Behavioral Characteristics of Ubiquitin-Specific Peptidase 46-Deficient Mice

We have previously identified Usp46, which encodes for ubiquitin-specific peptidase 46, as a quantitative trait gene affecting the immobility time of mice in the tail suspension test (TST) and forced swimming test. The mutation that we identified was a 3-bp deletion coding for lysine (Lys 92), and mice with this mutation (MT mice), as well as Usp46 KO mice exhibited shorter TST immobility times. Behavioral pharmacology suggests that the gamma aminobutyric acid A (GABA_A) receptor is involved in regulating TST immobility time. In order to understand how far Usp46 controls behavioral phenotypes, which could be related to mental disorders in humans, we subjected Usp46 MT and KO mice to multiple behavioral tests, including the open field test, ethanol preference test, ethanol-induced loss of righting reflex test, sucrose preference test, novelty-suppressed feeding test, marble burying test, and novel object recognition test. Although behavioral phenotypes of the Usp46 MT and KO mice were not always identical, deficiency of Usp46 significantly affected performance in all these tests. In the open field test, activity levels were lower in Usp46 KO mice than wild type (WT) or MT mice. Both MT and KO mice showed lower ethanol preference and shorter recovery times after ethanol administration. Compared to WT mice, Usp46 MT and KO mice exhibited decreased sucrose preference, took longer latency periods to bite pellets, and buried more marbles in the sucrose preference test, novelty-suppressed feeding test, and marble burying test, respectively. In the novel object recognition test, neither MT nor KO mice showed an increase in exploration of a new object 24 hours after training. These findings indicate that Usp46 regulates a wide range of behavioral phenotypes that might be related to human mental disorders and provides insight into the function of USP46 deubiquitinating enzyme in the neural system.     

Caffeic acid production by simultaneous saccharification and fermentation of kraft pulp using recombinant Escherichia coli

Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

     

Brd4 is required for recovery from antimicrotubule drug-induced mitotic arrest: preservation of acetylated chromatin

The mammalian bromodomain protein Brd4 interacts with mitotic chromosomes by binding to acetylated histone H3 and H4 and is thought to play a role in epigenetic memory. Mitotic cells are susceptible to antimicrotubule drugs. These drugs activate multiple response pathways and arrest cells at mitosis. We found that Brd4 was rapidly released from chromosomes upon treatment with antimicrotubule drugs, including the reversible agent nocodazole. Yet, when nocodazole was withdrawn, Brd4 was reloaded onto chromosomes, and cells proceeded to complete cell division. However, cells in which a Brd4 allele was disrupted (Brd4+/-), and expressing only half of the normal Brd4 levels, were defective in reloading Brd4 onto chromosomes. Consequently, Brd4+/- cells were impaired in their ability to recover from nocodazole-induced mitotic arrest: a large fraction of +/- cells failed to reach anaphase after drug withdrawal, and those that entered anaphase showed an increased frequency of abnormal chromosomal segregation. The reloading defect observed in Brd4+/- cells coincided with selective hypoacetylation of lysine residues on H3 and H4. The histone deacetylase inhibitor trichostatin A increased global histone acetylation and perturbed nocodazole-induced Brd4 unloading. Brd4 plays an integral part in a cellular response to drug-induced mitotic stress by preserving a properly acetylated chromatin status.     

Cone-Beam Computed Tomography Correlates with Conventional Helical Computed Tomography in Evaluation of Lipiodol Accumulation in HCC after Chemoembolization

Background & Aims

The amount of drug-loaded lipiodol in an HCC tumor post-transarterial chemoembolization (TACE) correlates with the risk of local tumor recurrence. Lipiodol enhancement of a tumor on conventional CT, measured in Hounsfield units (HU), can predict tumor response. Here we investigate whether cone-beam CT (CBCT) can also be used to predict tumor response, providing the benefit of being able to optimize the patient’s treatment plan intra-procedurally.

