Restraint stress augments postprandial gastric contractions but impairs antropyloric coordination in conscious rats

Central corticotropin-releasing factor (CRF) plays an important role in mediating restraint stress-induced delayed gastric emptying. However, it is unclear how restraint stress modulates gastric motility to delay gastric emptying. Inasmuch as solid gastric emptying is regulated via antropyloric coordination, we hypothesized that restraint stress impairs antropyloric coordination, resulting in delayed solid gastric emptying in conscious rats. Two strain gauge transducers were sutured onto the serosal surface of the antrum and pylorus, and postprandial gastric motility was monitored before, during, and after restraint stress. Antropyloric coordination, defined as a propagated single contraction from the antrum to the pylorus within 10 s, was followed by > or = 20 s of quiescence. Restraint stress enhanced postprandial gastric motility in the antrum and pylorus to 140 +/- 9% and 134 +/- 9% of basal, respectively (n = 6). The number of episodes of antropyloric coordination before restraint stress, 2.4 +/- 0.4/10 min, was significantly reduced to 0.6 +/- 0.3/10 min by restraint stress. Intracisternal injection of the CRF type 2 receptor antagonist astressin 2B (60 microg) or guanethidine partially restored restraint stress-induced impairment of antropyloric coordination (1.6 +/- 0.3/10 min, n = 6). The restraint stress-induced augmentation of antral and pyloric contractions was increased by astressin 2B and guanethidine but abolished by atropine, hexamethonium, and vagotomy. Restraint stress enhanced postprandial gastric motility via a vagal cholinergic pathway. Restraint stress-induced delay of solid gastric emptying is due to impairment of antropyloric coordination. Restraint stress-induced impairment of antropyloric coordination might be mediated via a central CRF pathway.     

Time-dependent modulation of arterial baroreflex control of muscle sympathetic nerve activity during isometric exercise in humans

We investigated the time-dependent modulation of arterial baroreflex (ABR) control of muscle sympathetic nerve activity (MSNA) that occurs during isometric handgrip exercise (IHG). Thirteen healthy subjects performed a 3-min IHG at 30% maximal voluntary contraction, which was followed by a period of imposed postexercise muscle ischemia (PEMI). The ABR control of MSNA (burst incidence and strength and total activity) was evaluated by analyzing the relationship between spontaneous variations in diastolic arterial pressure (DAP) and MSNA during supine rest, at each minute of IHG, and during PEMI. We found that 1) the linear relations between DAP and MSNA variables were shifted progressively rightward until the third minute of IHG (IHG3); 2) 2 min into IHG (IHG2), the DAP-MSNA relations were shifted upward and were shifted further upward at IHG3; 3) the sensitivity of the ABR control of total MSNA was increased at IHG2 and increased further at IHG3; and 4) during PEMI, the ABR operating pressure was slightly higher than at IHG2, and the sensitivity of the control of total MSNA was the same as at IHG2. During PEMI, the DAP-burst strength and DAP-total MSNA relations were shifted downward from the IHG3 level to the IHG2 level, whereas the DAP-burst incidence relation remained at the IHG3 level. These results indicate that during IHG, ABR control of MSNA is modulated in a time-dependent manner. We suggest that this modulation of ABR function is one of the mechanisms underlying the progressive increase in blood pressure and MSNA during the course of isometric exercise.     

Possible mechanism of adenovirus generation from a cloned viral genome tagged with nucleotides at its ends

The entire cloned human adenovirus type 5 (Ad5) genome is known to be able to generate infectious virus after transfection into 293 cells when the both ends of the genome are exposed by digestion with appropriate restriction enzymes. However, when one or both ends of the genome are tagged with nucleotides and are not intact, whether the tagged end of the viral genome was remained tagged or corrected to be intact during the generation of viral clones has been unclear and, if such oligonucleotide removal occurs, how does the virus remove these tagged sequences and thereby restore its proper structure? Here, we show in our semi‐quantitative study that the generation efficiency of virus clones decreases depending on the length of nucleotide tags at the both ends and that both the oligonucleotide tags were precisely removed during virus generation with restoration of the proper terminal sequences. Interestingly the viral genome of which one end was tagged, while the other was attached about 12‐kb sequences, did generate intact viral clones at a reduced but significant efficiency. From these results, we here propose a possible mechanism whereby the terminal‐protein‐deoxycytidine complex enters from the enzyme‐cleaved end and reaches deoxyguanine at the initiating position of DNA synthesis in vivo. A replication origin at one end, embedded deeply in double‐stranded DNA, can be activated by two cycles of one‐directional full‐length DNA synthesis initiated by the other exposed replication origin about 30 kilobases away. We also describe new cassette cosmids which can use not only PacI but also BstBI for construction of an adenovirus vector, without reducing construction efficiency.     

Determination of antifungal activities in serum samples from mice treated with different antifungal drugs allows detection of an active metabolite of itraconazole

To establish an in vitro method of predicting in vivo efficacy of antifungal drugs against Candida albicans and Aspergillus fumigatus, the antifungal activities of fluconazole, itraconazole, and amphotericin B were determined in mouse serum. The minimum inhibitory concentration (MIC) of each drug was measured using mouse serum as a diluent. For C. albicans, the assay endpoint of azoles was defined as inhibition of mycelial extension (mMIC) and for A. fumigatus, as no growth (MIC). The MICs of amphotericin B for both pathogens were defined as the MIC at which no mycelial growth occurred. Serum MIC or mMIC determinations were then used to estimate the concentration of the drugs in serum of mice treated with antifungal drugs by multiplying the antifungal titer of the serum samples by the serum (m)MIC. The serum drug concentrations were also determined by HPLC. The serum concentrations estimated microbiologically showed good agreement with those determined by HPLC, except for itraconazole. Analysis of the serum samples from itraconazole-treated mice by a sensitive bioautography revealed the presence of additional spots, not seen in control samples of itraconazole. The bioautography assay demonstrated that the additional material detected in serum from mice treated with itraconazole was an active metabolite of itraconazole. The data showed that the apparent reduction in the itraconazole serum concentration as determined by HPLC was the result of the formation of an active metabolite, and that the use of a microbiological method to measure serum concentrations of drugs can provide a method for prediction of in vivo efficacy of antifungal drugs.     

