Sh3glb1/Bif-1 and mitophagy

Evasion of apoptosis, which enables cells to survive and proliferate under metabolic stress, is one of the hallmarks of cancer. We have recently reported that SH3GLB1/Bif-1 functions as a haploinsufficient tumor suppressor to prevent the acquisition of apoptosis resistance and malignant transformation during Myc-driven lymphomagenesis. SH3GLB1 is a membrane curvature-inducing protein that interacts with BECN1 though UVRAG and regulates the post-Golgi trafficking of membrane-integrated ATG9A for autophagy. At the premalignant stage, allelic loss of Sh3glb1 enhances Myc-induced chromosomal instability and results in the upregulation of anti-apoptotic proteins, including MCL1 and BCL2L1. Notably, we found that Sh3glb1 haploinsufficiency increases mitochondrial mass in overproliferated prelymphomatous Eμ-Myc cells. Moreover, loss of Sh3glb1 suppresses autophagy-dependent mitochondrial clearance (mitophagy) in PARK2/Parkin-expressing mouse embryonic fibroblasts (MEFs) treated with the mitochondrial uncoupler CCCP. Interestingly, PARK2-expressing Sh3glb1-deficient cells accumulate ER-associated immature autophagosome-like structures after treatment with CCCP. Taken together, we propose a model of mitophagy in which SH3GLB1 together with the class III phosphatidylinositol 3-kinase complex II (PIK3C3CII) (PIK3R4-PIK3C3-BECN1-UVRAG) regulates the trafficking of ATG9A-containing Golgi-derived membranes (A9⁺GDMs) to damaged mitochondria for autophagosome formation to counteract oncogene-driven tumorigenesis.     

Development of a System to Monitor Laryngeal Movement during Swallowing Using a Bend Sensor

Background

Swallowing dysfunction (also known as dysphagia), which results in a deterioration of nutritional intake, slows rehabilitation and causes aspiration pneumonia, is very common following neurological impairments. Although videofluorographic (VF) examination is widely used for detecting aspiration, an objective and non-invasive method for assessing swallowing function has yet to be established because of a lack of adequate devices and protocols. In this paper, a bend sensor whose resistance is altered by bending was introduced to monitor swallowing-related laryngeal movement.

Methods

Six healthy male volunteers were recruited in the present study. Specific time points on the signal waveform produced by the bend sensor were defined to describe laryngeal movement by differential analysis. Additionally, the physiological significance of the obtained waveform was confirmed by analyzing the sequential correlations between the signal waveform from the bend sensor and hyoid bone kinetics simultaneously recorded by VF.

Results

Seven time points were successfully defined on the signal waveform to reference laryngeal movement. Each time point was well correlated with certain VF events, with evidence of no significant time lags, and there were positive correlations between waveform time points and matched VF events. Furthermore, obvious similarities were noticed between the duration of each phase on the signal waveform and the duration of the matched hyoid bone activity.

Conclusions

The present monitoring system using a bend sensor might be useful for observing the temporal aspects of laryngeal movement during swallowing, and it was well coordinated with hyoid bone movement.     

Variation in life-history traits of male Japanese fluvial sculpin Cottus pollux in relation to nest abundance along a stream course

Life-history theory predicts the occurrence of variation in the life-history traits of fish populations under different environmental conditions; however, most studies have focused on such variation between geographically separated populations. We compared breeding characteristics and life-history traits of the Japanese fluvial sculpin (Cottus pollux), a bottom-dwelling nest-holding fish, between two adjacent sites sub-divided by a weir along a stream course in central Japan. Males in the area with a lower abundance of nest sites reached sexual maturity at an earlier age and had a shorter life span than males in the area with sufficient nest abundance. Size-dependent male reproduction was found only in areas with a shortage of nest sites, supporting the assumption of competitive exclusion among males for nests. Females matured at the same age in both sites with no differences in age-specific growth rates and mortality. Our results provide evidence for life-history variation in age and size at maturity and age-specific mortality schedule of males in nest-holding fishes in a single stream population via different sexual selection regimes related to differences in nest abundance between sites.     

