Evaluation of early gastric mucosal permeability induced by central thyrotropin-releasing hormone administration

Accumulating evidence suggests that central thyrotropin-releasing hormone (TRH) administration induces gastric erosion 4 h after administration through the vagal nerves. However, early changes in the gastric mucosa during these 4 h have not been described. To assess early changes in the gastric mucosa after intracisternal injection of a stable TRH analog, pGlu-His-(3,3'-dimethyl)-ProNH2 (RX-77368), we measured the blood-to-lumen 51Cr-labeled EDTA clearance and examined the effects of vagotomy, atropine, omeprazole, and hydrochloric acid (HCl) on RX-77368-induced mucosal permeability. A cytoprotective dose of RX-77368 (1.5 ng) did not increase mucosal permeability. However, higher doses significantly increased mucosal permeability. Permeability peaked within 20 min and gradually returned to control levels in response to a 15-ng dose (submaximal dose). Increased mucosal permeability was not recovered after a 150-ng dose (ulcerogenic dose). This increase in permeability was inhibited by vagotomy or atropine. Intragastric perfusion with HCl did not change the RX-77368 (15 ng)-induced increase in permeability, but completely inhibited the recovery of permeability after the peak. Pretreatment with omeprazole did not change the RX-77368 (15 ng)-induced increase in permeability, but quickened the recovery of permeability after the peak. These data indicate that the RX-77368-induced increase in permeability is mediated via the vagal-cholinergic pathway and is not a secondary change in RX-77368-induced acid secretion. Inhibited recovery of permeability on exposure to an ulcerogenic RX-77368 dose or on exposure to HCl plus a submaximal dose of RX-77368 may be crucial for the induction of gastric mucosal lesions by central RX-77368 administration.     

Influence of sound and light on heart rate variability

The effects of acoustic and visual stimuli and their synergistic effects on heart rate variability including gender differences were investigated. Of particular interest was the influence of visual stimulus on heart rate variability during listening to simple sounds of different characters. Twelve male and 12 female university students were selected as subjects. The subjects listened at rest to 7 different figures of sound at loudness levels averaging 60 dB. Beat-to-beat R-R intervals were continuously recorded under the closed-eye condition (CEC) and the open-eye condition (OEC) prior to, during, and immediately after the exposure to acoustic stimuli. Low frequency (LF) power was defined over 0.04-0.15 Hz and high frequency (HF) power over 0.15-0.40 Hz. Cardiac autonomic function was estimated by plotting LF/HF in standard measure against HF in standard measure and by plotting LF/HF (%) against HF (%), accompanied by a demarcated central area. Values of LF/HF tended to be smaller under CEC than under OEC. Values of HF while listening to a 110 Hz sine wave under CEC were significantly greater than values for 880 Hz and 3520 Hz sine waves, or for 110 Hz or 880 Hz sawtooth waves, under OEC. Under CEC, values of HF for 7 figures of sound were greater in females than in males. The value of HF of sine wave for 110 Hz under CEC and OEC was significantly greater than that for white noise under the OEC. The results suggest that the cardiac parasympathetic nervous activity during auditory excitation increases with elimination of visual stimuli and tends to be greater in females than in males.     

Formaldehyde activating enzyme (Fae) and hexulose-6-phosphate synthase (Hps) in <Emphasis Type="Italic">Methanosarcina barkeri</Emphasis>: a possible function in ribose-5-phosphate biosynthesis

Formaldehyde activating enzyme (Fae) was first discovered in methylotrophic bacteria, where it is involved in the oxidation of methanol to CO₂ and in formaldehyde detoxification. The 18 kDa protein catalyzes the condensation of formaldehyde with tetrahydromethanopterin (H₄MPT) to methylene-H₄MPT. We describe here that Fae is also present and functional in the methanogenic archaeon Methanosarcina barkeri. The faeA homologue in the genome of M. barkeri was heterologously expressed in Escherichia coli and the overproduced purified protein shown to actively catalyze the condensation reaction: apparent V _max=13 U/mg protein (1 U=μmol/min); apparent Km for H₄MPT=30 μM; apparent Km for formaldehyde=0.1 mM. By Western blot analysis the concentration of Fae in cell extracts of M. barkeri was determined to be in the order of 0.1% of the soluble cell proteins. Besides the faeA gene the genome of M. barkeri harbors a second gene, faeB-hpsB, which is shown to code for a 42 kDa protein with both Fae activity (3.6 U/mg) and hexulose-6-phosphate synthase (Hps) activity (4.4 U/mg). The results support the recent proposal that in methanogenic archaea Fae and Hps could have a function in ribose phosphate synthesis.     

