Involvement of autophagy in the maintenance of rat intervertebral disc homeostasis: an in-vitro and in-vivo RNA interference study of Atg5.

OBJECTIVE: In the largest avascular low-nutrient intervertebral disc, resident cells would utilize autophagy, a stress-response survival mechanism by self-digestion and recycling wastes. Our goal was to elucidate the involvement of autophagy in disc homeostasis through RNA interference of autophagy-related gene 5 (Atg5). DESIGN: In vitro, small interfering RNAs (siRNAs) targeting autophagy-essential Atg5 were transfected into rat disc cells. Cell viability with levels of autophagy including Atg5 expression, apoptosis, and senescence was assessed under serum starvation and/or pro-inflammatory interleukin-1 beta (IL-1ß) stimulation. In vivo, time-course autophagic flux was monitored following Alexa Fluor® 555-labeled Atg5-siRNA injection into rat tail discs. Furthermore, 24-h temporary static compression-induced disruption of Atg5 siRNA-injected discs was observed by radiography, histomorphology, and immunofluorescence. RESULTS: In disc cells, three different Atg5 siRNAs consistently suppressed autophagy with Atg5 protein knockdown (mean 44.4% [95% confidence interval: -51.7, -37.1], 51.5% [-80.5, -22.5], 62.3% [-96.6, -28.2]). Then, Atg5 knockdown reduced cell viability through apoptosis and senescence not in serum-supplemented medium (93.6% [-0.8, 21.4]) but in serum-deprived medium (66.4% [-29.8, -8.6]) further with IL-1ß (44.5% [-36.9, -23.5]). In disc tissues, immunofluorescence detected intradiscal signals for the labeled siRNA even at 56-d post-injection. Immunoblotting found 56-d autophagy suppression with prolonged Atg5 knockdown (33.2% [-52.8, -5.3]). With compression, Atg5 siRNA-injected discs presented radiographic height loss ([-43.9, -0.8]), histological damage ([-5.5, -0.2]), and immunofluorescent apoptosis ([2.2, 22.2]) and senescence ([4.1, 19.9]) induction compared to control siRNA-injected discs at 56 d. CONCLUSIONS: This loss-of-function study suggests Atg5-dependent autophagy-mediated anti-apoptosis and anti-senescence. Autophagy could be a molecular therapeutic target for degenerative disc disease.

Pharmacological inhibition of mTORC1 but not mTORC2 protects against human disc cellular apoptosis, senescence, and extracellular matrix catabolism through Akt and autophagy induction.

OBJECTIVE: The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth. We hypothesized that mTOR is influential in the intervertebral disc-largest avascular, low-nutrient organ. Our objective was to identify the optimal mTOR inhibitor for treating human degenerative disc disease. DESIGN: mTOR complex 1 (mTORC1) regulates p70/ribosomal S6 kinase (p70/S6K), negatively regulates autophagy, and is controlled by Akt. Akt is controlled by phosphatidylinositol 3-kinase (PI3K) and mTOR complex 2 (mTORC2). mTORC1 inhibitors-rapamycin, temsirolimus, everolimus, and curcumin, mTORC1&mTORC2 inhibitor-INK-128, PI3K&mTOR inhibitor-NVP-BEZ235, and Akt inhibitor-MK-2206-were applied to human disc nucleus pulposus (NP) cells. mTOR signaling, autophagy, apoptosis, senescence, and matrix metabolism were evaluated. RESULTS: mTORC1 inhibitors decreased p70/S6K but increased Akt phosphorylation, promoted autophagy with light chain 3 (LC3)-II increases and p62/sequestosome 1 (p62/SQSTM1) decreases, and suppressed pro-inflammatory interleukin-1 beta (IL-1ß)-induced apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity (versus rapamycin, 95% confidence interval (CI) -0.431 to -0.194; temsirolimus, 95% CI -0.529 to -0.292; everolimus, 95% CI -0.477 to -0.241; curcumin, 95% CI -0.248 to -0.011) and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage, senescent senescence-associated beta-galactosidase (SA-ß-gal) positivity (versus rapamycin, 95% CI -0.437 to -0.230; temsirolimus, 95% CI -0.534 to -0.327; everolimus, 95% CI -0.485 to -0.278; curcumin, 95% CI -0.210 to -0.003) and p16/INK4A expression, and catabolic matrix metalloproteinase (MMP) release and activation. Meanwhile, dual mTOR inhibitors decreased p70/S6K and Akt phosphorylation without enhanced autophagy and suppressed apoptosis, senescence, and matrix catabolism. MK-2206 counteracted protective effects of temsirolimus. Additional disc-tissue analysis found relevance of mTOR signaling to degeneration grades. CONCLUSION: mTORC1 inhibitors-notably temsirolimus with an improved water solubility-but not dual mTOR inhibitors protect against inflammation-induced apoptosis, senescence, and matrix catabolism in human disc cells, which depends on Akt and autophagy induction.