Isolation of porphyran-degrading marine microorganisms from the surface of red alga, Porphyra yezoensis

Marine microorganisms degrading porphyran (POR) were found on the surface of thalli of Porphyra yezoensis. Fifteen crude microorganism groups softened and liquefied the surface of agar-rich plate medium. Among these, 11 microorganism groups degraded porphyran that consisted of sulfated polysaccharide in Porphyra yezoensis. Following isolation, 7 POR-degradable microorganisms were isolated from the 11 POR-degradable microorganism groups.     

Regulation of the body fat percentage in developmental-stage rats by methylxanthine derivatives in a high-fat diet

We investigated the regulatory effects of structural differences among methylxanthine derivatives on the elevation of body fat percentage in developmental-stage rats. Caffeine, theophylline and theobromine were used as the methylxanthines. High-fat diets (20% lard) containing each methylxanthine (0.025%) were administered to male Sprague-Dawley rats for 12 weeks, with the result that the body fat percentage was generally reduced in each methylxanthine-fed group. The abdominal adipose tissue weight in the caffeine group was also significantly lower than that in the control group, the serum cholesterol and triglyceride levels in the caffeine group also being significantly lower than the levels in the control group. The study results suggest that caffeine could contribute most to preventing arteriosclerotic diseases.     

Induction of multidrug resistance-associated protein MRP3 in the liver of rats fed with docosahexaenoic acid

To clarify the alternative mechanisms to vitamin E (VE) regulating lipid peroxide accumulation in the liver after docosahexaenoic acid (DHA) ingestion, we examined the relationship between the DHA-induced lipid peroxide formation and induction of the xenobiotic transporters, Ral-binding GTPase-activating protein (RalBP1) and multidrug resistance-associated proteins 1, 2 and 3 (MRP1-3), in the liver of rats fed with DHA. The test diets contained DHA and linoleic acid (LA) (8.7% and 2.1% of total energy, respectively) with different levels of dietary VE (normal and low: 68 and 7.7 mg of alpha-tocopherol equivalent per kg diet, respectively), and the control diet contained LA alone (11.5% of total energy). The rats were fed with these experimental diets for 14 d. The proportions of DHA in the liver, kidney and heart were higher in the DHA-fed groups than in the LA-fed group. The tissue thiobarbituric acid values as an index of lipid peroxidation were also significantly higher in the DHA-fed groups, but the value did not differ between the DHA-fed groups with different VE levels. In the liver, there were no significant differences in the glutathione S-transferase (GST) and aldehyde dehydrogenase (ALDH) activities or in the expression of GST M2, RalBP1, MRP1 and MRP2 mRNA. However, the obvious induction of expression of liver MRP3 mRNA and tendency to produce the protein were recognized after DHA ingestion. This study is the first to report the gene expression of MRP3 by DHA ingestion. There might exist, therefore, some relationship between the DHA intake and MRP3 induction in regulating lipid peroxide accumulation in the liver.     

Mouse epidermal keratinocytes in three-dimensional organotypic coculture with dermal fibroblasts form a stratified sheet resembling skin

We describe an organotypic model of mouse skin consisting of a stratified sheet of epidermal keratinocytes and dermal fibroblasts within a contracted collagen gel. The model was designed to maintain the polarity of stratified keratinocytes and permit their long-term culture at an air-liquid interface. After air exposure, the thickness of the keratinocyte sheet transiently increased and then decreased to two cell layers at 2 weeks. The two-cell-layer structure is similar to that of the adult mouse epidermis. Cytokeratin 5 was localized in the lowest cell layer in the epithelial sheet, but cytokeratin 1 and loricrin were localized in the outer cell layers, resembling mouse skin. The expressions of interleukin 1alpha and 1beta in the keratinocytes and of keratinocyte growth factor 1 and 2 in the fibroblasts correlated with keratinocyte stratification. The mouse organotypic coculture is useful in studying epithelial cell-mesenchymal cell interactions in vitro.     

A plant growth retardant, uniconazole, is a potent inhibitor of ABA catabolism in Arabidopsis

Plant growth retardants (PGRs) reduce the shoot growth of plants by inhibiting gibberellin biosynthesis. In this study, we performed detailed analyses of the inhibitory effects of PGRs on Arabidopsis abscisic acid (ABA) 8'-hydroxylase, a major ABA catabolic enzyme, recently identified as CYP707As. In an in vitro assay with CYP707A3 microsomes expressed in insect cells, uniconazole-P inhibited CYP707A3 activity more effectively than paclobutrazol or tetcyclacis, whereas the other PGRs tested did not inhibit it significantly. Uniconazole-P was found to be a strong competitive inhibitor (K(i)=8.0 nM) of ABA 8'-hydroxylase. Uniconazole-P-treated Arabidopsis plants showed enhanced drought tolerance. In uniconazole-P-treated plants, endogenous ABA levels increased 2-fold as compared with the control, and co-application of GA(4) did not suppress the effects, indicating that the effects were not due to gibberellin deficiency. Thus uniconazole-P effectively inhibits ABA catabolism in Arabidopsis plants. We also discuss the structure-activity relationship of the azole-type compounds on ABA 8'-hydroxylase inhibitory activity.