Methods

A total of 82 HCC nodules (82 patients), ≤5 cm in diameter, were treated with balloon-occluded TACE using miriplatin between December 2013 and November 2014. For each patient, both CBCT and conventional CT images were obtained post-TACE. The degree of correlation between CBCT and conventional CT was determined by comparing identical regions of interest for each imaging modality using pixel values.

Results

The pixel values from conventional CT and CBCT were highly correlated, with a Pearson correlation coefficient of 0.912 (p<0.001). The location of the nodules within the liver did not affect the results; the correlation coefficient was 0.891 (p<0.001) for the left lobe and 0.926 (p<0.001) for the right lobe. The mean pixel value for conventional CT was 439 ± 279 HU, and the mean pixel value for CBCT was 416 ± 311 HU.

Conclusions

CBCT may be used as a substitute for conventional CT to quantitatively evaluate the amount of drug-loaded lipiodol within an HCC nodule and, hence, the efficacy of TACE treatment. The major benefit of using CBCT is the ability to predict the likelihood of local recurrence intra-procedurally, enabling subsequent treatment optimization.     

Rab35 GTPase recruits NDP52 to autophagy targets

Autophagy targets intracellular molecules, damaged organelles, and invading pathogens for degradation in lysosomes. Recent studies have identified autophagy receptors that facilitate this process by binding to ubiquitinated targets, including NDP52. Here, we demonstrate that the small guanosine triphosphatase Rab35 directs NDP52 to the corresponding targets of multiple forms of autophagy. The active GTP‐bound form of Rab35 accumulates on bacteria‐containing endosomes, and Rab35 directly binds and recruits NDP52 to internalized bacteria. Additionally, Rab35 promotes interaction of NDP52 with ubiquitin. This process is inhibited by TBC1D10A, a GAP that inactivates Rab35, but stimulated by autophagic activation via TBK1 kinase, which associates with NDP52. Rab35, TBC1D10A, and TBK1 regulate NDP52 recruitment to damaged mitochondria and to autophagosomes to promote mitophagy and maturation of autophagosomes, respectively. We propose that Rab35‐GTP is a critical regulator of autophagy through recruiting autophagy receptor NDP52.     

Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition

The antiviral protein kinase PKR inhibits protein synthesis by phosphorylating the translation initiation factor eIF2alpha on Ser51. Binding of double-stranded RNA to the regulatory domains of PKR promotes dimerization, autophosphorylation, and the functional activation of the kinase. Herein, we identify mutations that activate PKR in the absence of its regulatory domains and map the mutations to a recently identified dimerization surface on the kinase catalytic domain. Mutations of other residues on this surface block PKR autophosphorylation and eIF2alpha phosphorylation, while mutating Thr446, an autophosphorylation site within the catalytic-domain activation segment, impairs eIF2alpha phosphorylation and viral pseudosubstrate binding. Mutational analysis of catalytic-domain residues preferentially conserved in the eIF2alpha kinase family identifies helix alphaG as critical for the specific recognition of eIF2alpha. We propose an ordered mechanism of PKR activation in which catalytic-domain dimerization triggers Thr446 autophosphorylation and specific eIF2alpha substrate recognition.     

The role of tropomyosin domains in cooperative activation of the actin-myosin interaction

To establish α-tropomyosin (Tm)'s structure–function relationships in cooperative regulation of muscle contraction, thin filaments were reconstituted with a variety of Tm mutants (Δ2Tm, Δ3Tm, Δ6Tm, P2sTm, P3sTm, P2P3sTm, P1P5Tm, and wtTm), and force and sliding velocity of the thin filament were studied using an in vitro motility assay. In the case of deletion mutants, Δ indicates which of the quasi-equivalent repeats in Tm was deleted. In the case of period (P) mutants, an Ala cluster was introduced into the indicated period to strengthen the Tm–actin interaction. In P1P5Tm, the N-terminal half of period 5 was substituted with that of period 1 to test the quasi-equivalence of these two Tm periods. The reconstitution included bovine cardiac troponin. Deletion studies revealed that period 3 is important for the positive cooperative effect of Tm on actin filament regulation and that period 2 also contributes to this effect at low ionic strength, but to a lesser degree. Furthermore, Tm with one extra Ala cluster at period 2 (P2s) or period 3 (P3s) did not increase force or velocity, whereas Tm with two extra Ala clusters (P2P3s) increased both force and velocity, demonstrating interaction between these periods. Most mutants did not move in the absence of Ca²⁺. Notable exceptions were Δ6Tm and P1P5Tm, which moved near at the full velocity, but with reduced force, which indicate impaired relaxation. These results are consistent with the mechanism that the Tm–actin interaction cooperatively affects actin to result in generation of greater force and velocity.     