Selective interference of mTORC1/RAPTOR protects against human disc cellular apoptosis, senescence, and extracellular matrix catabolism with Akt and autophagy induction.

OBJECTIVE: The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth and protein synthesis. We hypothesized that mTOR is essential for the intervertebral disc, the largest avascular, low-nutrient organ. Our objective was to elucidate roles of mTOR signaling in human disc cells. DESIGN: The mTOR exists in two complexes: mTORC1 containing the regulatory-associated protein of mTOR (RAPTOR) and mTORC2 containing the rapamycin-insensitive companion of mTOR (RICTOR). To analyze their functions in human disc nucleus pulposus cells, RNA interference (RNAi) of mTOR targeting mTORC1 and mTORC2, RAPTOR targeting mTORC1, or RICTOR targeting mTORC2 or rapamycin, a pharmacological mTORC1 inhibitor, was applied. First, mTOR signaling including Akt, p70/ribosomal S6 kinase (p70/S6K), and autophagy were assessed. Then, apoptosis, senescence, and matrix metabolism were evaluated under pro-inflammatory interleukin-1 beta (IL-1ß) stimulation. RESULTS: Western blotting showed significant decreases in specific proteins by each RNAi (all P < 0.0001). In mTOR signaling, RNAi of mTOR and RICTOR decreased p70/S6K and Akt phosphorylation, whereas RAPTOR RNAi decreased p70/S6K but increased Akt phosphorylation. All RNAi treatments increased light chain 3 (LC3)-II and decreased p62/sequestosome 1 (p62/SQSTM1), indicating enhanced autophagy. In apoptosis, IL-1ß-induced terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage decreased by RAPTOR RNAi. In senescence, IL-1ß-induced senescence-associated beta-galactosidase (SA-ß-gal)-positive cells and p16/INK4A expression also decreased by RAPTOR RNAi. In matrix metabolism, RAPTOR RNAi reduced IL-1ß-induced catabolic matrix metalloproteinase (MMP) release and activation and up-regulated anabolic gene expression. These findings were all consistent with rapamycin administration. Additional disc-tissue analysis detected expression and phosphorylation of mTOR-signaling molecules in varying ages. CONCLUSION: Selective interference of mTORC1/RAPTOR protects against inflammation-induced apoptosis, senescence, and matrix catabolism possibly through Akt and autophagy induction in human disc cells.

Targeted destruction of fungal structures of Erysiphe trifoliorum on flat leaf surfaces of Marchantia polymorpha.

In this study, we observed the germination behaviour of airborne conidia from powdery mildews that settle on thalloid surfaces. We inoculated thalli (flat, sheet-like leaf tissues) and gemmae (small, flat, sheet-like leaf tissues that propagate asexually via bud-like structures) of the common liverwort (Marchantia polymorpha) with conidia from tomato powdery mildew (Oidium neolycopersici; KTP-02) and red clover powdery mildew (Erysiphe trifoliorum; KRCP-4N) and examined their germination and subsequent appressorium formation under a high-fidelity digital microscope. Conidial bodies and germ tubes of the inoculated KRCP-4N conidia were destroyed on both the thalli and gemmae. The destruction of these fungal structures was observed only for KRCP-4N conidia inoculated onto M. polymorpha on both leaf surfaces. No differences in destruction of the KRCP-4N fungal structures between thalli and gemmae were observed. At 4 h post-inoculation, destruction of the germ tube tip was observed when it reached the gemmae leaf surface. At 6 h post-inoculation, the conidial bodies and germ tubes were destroyed. In contrast, KTP-02 conidia were not destroyed and formed normal, well-lobed appressoria on the surface of M. polymorpha gemmae.

The isolation and identification of a light-induced protein in alfalfa sprouts and the cloning of its specific promoter.

We used 2D-PAGE to isolate a light-induced protein (AL-A) that is expressed abundantly in light-growth alfalfa sprouts. The seven amino acids of the N-terminal region of the protein were identified, and we searched for the protein in GenBank using the BLAST program. The results of the homology analysis showed that the amino acid sequence of the isolated protein is most similar to one from a pea plastocyanin. To identify the protein, we amplified and sequenced the DNA fragment encoding AL-A from genomic alfalfa DNA. We found that the AL-A gene was highly homologous (90%) to the sequences from the pea plastocyanin via multiple alignments, and the deduced protein precursor was predicted to be chloroplast-specific via the ChloroP computer program. The protein was named alfalfa-plastocyanin (AL-P). It was characterized as being a light-inducible protein, and RT-PCR analysis showed that AL-P mRNA transcription only occurred in the leaves of the alfalfa plant and the alfalfa seedlings growth in lighted conditions. PCR was also used to amplify the DNA fragment encoding the AL-P promoter (AL-Pp) from genomic alfalfa DNA. PlantCARE analysis of the promoter sequence indicated that both a typical TATA box and a CAAT box were located in the promoter sequence, and some of the cis-elements that are responsible for light responsiveness were also identified within this promoter region. The AL-P gene promoter fused to the ß-glucuronidase (GUS) reporter gene has been examined for expression in transgenic alfalfa seedlings. Our findings have a potential application in plant genetic engineering; the AL-Pp may be used to drive the expression of heterologous genes in transgenic alfalfa plants.