Complicated water orientations in the minor groove of the B-DNA decamer d(CCATTAATGG)2 observed by neutron diffraction measurements

It has long been suspected that the structure and function of a DNA duplex can be strongly dependent on its degree of hydration. By neutron diffraction experiments, we have succeeded in determining most of the hydrogen (H) and deuterium (D) atomic positions in the decameric d(CCATTAATGG)₂ duplex. Moreover, the D positions in 27 D₂O molecules have been determined. In particular, the complex water network in the minor groove has been observed in detail. By a combined structural analysis using 2.0 Å resolution X-ray and 3.0 Å resolution neutron data, it is clear that the spine of hydration is built up, not only by a simple hexagonal hydration pattern (as reported in earlier X-ray studies), but also by many other water bridges hydrogen-bonded to the DNA strands. The complexity of the hydration pattern in the minor groove is derived from an extraordinary variety of orientations displayed by the water molecules.     

Pathophysiological studies of trinitrobenzene sulfonic acid-induced colitis in Syrian hamsters (Mesocricetus auratus)

We developed a colitis model in Syrian hamsters (Mesocricetus auratus) to investigate the relationship between colitis and neutrophil elastase (NE). Colitis was induced by a single intracolonic dose of trinitrobenzene sulfonic acid (TNBS; 90 mg/ml) dissolved in 15% (vol/vol) ethanol. The ulcer area, tissue myeloperoxidase (MPO) activity, and luminal NE activity all were increased on Days 1 and 5, corresponding with the acute inflammatory histopathological changes. These acute inflammatory parameters subsequently decreased by Day 14, and chronic inflammatory histopathological changes became evident. Recurrence of inflammation was not observed during the period up to Day 28. To evaluate our colitis model, the effects of prednisolone were examined. Prednisolone was administered orally once on the day before induction of colitis, and animals were treated twice daily thereafter. Although prednisolone had little effect on the tissue MPO activity, prednisolone inhibited the ulcer area and NE activity. In addition, the effects of an NE-specific inhibitor (ONO-6818) on our TNBS-induced colitis model were examined. In the subcutaneous treatment study, ONO-6818 was administered once before the induction of colitis. Although ONO-6818 had little effect on the tissue MPO activity, the ulcer area and NE activity were decreased in the ONO-6818-treated group. The inhibitory effects on the ulcer area and NE activity were confirmed after oral treatment with ONO-6818 after induction of colitis. We conclude that our colitis model is useful for investigating the relationship between colitis and NE, and inhibition of NE activity can prevent the progression of ulceration.     

The crystal structure of plant ATG12 and its biological implication in autophagy

Atg12 is a post-translational modifier that is activated and conjugated to its single target, Atg5, by a ubiquitin-like conjugation system. The Atg12-Atg5 conjugate is essential for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. Here, we demonstrate that the Atg12 conjugation system exists in Arabidopsis and is essential for plant autophagy as well as in yeast and mammals. We also report the crystal structure of Arabidopsis thaliana (At) ATG12 at 1.8 A resolution. Despite no obvious sequence homology with ubiquitin, the structure of AtATG12 shows a ubiquitin fold strikingly similar to those of mammalian homologs of Atg8, the other ubiquitin-like modifier essential for autophagy, which is conjugated to phosphatidylethanolamine. Two types of hydrophobic patches are present on the surface of AtATG12: one is conserved in both Atg12 and Atg8 orthologs, while the other is unique to Atg12 orthologs. Considering that they share Atg7 as an E1-like enzyme, we suggest that the first hydrophobic patch is responsible for the conjugation reaction, while the latter is involved in Atg12-specific functions.     

