Bromodeoxyuridine Immunohistochemistry for Evaluating Age-Related Changes in the Rat Mandibular Condyle Decalcified by Intravenous Infusion

Age-related changes in cell proliferation kinetics of the mandibular condyle were studied in Sprague-Dawley rats using bromodeoxyuridine (BrdUrd) immunohistochemistry in decalcified, paraffin-embedded tissues. Intraperitoneal injection of 40 mg/kg BrdUrd was given 1 hr before animal sacrifice. Continuous perfusion of EDTA solution via the left ventricle shortened the decalcification time. Immunohistochemical staining was performed with monoclonal anti-BrdUrd antibody. BrdUrd labeled cells, i.e., the S-phase cells, were clearly visible with well-preserved cytological detail. Their nuclei exhibited homogeneously stained granules. The labeling index in the intermediate zone of the condyle decreased with increasing age of the animals. This method is useful for evaluating physiological and pathological changes of the rat mandibular condyle as well as other bones and joints.     

Studies on the Utilization of Hydrocarbons by Microorganisms

Taxonomical studies on ten strains of hydrocarbon-utilizing bacteria reported in previous paper, which produced various kinds of amino acid, were carried out. They were Achromo-bacter cycloclastes, Achromobacter delmarvae, Bacillus species, Corynebacterium species, Micrococcus species. Many of them were not identical with the species which are described in Bergey’s Manual of 7th Edition.     

Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase

The breast and ovarian cancer suppressor BRCA1 acquires significant ubiquitin ligase activity when bound to BARD1 as a RING heterodimer. Although the activity may well be important for the role of BRCA1 as a tumor suppressor, the biochemical consequence of the activity is not yet known. Here we report that BRCA1-BARD1 catalyzes Lys-6-linked polyubiquitin chain formation. K6R mutation of ubiquitin dramatically reduces the polyubiquitin products mediated by BRCA1-BARD1 in vitro. BRCA1-BARD1 preferentially utilizes ubiquitin with a single Lys residue at Lys-6 or Lys-29 to mediate autoubiquitination of BRCA1 in vivo. Furthermore, mass spectrometry analysis identified the Lys-6-linked branched ubiquitin fragment from the polyubiquitin chain produced by BRCA1-BARD1 using wild type ubiquitin. The BRCA1-BARD1-mediated Lys-6-linked polyubiquitin chains are deubiquitinated by 26 S proteasome in vitro, whereas autoubiquitinated CUL1 through Lys-48-linked polyubiquitin chains is degraded. Proteasome inhibitors do not alter the steady state level of the autoubiquitinated BRCA1 in vivo. Hence, the results indicate that BRCA1-BARD1 mediates novel polyubiquitin chains that may be distinctly edited by 26 S proteasome from conventional Lys-48-linked polyubiquitin chains.     

Distinct contributions of vacuolar Qabc- and R-SNARE proteins to membrane fusion specificity

In eukaryotic endomembrane systems, Qabc-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) on one membrane and R-SNARE on the opposing membrane assemble into a trans-QabcR-SNARE complex to drive membrane fusion. However, it remains ambiguous whether pairing of Qabc- and R-SNAREs mediates membrane fusion specificity. Here, we explored the fusion specificity of reconstituted proteoliposomes bearing purified SNAREs in yeast vacuoles and other organelles. We found that not only vacuolar R-SNARE Nyv1p but also the non-cognate R-SNAREs, endosomal Snc2p, and endoplasmic reticulum-Golgi Sec22p caused efficient fusion with vacuolar Qabc-SNAREs. In contrast, their fusion is blocked completely by replacing vacuolar Qc-SNARE Vam7p with the non-cognate endosomal Tlg1p and Syn8p, although these endosomal Qc-SNAREs fully retained the ability to form cis-SNARE complexes with vacuolar SNAREs in solution and on membranes. Thus, our current study establishes that an appropriate assembly of Qabc-SNAREs is crucial for regulating fusion specificity, whereas R-SNARE itself has little contribution to specificity.     

Phenotypic plasticity in the range-margin population of the lycaenid butterfly <Emphasis Type="Italic">Zizeeria maha</Emphasis>

Background  

Many butterfly species have been experiencing the northward range expansion and physiological adaptation, probably due to climate warming. Here, we document an extraordinary field case of a species of lycaenid butterfly, Zizeeria maha, for which plastic phenotypes of wing color-patterns were revealed at the population level in the course of range expansion. Furthermore, we examined whether this outbreak of phenotypic changes was able to be reproduced in a laboratory.     

