Tumor suppressor IRF-1 mediates retinoid and interferon anticancer signaling to death ligand TRAIL

Retinoids and interferons are signaling molecules with pronounced anticancer activity. We show that in both acute promyelocytic leukemia and breast cancer cells the retinoic acid (RA) and interferon signaling pathways converge on the promoter of the tumoricidal death ligand TRAIL. Promoter mapping, chromatin immunoprecipitation and RNA interference reveal that retinoid-induced interferon regulatory factor-1 (IRF-1), a tumor suppressor, is critically required for TRAIL induction by both RA and IFNgamma. Exposure of breast cancer cells to both antitumor agents results in enhanced TRAIL promoter occupancy by IRF-1 and coactivator recruitment, leading to strong histone acetylation and synergistic induction of TRAIL expression. In coculture experiments, pre-exposure of breast cancer cells to RA and IFNgamma induced a dramatic TRAIL-dependent apoptosis in heterologous cancer cells in a paracrine mode of action, while normal cells were not affected. Our results identify a novel TRAIL-mediated tumor suppressor activity of IRF-1 and suggest a mechanistic basis for the synergistic antitumor activities of certain retinoids and interferons. These data argue for combination therapies that activate the TRAIL pathway to eradicate tumor cells.     

Role of the chemokine stromal cell-derived factor 1 in autoantibody production and nephritis in murine lupus

In normal mice, stromal cell-derived factor 1 (SDF-1/CXCL12) promotes the migration, proliferation, and survival of peritoneal B1a (PerB1a) lymphocytes. Because these cells express a self-reactive repertoire and are expanded in New Zealand Black/New Zealand White (NZB/W) mice, we tested their response to SDF-1 in such mice. PerB1a lymphocytes from NZB/W mice were exceedingly sensitive to SDF-1. This greater sensitivity was due to the NZB genetic background, it was not observed for other B lymphocyte subpopulations, and it was modulated by IL-10. SDF-1 was produced constitutively in the peritoneal cavity and in the spleen. It was also produced by podocytes in the glomeruli of NZB/W mice with nephritis. The administration of antagonists of either SDF-1 or IL-10 early in life prevented the development of autoantibodies, nephritis, and death in NZB/W mice. Initiation of anti-SDF-1 mAb treatment later in life, in mice with established nephritis, inhibited autoantibody production, abolished proteinuria and Ig deposition, and reversed morphological changes in the kidneys. This treatment also counteracted B1a lymphocyte expansion and T lymphocyte activation. Therefore, PerB1a lymphocytes are abnormally sensitive to the combined action of SDF-1 and IL-10 in NZB/W mice, and SDF-1 is key in the development of autoimmunity in this murine model of lupus.     

CodY-regulated aminotransferases AraT and BcaT play a major role in the growth of Lactococcus lactis in milk by regulating the intracellular pool of amino acids

Aminotransferases, which catalyze the last step of biosynthesis of most amino acids and the first step of their catabolism, may be involved in the growth of Lactococcus lactis in milk. Previously, we isolated two aminotransferases from L. lactis, AraT and BcaT, which are responsible for the transamination of aromatic amino acids, branched-chain amino acids, and methionine. In this study, we demonstrated that double inactivation of AraT and BcaT strongly reduced the growth of L. lactis in milk. Supplementation of milk with amino acids and keto acids that are substrates of both aminotransferases did not improve the growth of the double mutant. On the contrary, supplementation of milk with isoleucine or a dipeptide containing isoleucine almost totally inhibited the growth of the double mutant, while it did not affect or only slightly affected the growth of the wild-type strain. These results suggest that AraT and BcaT play a major role in the growth of L. lactis in milk by degrading the intracellular excess isoleucine, which is responsible for the growth inhibition. The growth inhibition by isoleucine is likely to be due to CodY repression of the proteolytic system, which is necessary for maximal growth of L. lactis in milk, since the growth of the CodY mutant was not affected by addition of isoleucine to milk. Moreover, we demonstrated that AraT and BcaT are part of the CodY regulon and therefore are regulated by nutritional factors, such as the carbohydrate and nitrogen sources.     

