Proteins Released from Myofibrils by CASF (Ca2+-activated sarcoplasmic factor) and Trypsin

The nature of the soluble proteins and peptides released from myofibrils by treatment with CASF (Ca²⁺-activated sarcoplasmic factor) was investigated by using Polyacrylamide gel electrophoresis in both a nondenaturing and a denaturing (sodium dodecyl sulfate=SDS) solvent and by using gel permeation chromatography on Sepharose 6B. Both CASF and trypsin treatment cause removal of Z-disks before causing other ultrastructurally detectable degradation of myofibrils. CASF treatment of myofibrils releases a protein that is identical to α-actinin, one of the known components of the Z-disk, on the basis of mobility in Polyacrylamide gel electrophoresis in a nondenaturing solvent and in SDS and on the basis of elution from gel permeation columns. Trypsin treatment of myofibrils releases a number of smaller molecular weight products that cannot be identified with any of the known myofibrillar proteins. Hence, CASF seems to remove Z-disks from myofibrils by means of a very specific proteolytic activity that releases α-actinin without extensively degrading it. Trypsin, on the other hand, probably seems to remove Z-disks by degrading α-actinin to peptides.     

Effect of Materials Released from Myofibrils by CAF (Ca2+-activated Factor) on Z-Disk Reconstitution

To investigate the constituents of Z-disk, reconstitution of Z-disk by incubating some proteins released from myofibrils by CAF(Ca²⁺-activated factor) with Z-disk-extracted fiber bundles was carried out and examined with electron microscope. The materials released from myofibrils by CAF have been bound in Z-disk region, and Z-disk extracted from myofibrils with a low ionic strength solution has been reconstituted. On the other hand, Z-disk removed from myofibrils by CAF has not been reconstituted by the same way.     

Synthesis of 3Z-Nonenal and 3Z,6Z-Nonadienal

3Z-Nonenal and 3Z, 6Z-nonadienal, potential biosynthetic precursors of 2E-nonenal and 2E, 6Z-nonadienal, were for the first time synthesized stereoseleclively.     

Lipase from Candida paralipolytica

The extracellular lipase from Candida paralipolytica required essential activators* (usually bile- or calcium salts) for the in vitro hydrolysis of triglycerides. The reaction systems emulsified with gum arabic, gelatin, lecithin, methyl cellulose, pectin, polyvinyl alcohol, sodium cholate, or without emulsifier were compared concerning requirement for essential activator, inhibition with sodium chloride and maximum reaction rate, and the following findings have been obtained. (1) The emulsions used can be classified into five groups by the essential activator requirement. (2) The inhibition with sodium chloride depended on reaction system. (3) Each reaction system gave a similar reaction rate at pH 8.2. (4) Long-chain fatty acid dissolved in substrate was necessary to the activation with calcium salts.     

Lipids and Related Substances Inducing the Lipase Production by Candida paralipolytica

Properties of autolytic breakdown of rat skeletal muscle proteins in the alkaline pH range have been reported. The activity is almost exclusively localized in the myofibrillar fraction, but is not solubilized with Triton X-100. The activity is affected by the KCI concentration in the reaction mixture. In 0.6 M and the more concentrated KCI solutions, the maximum activity is attained. The optimum pH of the activity is in the range of pH 7.5～9.5, and the optimum temperature is between 47～57°C.

This autolytic activity seems to be different from catheptic activity which shows its optimum pH in the acid pH range. Moreover, though more than half of the catheptic activity of rat skeletal muscle is recovered in the myofibrillar fraction, the catheptic activity in the myofibrillar fraction can be removed from the fraction by the extraction with dilute saline solution containing Triton X-100.     