Distinct tRNA modifications in the thermo-acidophilic archaeon, Thermoplasma acidophilum

Thermoplasma acidophilum is a thermo-acidophilic archaeon. We purified tRNA^Leu (UAG) from T. acidophilum using a solid-phase DNA probe method and determined the RNA sequence after determining via nucleoside analysis and m⁷G-specific aniline cleavage because it has been reported that T. acidophilum tRNA contains m⁷G, which is generally not found in archaeal tRNAs. RNA sequencing and liquid chromatography–mass spectrometry revealed that the m⁷G modification exists at a novel position 49. Furthermore, we found several distinct modifications, which have not previously been found in archaeal tRNA, such as 4-thiouridine9, archaeosine13 and 5-carbamoylmethyuridine34. The related tRNA modification enzymes and their genes are discussed.     

Structure-based design,synthesis, and evaluation of imidazo[1,2-b]pyridazine and imidazo[1,2-a]pyridine derivatives as novel dual c-Met and VEGFR2 kinase inhibitors

To identify compounds with potent antitumor efficacy for various human cancers, we aimed to synthesize compounds that could inhibit c-mesenchymal epithelial transition factor (c-Met) and vascular endothelial growth factor receptor 2 (VEGFR2) kinases. We designed para-substituted inhibitors by using co-crystal structural information from c-Met and VEGFR2 in complex with known inhibitors. This led to the identification of compounds 3a and 3b, which were capable of suppressing both c-Met and VEGFR2 kinase activities. Further optimization resulted in pyrazolone and pyridone derivatives, which could form intramolecular hydrogen bonds to enforce a rigid conformation, thereby producing potent inhibition. One compound of particular note was the imidazo[1,2-a]pyridine derivative (26) bearing a 6-methylpyridone ring, which strongly inhibited both c-Met and VEGFR2 enzyme activities (IC₅₀ = 1.9, 2.2 nM), as well as proliferation of c-Met-addicted MKN45 cells and VEGF-stimulated human umbilical vein endothelial cells (IC₅₀ = 5.0, 1.8 nM). Compound 26 exhibited dose-dependent antitumor efficacy in vivo in MKN45 (treated/control ratio [T/C] = 4%, po, 5 mg/kg, once-daily) and COLO205 (T/C = 13%, po, 15 mg/kg, once-daily) mouse xenograft models.     

Absorption,Translocation and Metabolism of Nicotinamide in Some Plants

The seedlings of rice, eggplant and tomato at the 5th leaf stage of growth readily absorbed exogenous ¹⁴C-nicotinamide through the root and the foliage in water culture. Within the 24 hr period after the bigining of cultivation, the radioactivity gradually translocated from the part treated with ¹⁴C-nicotinamide to the whole plant body. This compound was rapidly metabolised in the plants to at least six metabolites, in which three compounds were identified as nicotinic acid, NAD and NADP. ¹⁴C-Nicotinic acid was also taken up quickly through the root of rice and its metabolism showed a similar pattern to that of ¹⁴C-nicotinamide. The incorporation of radioactivity into NAD and NADP from ¹⁴C-nicotinamide added to cultivating solution at a concentration of 0.21 ppm was decreased to 10~20% by the simultaneous addition of unlabeled nicotinic acid at a concentration about 1000 times higher than that of the labeled one. It was concluded that the biosynthesis of these pyridine nucleotides from nicotinamide was chiefly via nicotinic acid. The formation of ¹⁴C-nicotinamide in the ¹⁴C-nicotinic acid metabolism suggested a breakdown of NAD. Three unknown compounds observed in both the metabolisms described above were not intermediates in the pyridine nucleotide biosynthesis.     

Polarographic Studies on Fat-soluble Vitamins in Nonaqueous Media

Polarographic reduction-wave of vitamin K₃ showed two inflexions in acetonitrile as solvent. The first step inflexion is caused by reduction of quinone to semiquinone stabilized as anion free-radical in acetonitrile, and the second by reduction of semiquinone to hydro-quinone dianion. With small addition of water, the dismutation of semiquinone is accelerated and the reduction-wave becomes only one step inflexion. The reduction-wave in acetonitrile is controlled by diffusion. The total wave-h eight is strictly proportional to the concentration of vitamin K₃. The measurement is not disturbed by coexistent fatty substances.     

