Blocking LC3 lipidation and ATG12 conjugation reactions by ATG7 mutant protein containing C572S

Autophagy, a system for the bulk degradation of intracellular components, is essential for homeostasis and the healthy physiology and development of cells and tissues. Its deregulation is associated with human disease. Thus, methods to modulate autophagic activity are critical for analysis of its role in mammalian cells and tissues. Here we report a method to inhibit autophagy using a mutant variant of the protein ATG7, a ubiquitin E1-like enzyme essential for autophagosome formation. During autophagy, ATG7 activates the conjugation of LC3 (ATG8) with phosphatidylethanolamine (PE) and ATG12 with ATG5. Human ATG7 interactions with LC3 or ATG12 require a thioester bond involving the ATG7 cysteine residue at position 572. We generated TetOff cells expressing mutant ATG7 protein carrying a serine substitution of this critical cysteine residue (ATG7C572S). Because ATG7C572S forms stable intermediate complexes with LC3 or ATG12, its expression resulted in a strong blockage of the ATG-conjugation system and suppression of autophagosome formation. Consequently, ATG7C572S mutant protein can be used as an inhibitor of autophagy.     

The establishment of IL-2 producing cells by genetic engineering

Expression plasmids containing human interleukin-2(IL-2) cDNA under the control of viral promoters (SV40 early region, MuLV LTR, HTLV-I LTR, and ASV (Y73) LTR) were introduced into TK- mouse L cells and human FL cells to establish IL-2 producing cells. The highest levels of IL-2 producing clones were obtained in TK+ mouse L cells transformed with a recombinant plasmid having MuLV LTR as a promoter, whereas transformed cells of human FL cells (G418r) were revealed to produce IL-2 at the highest level when the cells were transfected with a plasmid containing HTLV LTR as a promoter. These results suggest that these promoter/enhancer regions possess different cell specificities in gene expression. To obtain higher levels of IL-2 production using gene amplification, the hybrid plasmids containing the hamster DHFR and human IL-2 genes were constructed and transfected into DHFR- CHO cells. DHFR+ colonies produced IL-2 at about the same level as that produced by TK+ L cells transformed with the recombinants containing MuLV LTR. Selection of methotrexate-resistant cells resulted in a 5- to 30-fold increase of IL-2 production. These cells produced IL-2 stably for at least 3 months, even in the absence of methotrexate.     

Studies on rat liver argininosuccinate synthetase. Inhibition by various amino acids.

Inhibition studies of crystallized rat liver argininosuccinate synthetase [EC 6.3.4.5] are described. 1. L-Argininosuccinate, L-histidine, and L-tryptophan inhibited the enzyme activity at saturating amounts of the substrates. 2. L-Norvaline, L-argininosuccinate, L-arginine, L-isoleucine, and L-valine competitively inhibited the enzyme activity at a low concentration of L-citrulline, with Ki values of 1.3 x 10(4) M, 2.5 X 10(-4) M, 6.7 X 10(-4) M, 6.3 X 10(-4) M, and 6.0 x 10(-4) M, respectively. 3. L-Argininosuccinate and L-arginine competitively inhibited the enzyme activity at a low concentration of L-aspartate, with Ki values of 9.5 x 10(-4) M and 1.2 x 10(-3) M, respectively. 4. The modes of inhibition by L-histidine were mixed-noncompetitive, uncompetitive, and noncompetitive types with respect to L-citrulline, L-aspartate, and ATP, respectively. 5. When the enzyme was preincubated with L-citrulline, the enzyme activity was slightly increased in the presence of a low concentration of L-histidine in the assay mixture. 6. The conformation of the enzyme was markedly changed by the addition of L-histidine as judged from the CD spectrum. This change was partially reversed by incubation with L-citrulline.     

Wavefront-Guided versus Non-Wavefront-Guided Photorefractive Keratectomy for Myopia: Meta-Analysis of Randomized Controlled Trials

Purpose

To compare the efficacy, predictability, safety, and induced higher-order aberrations (HOAs) between wavefront-guided and non-wavefront-guided photorefractive keratectomy (PRK).

Methods

The Cochrane Central Register of Controlled Trials, PubMED, and EMBASE were searched for randomized controlled trials. Trials meeting the selection criteria were quality appraised, and data was extracted by 2 independent authors. Measures of association were pooled quantitatively using meta-analytical methods. Comparisons between wavefront-guided and non-wavefront-guided ablations were made as pooled odds ratios (ORs) or weighted mean differences. The pooled ORs and 95% confidence intervals (CIs) were computed for efficacy, safety, and predictability. The weighted mean differences and 95% CIs were used to compare induced HOAs.