Novel mutations of CDKN1C in Japanese patients with Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an imprinting-related human disease that is characterized by macrosomia, macroglossia, abdominal wall defects, and variable minor features. BWS is caused by several genetic/epigenetic alterations, such as loss of methylation at KvDMR1, gain of methylation at H19-DMR, paternal uniparental disomy of chromosome 11, CDKN1C mutations, and structural abnormalities of chromosome 11. CDKN1C is an imprinted gene with maternal preferential expression, encoding for a cyclin-dependent kinase (CDK) inhibitor. Mutations in CDKN1C are found in 40 % of familial BWS cases with dominant maternal transmission and in ~5 % of sporadic cases. In this study, we searched for CDKN1C mutations in 37 BWS cases that had no evidence for other alterations. We found five mutations—four novel and one known—from a total of six patients. Four were maternally inherited and one was a de novo mutation. Two frame-shift mutations and one nonsense mutation abolished the QT domain, containing a PCNA-binding domain and a nuclear localization signal. Two missense mutations occurred in the CDK inhibitory domain, diminishing its inhibitory function. The above-mentioned mutations were predicted by in silico analysis to lead to loss of function; therefore, we strongly suspect that such anomalies are causative in the etiology of BWS.     

Identification of Highly Selective and Potent Histone Deacetylase 3 Inhibitors Using Click Chemistry-Based Combinatorial Fragment Assembly

To find histone deacetylase 3 (HDAC3)-selective inhibitors, a series of 504 candidates was assembled using “click chemistry”, by reacting nine alkynes bearing a zinc-binding group with 56 azide building blocks in the presence of Cu(I) catalyst. Screening of the 504-member triazole library against HDAC3 and other HDAC isozymes led to the identification of potent and selective HDAC3 inhibitors T247 and T326. These compounds showed potent HDAC3 inhibition with submicromolar IC₅₀s, whereas they did not strongly inhibit other isozymes. Compounds T247 and T326 also induced a dose-dependent selective increase of NF-κB acetylation in human colon cancer HCT116 cells, indicating selective inhibition of HDAC3 in the cells. In addition, these HDAC3-selective inhibitors induced growth inhibition of cancer cells, and activated HIV gene expression in latent HIV-infected cells. These findings indicate that HDAC3-selective inhibitors are promising candidates for anticancer drugs and antiviral agents. This work also suggests the usefulness of the click chemistry approach to find isozyme-selective HDAC inhibitors.     

Daily consumption of tea catechins improves aerobic capacity in healthy male adults: a randomized double-blind,placebo-controlled,crossover trial

Our previous studies demonstrated that dietary supplementation with tea catechins combined with exercise improved endurance capacity in mice. This study aimed to demonstrate the effect of daily tea catechin consumption on aerobic capacity in humans. Sixteen Japanese non-athlete male subjects (aged 25–47 years) took 500 mL of a test beverage with or without tea catechins (570 mg) daily for 8 weeks and attended a training program twice a week. Aerobic capacity was evaluated by indirect calorimetry and near-infrared spectroscopy during graded cycle exercise. Catechin beverage consumption was associated with a significantly higher ventilation threshold during exercise and a higher recovery rate of oxygenated hemoglobin and myoglobin levels after graded cycle exercise when compared to subjects receiving the placebo beverage. These results indicate that daily consumption of tea catechins increases aerobic capacity when combined with semiweekly light exercise, which may be due to increased skeletal muscle aerobic capacity.     

Spring and summer microhabitat use by Schlegel’s Japanese gecko,Gekko japonicus (Reptilia: Squamata: Gekkonidae), in urban areas

Differential microhabitat use may be beneficial to achieving fitness in seasonally variable environmental conditions. To explore whether the microhabitat use of the nocturnal Schlegel’s Japanese gecko, Gekko japonicus, varies seasonally and depends on juvenile, male, and female reproductive groups, we investigated five categorical and five quantitative measure variables of microhabitat use in a wild population both in spring and summer. Most geckos were found on white, vertical planes of concrete and plastered brick walls. None of the categorical variables (type of location, substrate, substrate color, light source, and refuge) significantly differed according to season or group, while substrate temperature and irradiance at the location where geckos were observed and the distance from the nearest potential refuge were significantly greater in summer than in spring. The quantitative measure variables did not differ among the reproductive groups. These results suggest that G. japonicus seasonally adjusts its microhabitat use mainly in terms of quantitative measure variables rather than categorical variables.