No abstract

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer. A majority of these mutations are missense mutations in the DNAbinding domain. As a result, the mutated p53 gene encodes a full-length protein incapable of transactivating its target genes. In addition to this loss of function, mutant p53 can have a dominant negative effect over wild-type p53 and/or gain of function activity independently of the wild-type protein.To better understand the nature of the tumorigenic activity of mutant p53, we have investigated the mechanism by which mutant p53 can exert a dominant negative effect. We have established several stable cell lines capable of inducibly expressing a p53 mutant alone, wild-type p53 alone, or both proteins concurrently. In this context, we have used chromatin immunoprecipitation to determine the ability of wild-type p53 to bind to its endogenous target genes in the presence of various p53 mutants. We have found that p53 missense mutants markedly reduce the binding of wild-type p53 to the p53 responsive element in the target genes of p21, MDM2, and PIG3. These findings correlate with the reduced ability of wild-type p53 in inducing these and other endogenous target genes and growth suppression in the presence of mutant p53. We also showed that mutant p53 suppresses the ability of wild-type p53 in inducing cell cycle arrest. This highlights the sensitivity and utility of the dual inducible expression system because in previous studies, p53-mediated cell cycle arrest is not affected by transiently overexpressed p53 mutants. Together, our data showed that mutant p53 exerts its dominant negative activity by abrogating the DNA binding, and subsequently the growth suppression, functions of wild-type p53.

IntroductionDiabetic patients with a long disease duration usually accompanied complication such as diabetic retinopathy, but in some patients had no complication.ObjectivesWe analyzed differences in plasma metabolites according to the presence or absence of diabetic retinopathy (DR) in type 2 diabetic (T2D) patients with disease duration ≥ 15 years.MethodsA cohort of 183 T2D patients was established. Their biospecimens and clinical information were collected in accordance with the guidelines of the National Biobank of Korea, and the Korean Diabetes Association. DR phenotypes of the subjects were verified by ophthalmologic specialists. Plasma metabolites were analyzed using gas chromatography time-of-flight mass spectrometry and ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry. And these results were analyzed using multivariate statistics.ResultsFor metabolomic study, propensity score matched case and control subjects were chosen. Mean age of the subjects was 66.4 years and mean T2D duration was 22.2 years. Metabolomic identification revealed various carbohydrates, amino acids, and organic compounds that distinguished between age- and sex-matched non-diabetic controls and T2D subjects. Among these, glutamine and glutamic acid were suggested as the most distinctive metabolites for the presence of DR. Receiver operating characteristics curves showed an excellent diagnostic value of combined (AUC = 0.739) and the ratio (AUC = 0.742) of glutamine and glutamic acid for DR. And these results were consistent in validation analyses.ConclusionOur results imply that plasma glutamine, glutamic acid, and their ratio may be valuable as novel biomarkers for anticipating DR in T2D subjects.Electronic supplementary materialThe online version of this article (10.1007/s11306-018-1383-3) contains supplementary material, which is available to authorized users.

Calf diarrhea is associated with enteric infections, and also provokes the overuse of antibiotics. Therefore, proper treatment of diarrhea represents a therapeutic challenge in livestock production and public health concerns. Here, we describe the ability of a fecal microbiota transplantation (FMT), to ameliorate diarrhea and restore gut microbial composition in 57 growing calves. We conduct multi-omics analysis of 450 longitudinally collected fecal samples and find that FMT-induced alterations in the gut microbiota (an increase in the family Porphyromonadaceae) and metabolomic profile (a reduction in fecal amino acid concentration) strongly correlate with the remission of diarrhea. During the continuous follow-up study over 24 months, we find that FMT improves the growth performance of the cattle. This first FMT trial in ruminants suggest that FMT is capable of ameliorating diarrhea in pre-weaning calves with alterations in their gut microbiota, and that FMT may have a potential role in the improvement of growth performance.

Myosin VI is an unconventional motor protein, and its mutation is responsible for the familiar conditions sensorineural deafness and hypertrophic cardiomyopathy. Myosin VI is found to play a key role in the protein trafficking and homeostasis of the Golgi complex. However, very little is known about how myosin VI is regulated and whether myosin VI has a function in the DNA damage response. Here, we found that myosin VI is regulated by DNA damage in a p53-dependent manner and possesses a novel function in the p53-dependent prosurvival pathway. Specifically, we show that myosin VI is induced by p53 and DNA damage in a p53-dependent manner. We found that p53 directly binds to, and activates, the promoter of the myosin VI gene. We also show that the intracellular localization of myosin VI is substantially altered by p53 and DNA damage in a p53-dependent manner such that the pool of myosin VI in endocytic vesicles, membrane ruffles, and cytosol migrates to the Golgi complex, perinuclear membrane, and nucleus. Furthermore, we show that knockdown of myosin VI attenuates activation of p53 and impairs Golgi complex integrity, which makes myosin VI-deficient cells susceptible to apoptosis upon DNA damage. Taken together, we found a novel function for p53 in the maintenance of Golgi complex integrity and for myosin VI in the p53-dependent prosurvival pathway.p53, a tumor suppressor protein, exerts its tumor suppression by regulating a plethora of p53 target genes (22). Based on their biological functions, these target genes can be classified into the following categories based on their products: cell cycle arrest, such as p21; apoptosis, such as Bax and Fas; DNA repair, such as XPB and DDB2; and antiangiogenesis and antimetastasis, such as VEGF and maspin. Induction of these genes is necessary for the host to repair or eliminate damaged cells and to prevent cellular transformation during exposure to genotoxic stresses (14,22,28,43). Interestingly, recent evidence also indicates that p53 up-regulates a group of target genes that are prosurvival, such as those coding for DDR1, COX2, and HB-EGF (16,20,34). Presumably, induction of these prosurvival genes is necessary for the tissue homeostasis once the stress signals are subsided (22).Myosins are the primary motor protein that use the energy derived from ATP to move cargos along actin filaments (11,18,23). Myosin VI, a member of the myosin superfamily, is an unconventional actin-based motor protein. One of the unique features of myosin VI is that myosin VI moves toward the minus end of actin filaments and therefore moves away from the plasma membrane into the cell and away from the surface of internal organelles such as the Golgi complex (11,18,23). Due to its localization in endocytic vesicles, membrane ruffles, cytosol, the Golgi complex, and perinuclear membrane, the motor activity of myosin VI is required for several physiological functions of the cell, such as protein secretion (9, 44), endocytosis (2,3,8,10,44), cell migration (19), spindle orientation (37), and spermat...