2′-phosphodiesterase and 2′,5′-oligoadenylate synthetase activities in the lowest metazoans,sponge [porifera]

Sponges [porifera], the most ancient metazoans, contain modules related to the vertebrate immune system, including the 2′,5′-oligoadenylate synthetase (OAS). The components of the antiviral 2′,5′-oligoadenylate (2–5A) system (OAS, 2′-Phosphodiesterase (2′-PDE) and RNAse L) of vertebrates have not all been identified in sponges. Here, we demonstrate for the first time that in addition to the OAS activity, sponges possess a 2′-PDE activity, which highlights the probable existence of a premature 2–5A system. Indeed, Suberites domuncula and Crella elegans exhibited this 2–5A degrading activity. Upon this finding, two out of three elements forming the 2–5A system have been found in sponges, only a endoribonuclease, RNAse L or similar, has to be found. We suspect the existence of a complex immune system in sponges, besides the self/non-self recognition system and the use of phagocytosis and secondary metabolites against pathogens.     

Development of an Equine Groove Model to Induce Metacarpophalangeal Osteoarthritis: A Pilot Study on 6 Horses

The aim of this work was to develop an equine metacarpophalangeal joint model that induces osteoarthritis that is not primarily mediated by instability or inflammation. The study involved six Standardbred horses. Standardized cartilage surface damage or “grooves” were created arthroscopically on the distal dorsal aspect of the lateral and medial metacarpal condyles of a randomly chosen limb. The contralateral limb was sham operated. After 2 weeks of stall rest, horses were trotted 30 minutes every other day for 8 weeks, then evaluated for lameness and radiographed. Synovial fluid was analyzed for cytology and biomarkers. At 10 weeks post-surgery, horses were euthanized for macroscopic and histologic joint evaluation. Arthroscopic grooving allowed precise and identical damage to the cartilage of all animals. Under the controlled exercise regime, this osteoarthritis groove model displayed significant radiographic, macroscopic, and microscopic degenerative and reactive changes. Histology demonstrated consistent surgically induced grooves limited to non-calcified cartilage and accompanied by secondary adjacent cartilage lesions, chondrocyte necrosis, chondrocyte clusters, cartilage matrix softening, fissuring, mild subchondral bone inflammation, edema, and osteoblastic margination. Synovial fluid biochemistry and cytology demonstrated significantly elevated total protein without an increase in prostaglandin E2, neutrophils, or chondrocytes. This equine metacarpophalangeal groove model demonstrated that standardized non-calcified cartilage damage accompanied by exercise triggered altered osteochondral morphology and cartilage degeneration with minimal or inefficient repair and little inflammatory response. This model, if validated, would allow for assessment of disease processes and the effects of therapy.     

The Search for Therapeutic Bacteriophages Uncovers One New Subfamily and Two New Genera of Pseudomonas-Infecting Myoviridae

In a previous study, six virulent bacteriophages PAK_P1, PAK_P2, PAK_P3, PAK_P4, PAK_P5 and CHA_P1 were evaluated for their in vivo efficacy in treating Pseudomonas aeruginosa infections using a mouse model of lung infection. Here, we show that their genomes are closely related to five other Pseudomonas phages and allow a subdivision into two clades, PAK_P1-like and KPP10-like viruses, based on differences in genome size, %GC and genomic contents, as well as number of tRNAs. These two clades are well delineated, with a mean of 86% and 92% of proteins considered homologous within individual clades, and 25% proteins considered homologous between the two clades. By ESI-MS/MS analysis we determined that their virions are composed of at least 25 different proteins and electron microscopy revealed a morphology identical to the hallmark Salmonella phage Felix O1. A search for additional bacteriophage homologs, using profiles of protein families defined from the analysis of the 11 genomes, identified 10 additional candidates infecting hosts from different species. By carrying out a phylogenetic analysis using these 21 genomes we were able to define a new subfamily of viruses, the Felixounavirinae within the Myoviridae family. The new Felixounavirinae subfamily includes three genera: Felixounalikevirus, PAK_P1likevirus and KPP10likevirus. Sequencing genomes of bacteriophages with therapeutic potential increases the quantity of genomic data on closely related bacteriophages, leading to establishment of new taxonomic clades and the development of strategies for analyzing viral genomes as presented in this article.     