Results

The study covered five trials involving 298 eyes. After wavefront-guided PRK, the pooled OR of achieving an uncorrected distance visual acuity of 20/20 (efficacy) was 1.18 (95% CI, 0.53–2.60; p = 0.69), the pooled OR of achieving a result within ±0.50 diopter of the intended target (predictability) was 0.86 (95% CI, 0.40–1.84; p = 0.70). No study reported a loss of 2 or more lines of Snellen acuity (safety) with either modality. In eyes with wavefront-guided PRK, the postoperative trefoil aberrations (mean difference −0.02; 95% CI, −0.03 to −0.00; p = 0.03) were significantly lower. There were no significant differences between the two groups in the postoperative total HOAs (mean difference −0.04; 95% CI, −0.23 to 0.14; p = 0.63), spherical (mean difference 0.00; 95% CI, −0.08 to 0.09; p = 0.93), and coma (mean difference −0.06; 95% CI, −0.14 to 0.03; p = 0.20) aberrations.

Conclusions

According to the meta-analysis, wavefront-guided PRK offered no advantage in efficacy, predictability, or safety measures over non-wavefront-guided PRK, although it may have induced fewer trefoil aberrations.     

Fifteen weeks of dietary n-3 polyunsaturated fatty acid deprivation increase turnover of n-6 docosapentaenoic acid in rat-brain phospholipids

Docosapentaenoic acid (DPAn-6, 22:5n-6) is an n-6 polyunsaturated fatty acid (PUFA) whose brain concentration can be increased in rodents by dietary n-3 PUFA deficiency, which may contribute to their behavioral dysfunction. We used our in vivo intravenous infusion method to see if brain DPAn-6 turnover and metabolism also were altered with deprivation. We studied male rats that had been fed for 15weeks post-weaning an n-3 PUFA adequate diet containing 4.6% alpha-linolenic acid (α-LNA, 18:3n-3) or a deficient diet (0.2% α-LNA), each lacking docosahexaenoic acid (22:6n-3) and arachidonic acid (AA, 20:4n-6). [1-(14)C]DPAn-6 was infused intravenously for 5min in unanesthetized rats, after which the brain underwent high-energy microwaving, and then was analyzed. The n-3 PUFA deficient compared with adequate diet increased DPAn-6 and decreased DHA concentrations in plasma and brain, while minimally changing brain AA concentration. Incorporation rates of unesterified DPAn-6 from plasma into individual brain phospholipids were increased 5.2-7.7 fold, while turnover rates were increased 2.1-4.7 fold. The observations suggest that increased metabolism and brain concentrations of DPAn-6 and its metabolites, together with a reduced brain DHA concentration, contribute to behavioral and functional abnormalities reported with dietary n-3 PUFA deprivation in rodents. (196 words).     

Distribution and variable expression of secretory pathway protein reticulocalbin in normal human organs and non-neoplastic pathological conditions.

Reticulocalbin (RCN) is one member of the Ca(2+)-binding proteins in the secretory pathway and is localized in the endoplasmic reticulum. RCN may play a role in the normal behavior and life of cells, although its detailed role remains unknown. Overexpression of RCN may also play a role in tumorigenesis, tumor invasion, and drug resistance. The new antibody for human RCN is used in the distribution of RCN in normal human organs of fetuses and adults with or without inflammation. Immunohistochemical examination demonstrated a broad distribution of RCN in various organs of fetuses and adults, predominantly in the endocrine and exocrine organs. However, RCN expression was heterogeneous in each constituent cell of some organs. Among non-epithelial organs, vascular endothelial cells, testicular germ cells, neurons, and follicular dendritic cells showed strong staining. Plasma cells were the only RCN-positive cells among hematopoietic and lymphoid cells. In inflammatory conditions, RCN expression was enhanced in both epithelial and non-epithelial cells. Heterogeneous expression of RCN indicates that the amount of RCN needed for cell behavior and life may be variable, depending on each cell type and, therefore, RCN may be helpful in establishing the cell origin of neoplasms in some organs. However, further study is needed to establish the significance of RCN in tumorigenesis and in some peculiar features of neoplasms.     

Rice straw fermentation using lactic acid bacteria

To efficiently utilize rice straw and lessen its disposal problem on the environment, a lactic acid bacteria community, SFC-2 was developed from natural fermentation products of rice straw by continuous enrichment with the MRS-S broth (MRS broth with sucrose), and used to accelerate the fermentation of air-dried straws. The SFC-2 could rapidly lower the pH of the broth and produce high levels of lactic acid. Using a combination of plate isolation, denaturing gradient gel electrophoresis (DGGE) and 16S rDNA sequencing, the microbial composition of the SFC-2 was classified into Lactobacillus, mainly comprised of L. fermentum, L. plantarum and L. paracacei. An evaluation of the fermentation effect of SFC-2 on rice straw showed that it lowered the pH and significantly (P<0.05) increased lactic acid concentration in the straw. Further analysis with DGGE indicated that L. plantarum, L. fermentum and L. paracasei were the dominant species during fermentation.