Skin temperature changes induced by strong static magnetic field exposure

High intensity static magnetic fields, when applied to the whole body of the anesthetized rat, have previously been reported to decrease skin temperature. The hypothesis of the present study was that in diamagnetic water, molecules in the air play significant roles in the mechanism of skin temperature decrease. We used a horizontal cylindrical superconducting magnet. The magnet produced 8 T at its center. A thermistor probe was inserted in a subcutaneous pocket of the anesthetized rats to measure skin temperature. Animals (n=10) were placed in an open plastic holder in which the ambient air was free to move in any direction (group I). Animals (n=10) were placed in a closed holder in which the air circulation toward the direction of weak magnetic field was restricted (group II). Each holder was connected to a hydrometer to measure humidity around the animal in the holder. The data acquisition phase consisted of a 5 min baseline interval, followed by inserting the animal together with the holder into the center of the magnet bore for a 5 min exposure and a 5 min postexposure period outside the bore. In group I, skin temperature and humidity around the animal significantly decreased during exposure, followed by recovery after exposure. In group II, skin temperature and humidity did not decrease during the measurement. The skin temperature decrease was closely related to the decrease in humidity around the body of the animal in the holder, and the changes were completely blocked by restricting the air circulation in the direction of the bore entrance. Possible mechanisms responsible for the decrease in skin temperature may be associated with magnetically induced movement of water vapor at the skin surface, leading to skin temperature decrease.     

Sampling efficiency of the pale grass blue butterfly Zizeeria maha (Lepidoptera: Lycaenidae): A versatile indicator species for environmental risk assessment in Japan

The pale grass blue butterfly, Zizeeria maha (Lepidoptera, Lycaenidae), has successfully been employed as an indicator species to evaluate acute biological changes in polluted human-living environments after the Fukushima nuclear accident. Here, we quantitatively examined the field sampling efficiency of the butterfly and its associated sexual sampling bias under different conditions, which may provide important information for environmental risk assessment. Sampling data were obtained in 2014 and 2015 from 87 localities across Japan. Across the localities, the mean capture rate was 17 individuals per hour per person, and males represented approximately 80% of the collected butterflies. No significant difference was detected in the capture rate under different weather conditions. Among the four habitats, the riverside showed a significantly higher capture rate than the city park. A shorter sampling time tended to yield a higher capture rate. Comparisons among prefectures and districts of collection localities revealed that the Kanto district had significantly higher percentages of collected males than 4 other districts. Fukushima prefecture also had high percentage, although not significant. A generalized linear mixed model indicated that the capture rate was significantly negatively affected by the agricultural village, the city park, the rainy weather, and latitude and positively by longitude. Together, the present study showed high sampling efficiency and versatility of this butterfly as an indicator for environmental risk assessment but simultaneously suggested preferable conditions for a field sampling design. This study also suggested a possibility that the Fukushima nuclear accident or other factors might have affected behavioral or population dynamics of the butterfly.     

Electron microscopical findings with special reference to cancer in rats caused by inhalation of nickel oxide

A special exposure system was used for the inhalation of nickel oxide (NiO) aerosol by Wistar male rats. The median aerodynamic diameter and the geometric standard deviation were 1.2 μm and 2.2, respectively. A histopathological study of the rats was performed immediately, and at intervals of 12 and 20 mo after a 1-mo expsoure to NiO. Electron microscopy showed that localization of NiO particles was restricted to the lungs and that each particle had been engulfed by the alveolar macrophages. Type II pneumocytes and nonciliated bronchiolar epithelial cells (Clara cells), as well as numerous tubular myelin (surfactant) in the alveoli were prominent. In rats dissected after 12 mo, clusters of NiO particles were still present within the terminal bronchioli, alveolar walls, and lysosomes of the alveolar macrophages. Pools of tubular myelin were observed in the peribron-chial lymphatics. The Clara cells, which project into the lumen of bronchioli, showed active secretion and were filled with smooth en-doplasmic reticulum (SER) in the apical cytoplasm. In the experimental group sacrificed after 20 mo, one rat had papillary adenocarcinoma and two rats showed adenomatosis in the peripheral portion of the lung, but none in the upper respiratory tract.