Powdery mildew pathogens can suppress the chitinase gene expression induced in detached inner epidermis of barley coleoptile.

Two-step PCR (RT-PCR and nested PCR) was used to detect gene expression in powdery mildew pathogen-infected cells of detached inner epidermis of barley coleoptiles. Cellular contents of infected cells were microscopically suctioned with a micropipette and subjected to PCR. Triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase genes involved in the glycolytic pathway and a stimulus-induced endochitinase gene were targeted, and their expression was determined by detecting cDNAs derived from spliced transcripts. The two gycolysis-related genes were constantly expressed in the tissue irrespective of pathogen inoculation. In contrast, chitinase gene expression was induced in non-infected inner epidermis after detachment. After inoculation, this expression was selectively suppressed in pathogen-invaded cells, in spite of continuous expression in non-invaded cells of the same epidermis. Thus, the present method enabled us to directly analyze transcripts in individual cells at the infection site and assess the capability of the pathogen to regulate host gene expression.

Antigen selectivity characteristic of polyclonal antibodies against omega-conotoxin GVIA and N-type voltage-dependent calcium channels.

The antibodies against omega-conotoxin GVIA (omega-CTX GVIA; N-type voltage-dependent calcium channel [VDCC] blocker) and B1Nt (N-terminal segment [residues 1-13] of BI alpha1 subunits of VDCCs) were prepared, and the selectivity for each antigen omega-CTX GVIA and B1Nt was investigated. For the antigen selectivity of anti-omega-CTX GVIA antibody against omega-CTX GVIA, ELISA, and immunoprecipitation were used. The reactions for ELISA and immunoprecipitation were observed except when antibody IgG purified by Protein A-Sepharose CL-4B from nonimmunized serum (purified NI-Ab) was used. The specific reactions were inhibited by 10 nM omega-CTX GVIA, but not by omega-CTX SVIB (N-type VDCC blocker), omega-CTX MVIIC (N- and P-type VDCC blocker), or omega-Aga IVA (P-type VDCC blocker). For the antigen selectivity of the anti-B1Nt antibody, analyses by ELISA, immunoprecipitation, and Western blotting were conducted. The reactions were observed except when NI-Ab was used. The ELISA and immunoprecipitation reactions were inhibited by the antigen peptide B1Nt, and the IC50 values were about 1.2 x 10(-8) and 1.3 x 10(-8) M, respectively. The bands of 210 and 190 kD by Western blotting of crude membranes from chick brain were also inhibited by 1 microM B1Nt. These results suggest that the antibodies prepared against omega-CTX GVIA and B1Nt in this work have high selectivity for their antigen. Therefore we assume that the antibodies against omega-CTX GVIA and B1Nt are useful tools for the analyses of the function and distribution of N-type VDCCs. The anti omega-CTX GVIA antibody must also be useful for the radioimmunoassay of omega-CTX GVIA.

Rapid detection of phylloplane bacterium Enterobacter cloacae based on chitinase gene transformation and lytic infection by specific bacteriophages.

AIMS: To establish a rapid and efficient method for detecting Enterobacter cloacae based on chitinase gene transformation and lytic infection by virulent bacteriophages. METHODS AND RESULTS: A phylloplane strain of E. cloacae was isolated from tomato leaves and transformed with a chitinase gene. Transformed bacteria were collected from single colonies and infected with newly isolated, virulent bacteriophages in the presence of the chitinase substrate 4-methylumbelliferon (4MU)-(GlcNac)3. To assay chitinase activity in the lysates, the product 4MU was measured spectrofluorophotometrically or visibly detected under u.v. irradiation. Chitinase gene-transformed bacteria obtained from single colonies could be specifically identified in 30 min by the emission of 4MU fluorescence following lysis caused by phage infection. CONCLUSIONS: The chitinase gene was used as a reporter gene to construct a new system for easy and rapid monitoring of transgenic strains of E. cloacae released in the environment, in combination with specific recognition by virulent bacteriophages. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay is simple, rapid, inexpensive, easy to perform and applicable to other strains. The system can be used for the routine monitoring of bacteria, which is important because of the increased use of transgenic strains of E. cloacae as an antagonistic biological control agent for plant diseases.