantly by restraint. However, fractional distribution of cardiac output increased greatly to the skeletal muscles. The restraint-hypermetabolism may be attributed to the increased metabolism of the skeletal muscles supported with an increased availability of oxygen. With a slight increase in mean aortic pressure, total peripheral resistance increased greatly as observed during Valsalva-like maneuver. The decrease in cardiac output and stroke volume during restraint may be caused by obstruction to the systemic venous return.