Polarographic Studies on Fat-soluble Vitamins in Nonaqueous Media

Vitamin D₂ and vitamin D_s gave a single reduction-wave, respectively, in polarography in acetonitrile containing 0.05 M-tetrabutylammonium iodide. The eduction-wave of vitamin D_s was indistinguishable from that of vitamin D₂, whose half-wave potentials were evaluated identically at -2.1₀ V vs. Hg. Proportionalities between wave-height and concentration were held in the concentration range fronl 4× 10^-4 M (ca. 6000 IU·ml^-1) to 1.2×10 M (ca. 18000 IUD·ml^-1)

In both cases, the conjugated three double-bond system may be reduced to two double-bond system, which corresponds to the third reduction-step of vitamin A.

Ergosterol which has a conjugated two double-bond system and an isolated double-bond gave no reduction-wave under the present conditions.     

Studies on the Mode of Action of Polyoxins

(1) Chitin-UDP acetylglucosaminyltransferase (E.C. 2.4.1.16., chitin synthetase) in the cell-free system from phytopathogenic fungus Piricularia oryzae, and effects of various polyoxins and related compounds on the enzyme activity were studied. Polyoxins A~M, polyoxin A derivatives, polyoxin C derivatives, 5′-amino-5′-deoxyuridine, uridine and thymidine inhibited equally the incorporation of N-acetylglucosamine (GlcNAc) from UDP-N-acetylglucosamine (UDP-GlcNAc) into chitin.

(2) Competition between the above inhibitors and UDP-GlcNAc was observed by kinetic studies. The Km for UDP-GlcNAc was determined to be 3.3 × 10^?3 m and the Ki values for polyoxins A~M, except polyoxin C, were found to be in the range of 3.3 × 10^?5 m to 3.4 × 10^?6 m. For polyoxin C, 5′-amino-5′-deoxyuridine and uridine, the Ki values of 2.7 × 10^?3 m, 8.0 × 10^?3 m and 3.0 × 10^?3 m were given, respectively. The inhibitor constants for other related compounds were also calculated.

(3) The values of binding affinity, ?ΔG, for formation of substrate- or inhibitor-enzyme complexes were calculated from the Km or Ki values. In addition, partial binding affinities, ?Δg, for certain moieties or groups of polyoxins were estimated from the ?ΔG. For instance, the ?ΔG values for UDP-GlcNAc and polyoxin L were 5.7 kcal/mole and 9.2 kcal/mole, respectively. And the ?Δg values for the nucleoside moiety (part I), the carbamylpolyoxamic acid moiety (part II) and the carboxyl group at C5′ position of polyoxin L were 5.2, 3.5 and 0.7 kcal/mole, respectively.

(4) From the results obtained, the mechanism of action and relation between chemical structure and competitive inhibition of chitin synthetase were discussed.

     

Further Study on the Relation of Polyoxin Structure to Chitin Synthetase Inhibition

As a part of studies on the mechanism of action of antibiotics polyoxins, effects of various N-aminoacyl derivatives of polyoxin C and other polyoxins on chitin-UDP acetylglucosaminyl-transferase (EC 2.4.1.16, chitin synthetase) prepared from phytopathogenic fungus Piricularia oryzae were investigated. It was found that they inhibited the enzyme in competition with the substrate UDP-N-acetylglucosamine, Inhibitor constants, Ki, for these polyoxins were determined and the values of binding affinity, ?ΔG_bind of the inhibitors to the enzyme were calculated from the Ki values. In addition, by using these ?ΔG_bind values the values of partial binding affinity, ?Δg for the several atoms and atomic groups or the several moieties contained in the polyoxin J molecule were estimated. From the results obtained, it was concluded that the carbamoylpolyoxamic acid moiety of polyoxins helps to stabilize the polyoxin-enzyme complex through the contributions of its oxygen atom at C?1″, amino group at C?2″, hydroxyl groups at C?3″ and C?4″, aliphatic carbon chain and terminal carbamoyloxy group.

The results obtained by the kinetic investigation using various nucleotides and nucleotide sugars suggested that there was a specific binding site on the enzyme corresponding to the uridine moiety of UDP-N-acetylglucosamine, and that the pyrimidine nucleoside moiety of polyoxins was also bound to this site.