Enzymatic diagnosis of medium-chain acyl-CoA dehydrogenase deficiency by detecting 2-octenoyl-CoA production using high-performance liquid chromatography: a practical confirmatory test for tandem mass spectrometry newborn screening in Japan

Many of the previously described enzymatic assay methods for the diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency have been dependent upon the measurement of radioisotope-labeled co-products or reduction of electron acceptors. We have developed a direct assay method to detect 2-enoyl-CoA production using high-performance liquid chromatography (HPLC). Crude cell lysate prepared from lymphocytes were incubated with n-octanoyl-CoA and ferrocenium hexafluorophosphate. The detection of 2-octenoyl-CoA was significantly reproducible. We applied the assay to samples from four infants suspected to have MCAD deficiency by tandem mass spectrometry (MS/MS) newborn screening conducted in the Hiroshima area of Japan. Three of them were proved to have pathologically reduced residual enzyme activities, although they were associated with various clinical and biochemical phenotypes. In addition, another symptomatic Japanese patient and her presymptomatic sibling who were detected by MS/MS selective screening were successfully diagnosed by our enzymatic assay. These results indicate that the method can be a useful confirmatory test for MS/MS screening of MCAD deficiency.     

Variation in maxillary sinus anatomy among platyrrhine monkeys

Variations in the maxillary sinus anatomy of extant and fossil catarrhine primates have been extensively examined using computed tomography (CT), and have potential utility for phylogenetic analyses. This approach has also been used to demonstrate its anatomical variation in eight of the 16 extant genera of platyrrhines and the absence of the sinus in Saimiri and Cacajao. We used this approach to evaluate the three-dimensional anatomy of the maxillary sinus in all extant platyrrhine genera, and here argue the phylogenic implications of this variation. This study confirms, for the most part, previous CT studies and augments them with the six genera not studied previously: Ateles, Lagothrix, Callithrix, Cebuella, Pithecia and Chiropotes. The entire maxilla is pneumatized by the sinus in the atelines, Cebus, and Callicebus, whereas the sinus pneumatizes only the medial part of the maxilla in the callitrichines and Aotus. Pithecia has a unique conformation in which the maxillary sinus and the expanded inferior meatus pneumatize the posteromedial and anterolateral parts of the entire maxilla, respectively. Chiropotes has no sinus, and the inferior meatus possibly expands into the area between the middle meatus and medial surface of the maxilla to disturb sinus formation, as in the case of its close relative Cacajao. Finally, we argue that the sinus that pneumatizes the entire maxilla is a primitive feature in extant platyrrhines and was probably shared by the last common ancestor of the anthropoids.     

DNA-collagen complex as a carrier for Ag+ delivery

The possibility of DNA-collagen complex as a drug carrier was investigated. The interaction between DNA and silver ions was proved by CD spectra. The release property of the complex of DNA-Ag+ was measured through turbidity of PBS solution to indicate that silver ions could coordinate with base pairs of DNA, and be released slowly from the complex of DNA-Ag+. Collagen film, collagen-Ag+ film, DNA-collagen film and DNA-collagen-Ag+ film were prepared, and studied through SEM. Particles were found present in DNA-collagen-Ag+ film by SEM. These show that silver ions may be enclosed inside these particles, which led to the slow release of Ag+ to the environments. Two bacteria, Escherichia coli and Staphylococcus aureus, were used to study the antibiotic properties of the complex films. The growth of E. coli and S. aureus could be inhibited by these films. It indicates that DNA-collagen may be a good drug carrier for the drug-controlled release.     

Monomeric and dimeric dibenzofurans from cultured mycobionts of Lecanora iseana

Spore-derived mycobionts of the lichen Lecanora iseana were cultivated on a malt-yeast extract medium supplemented with 10% sucrose and their metabolites were investigated. Four 3,7-dihydroxy-1,9-dimethyldibenzofuran derivatives along with the known 3,7-dihydroxy-1,9-dimethyldibenzofuran and five norlichexanthone derivatives were isolated. Their structures were determined by spectroscopic methods.