Changes in insulin-stimulated glucose transport and GLUT-4 protein in rat skeletal muscle after training

After running training, which increased GLUT-4 protein content in rat skeletal muscle by <40% compared with control rats, the training effect on insulin-stimulated maximal glucose transport (insulin responsiveness) in skeletal muscle was short lived (24 h). A recent study reported that GLUT-4 protein content in rat epitrochlearis muscle increased dramatically ( approximately 2-fold) after swimming training (J.-M. Ren, C. F. Semenkovich, E. A. Gulve, J. Gao, and J. O. Holloszy. J. Biol. Chem. 269, 14396-14401, 1994). Because GLUT-4 protein content is known to be closely related to skeletal muscle insulin responsiveness, we thought it possible that the training effect on insulin responsiveness may remain for >24 h after swimming training if GLUT-4 protein content decreases gradually from the relatively high level and still remains higher than control level for >24 h after swimming training. Therefore, we examined this possibility. Male Sprague-Dawley rats swam 2 h a day for 5 days with a weight equal to 2% of body mass. Approximately 18, 42, and 90 h after cessation of training, GLUT-4 protein concentration and 2-[1,2-3H]deoxy-D-glucose transport in the presence of a maximally stimulating concentration of insulin (2 mU/ml) were examined by using incubated epitrochlearis muscle preparation. Swimming training increased GLUT-4 protein concentration and insulin responsiveness by 87 and 85%, respectively, relative to age-matched controls when examined 18 h after training. Forty-two hours after training, GLUT-4 protein concentration and insulin responsiveness were still higher by 52 and 51%, respectively, in muscle from trained rats compared with control. GLUT-4 protein concentration and insulin responsiveness in trained muscle returned to sedentary control level within 90 h after training. We conclude that 1) the change in insulin responsiveness during detraining is directly related to muscle GLUT-4 protein content, and 2) consequently, the greater the increase in GLUT-4 protein content that is induced by training, the longer an effect on insulin responsiveness persists after the training.     

Direct intracerebral invasion from skull metastasis of large cell lung cancer

A 56-year-old Japanese woman was referred to us for the treatment of lung cancer. On admission, the patient showed multiple bone metastases, including the skull, without brain metastasis. During chemoradiotherapy for the primary tumor and bone metastasis involving the thoracic spine, she suffered a fatal intracerebral hemorrhage. Since the patient had no risk factors for intracerebral hemorrhage, the skull bone metastasis was thought to be responsible for this event. At autopsy, penetration of the metastatic tumor from the skull bone into the dura, with direct invasion of the brain tissue, was confirmed histologically. A hematoma also was identified at the same site adjacent to the skull bone metastasis. To our knowledge, direct tumor invasion to the brain from a skull metastasis of non-small cell lung cancer has not been previously reported.     

Estimation of endotoxin-like substances in deep seawater by using bioassay.

Deep seawater has recently been under trial as a fundamental material for mineral water, food, face lotion and an efficacious reagent for the cure of atopic dermatitis in Japan. However, little is known about the biologically effective substances, including toxic compounds in deep seawater. In this study, we investigated the effects of deep seawater on the function of murine macrophages in vitro, and examined the endotoxin-like substances in seawater. Mitochondrial activity and NO production in macrophage cells cultured with stimulants were enhanced in a depth dependent manner by pretreatment with deep seawater. In addition, fractions from deep seawater, enriched by hydrophobic column chromatography, activated the macrophage cells much more than the corresponding fractions from surface seawater. Furthermore, the effects of the fractions on macrophage cells remained significant, even with the addition of polymyxin B. which is a specific inhibitor of endotoxins. These results indicate that endotoxins and unknown substances, which affect macrophage functions, exist in a depth dependent manner in seawater.     