Age-related changes of dietary intake and blood eicosapentaenoic acid, docosahexaenoic acid, and arachidonic acid levels in Japanese men and women

We studied the relationship between dietary intake and the blood compositions of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (ARA) in four study groups with different ages and sexes. One hundred and four subjects were recruited. Dietary records together with photographic records from 28 consecutive days were amassed and the fatty acid composition in erythrocyte membranes and plasma lipid fractions was analyzed. Fish intake in the elderly group was significantly higher than that in the young group in both men and women. The compositions of ARA in erythrocytes and plasma phospholipids in the elderly were lower than those in the young, but the ARA intake was nearly identical. In the elderly group, the percentage of dietary ARA consumed at the same time as EPA and DHA derived from fish was high. We considered that these fatty acids markedly inhibited the incorporation of dietary ARA into blood phospholipids.     

Biochemical investigation and gene expression analysis of the immunostimulatory functions of an edible Salacia extract in rat small intestine

Roots and bark from plants belonging to genus Salacia of the family Hippocrateaceae (Salacia reticulata, Salacia oblonga, etc.) have been used for traditional Ayurvedic medicine, particularly for the treatment of diabetes. In our study, we evaluated the gene expression profiles in the small intestinal epithelium of rats that were given a Salacia plant extract to gain insight into its effects on the small intestine. In detail, DNA microarray analysis was performed to evaluate the gene expression profiles in the rat ileal epithelium. The intestinal bacterial flora was also studied using T-RFLP (Nagashima method) in these rats. Expressions of many immune-related genes, especially Th1-related genes associated with cell-mediated immunity, were found to increase in the small intestinal epithelium and the intestinal bacterial flora became similar to those in the case with Salacia plant extract administration. Our study thus revealed that Salacia plant extract exerts bioregulatory functions by boosting intestinal immunity.     

Transplantation of bone marrow-derived mononuclear cells improves mechanical hyperalgesia, cold allodynia and nerve function in diabetic neuropathy

Relief from painful diabetic neuropathy is an important clinical issue. We have previously shown that the transplantation of cultured endothelial progenitor cells or mesenchymal stem cells ameliorated diabetic neuropathy in rats. In this study, we investigated whether transplantation of freshly isolated bone marrow-derived mononuclear cells (BM-MNCs) alleviates neuropathic pain in the early stage of streptozotocin-induced diabetic rats. Two weeks after STZ injection, BM-MNCs or vehicle saline were injected into the unilateral hind limb muscles. Mechanical hyperalgesia and cold allodynia in SD rats were measured as the number of foot withdrawals to von Frey hair stimulation and acetone application, respectively. Two weeks after the BM-MNC transplantation, sciatic motor nerve conduction velocity (MNCV), sensory nerve conduction velocity (SNCV), sciatic nerve blood flow (SNBF), mRNA expressions and histology were assessed. The BM-MNC transplantation significantly ameliorated mechanical hyperalgesia and cold allodynia in the BM-MNC-injected side. Furthermore, the slowed MNCV/SNCV and decreased SNBF in diabetic rats were improved in the BM-MNC-injected side. BM-MNC transplantation improved the decreased mRNA expression of NT-3 and number of microvessels in the hind limb muscles. There was no distinct effect of BM-MNC transplantation on the intraepidermal nerve fiber density. These results suggest that autologous transplantation of BM-MNCs could be a novel strategy for the treatment of painful diabetic neuropathy.