Lipidomic Analysis Links Mycobactin Synthase K to Iron Uptake and Virulence in M. tuberculosis

The prolonged survival of Mycobacterium tuberculosis (M. tb) in the host fundamentally depends on scavenging essential nutrients from host sources. M. tb scavenges non-heme iron using mycobactin and carboxymycobactin siderophores, synthesized by mycobactin synthases (Mbt). Although a general mechanism for mycobactin biosynthesis has been proposed, the biological functions of individual mbt genes remain largely untested. Through targeted gene deletion and global lipidomic profiling of intact bacteria, we identify the essential biochemical functions of two mycobactin synthases, MbtK and MbtN, in siderophore biosynthesis and their effects on bacterial growth in vitro and in vivo. The deletion mutant, ΔmbtN, produces only saturated mycobactin and carboxymycobactin, demonstrating an essential function of MbtN as the mycobactin dehydrogenase, which affects antigenicity but not iron uptake or M. tb growth. In contrast, deletion of mbtK ablated all known forms of mycobactin and its deoxy precursors, defining MbtK as the essential acyl transferase. The mbtK mutant showed markedly reduced iron scavenging and growth in vitro. Further, ΔmbtK was attenuated for growth in mice, demonstrating a non-redundant role of hydroxamate siderophores in virulence, even when other M. tb iron scavenging mechanisms are operative. The unbiased lipidomic approach also revealed unexpected consequences of perturbing mycobactin biosynthesis, including extreme depletion of mycobacterial phospholipids. Thus, lipidomic profiling highlights connections among iron acquisition, phospholipid homeostasis, and virulence, and identifies MbtK as a lynchpin at the crossroads of these phenotypes.     

Insulin Resistance Is Not Associated with an Impaired Mitochondrial Function in Contracting Gastrocnemius Muscle of Goto-Kakizaki Diabetic Rats In Vivo

Insulin resistance, altered lipid metabolism and mitochondrial dysfunction in skeletal muscle would play a major role in type 2 diabetes mellitus (T2DM) development, but the causal relationships between these events remain conflicting. To clarify this issue, gastrocnemius muscle function and energetics were investigated throughout a multidisciplinary approach combining in vivo and in vitro measurements in Goto-Kakizaki (GK) rats, a non-obese T2DM model developing peripheral insulin resistant without abnormal level of plasma non-esterified fatty acids (NEFA). Wistar rats were used as controls. Mechanical performance and energy metabolism were assessed strictly non-invasively using magnetic resonance (MR) imaging and 31-phosphorus MR spectroscopy (³¹P-MRS). Compared with control group, plasma insulin and glucose were respectively lower and higher in GK rats, but plasma NEFA level was normal. In resting GK muscle, phosphocreatine content was reduced whereas glucose content and intracellular pH were both higher. However, there were not differences between both groups for basal oxidative ATP synthesis rate, citrate synthase activity, and intramyocellular contents for lipids, glycogen, ATP and ADP (an important in vivo mitochondrial regulator). During a standardized fatiguing protocol (6 min of maximal repeated isometric contractions electrically induced at a frequency of 1.7 Hz), mechanical performance and glycolytic ATP production rate were reduced in diabetic animals whereas oxidative ATP production rate, maximal mitochondrial capacity and ATP cost of contraction were not changed. These findings provide in vivo evidence that insulin resistance is not caused by an impairment of mitochondrial function in this diabetic model.