Photoluminescence of Eu<Superscript>3+</Superscript> ion in SnO<Subscript>2</Subscript> obtained by sol–gel

Photoluminescence data of Eu-doped SnO₂ xerogels are presented, yielding information on the symmetry of Eu³⁺ luminescent centers, which can be related to their location in the matrix: at lattice sites, substituting to Sn⁴⁺, or segregated at particles surface. Influence of doping concentration and/or particle size on the photoluminescence spectra obtained by energy transfer from the matrix to Eu³⁺ sites is investigated. Results show that a better efficiency in the energy transfer processes is obtained for high symmetry Eu³⁺ sites and low doping levels. Emission intensity from ⁵D₀→⁷F₁ transition increases as the temperature is raised from 10 to 240 K, under excitation at 266 nm laser line, because in this transition the multiphonon emission becomes significant only above 240 K. As an extension of this result, we predict high effectiveness for room temperature operation of Eu-based optical communication devices. X-ray diffraction data show that the impurity excess inhibits particle growth, which may influence the asymmetry ratio of luminescence spectra.     

A quartz crystal microbalance simulation to examine the effect of ultraviolet light treatment on characteristics of polyethylene surface

The effect of ultraviolet light irradiation on the characteristics of the polyethylene (PE) surface was investigated by the quartz crystal microbalance (QCM) technique. The PE film was prepared on the gold electrodes of the QCM by spin-coating from the solution and then was treated by the excimer UV lamp in ambient air. The changes in the hydrophilic properties, moisture adsorption, and water retention of the PE film due to the UV irradiation were determined from the frequency change of the QCM. To evaluate the detergency of the PE film, stearic acid as model oily soil was deposited onto the PE film formed on the QCM by the Langmuir-Blodgett (LB) technique, and was ultrasonically cleaned in aqueous detergent solutions containing ethanol or surfactant. The removal efficiency obtained from the frequency change of the QCM was found to increase considerably after the UV irradiation. From independently determined contact angles and the surface free energy components of the PE film, the free energy change resulting from the penetration of the detergent solution between stearic acid and PE in the zone of contact was calculated. Good relation was found between the removal efficiency and the free energy change, indicating that the increase in the detergency of the PE surface by UV irradiation was explained by surface energetics.     

Pseudo internal standard approach for label-free quantitative proteomics

Quantitative proteome analysis has become a versatile tool to understand biological functions. Although stable isotope labeling is the most reliable method for quantitative mass spectrometry, preparation of isotope-labeled compounds is time-consuming and expensive. Simple label-free approaches have been introduced, but intensity-based quantitation without standards is not generally accepted as reliable, especially for small molecules. We have developed a novel label-free quantitative proteome analysis using pseudo internal standards (PISs). This idea was derived from northern blotting analysis, in which housekeeping genes are used as standards to normalize and compare target gene expression levels in different samples. In many proteomics studies, most proteins do not change their expression levels under different conditions, and therefore, these proteins can be employed as pseudo internal standards. This new approach is simple and does not require additional standards or labeling reagents. The PIS method represents a novel approach for mass spectrometry-based comprehensive quantitatitation and may also be applicable to quantitative metabolome analysis.     

Sex Pheromone of the Cotton Mealybug, <Emphasis Type="Italic">Phenacoccus solenopsis</Emphasis>, with an Unusual Cyclobutane Structure

The cotton mealybug, Phenacoccus solenopsis, the distribution of which was formerly limited to Nearctic and Neotropical regions, recently invaded many countries in various regions including Asia, Africa, and the Pacific. More recently, P. solenopsis was newly recorded in Japan and is currently an emerging pest of agricultural crops. In this study, we determined the structure of a sex pheromone of P. solenopsis in order to develop an effective lure for monitoring this pest. From volatiles emitted by virgin adult females, we isolated a compound attractive to males. By means of coupled gas chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy, we identified this as (2,2-dimethyl-3-isopropylidenecyclobutyl)methyl 3-methylbut-2-enoate. This compound was synthesized and shown to be attractive to male P. solenopsis. Analysis by gas chromatography using an enantioselective stationary phase and polarimetry analyses of the natural pheromone and synthetic enantiomers showed the natural compound to be the (R)-(?)-enantiomer. This compound is an ester of maconelliol, which has an unusual cyclobutane structure found in sex pheromones of other mealybug species, and senecioic acid, also found in the pheromones of other mealybug species. However, this is the first example of the ester of maconelliol and senecioic acid as a natural product.     

Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.