Two proteolytic pathways regulate DNA repair by cotargeting the Mgt1 alkylguanine transferase

O^6-methylguanine (O^6meG) and related modifications of guanine in double-stranded DNA are functionally severe lesions that can be produced by many alkylating agents, including N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a potent carcinogen. O^6meG is repaired through its demethylation by the O^6-alkylguanine-DNA alkyltransferase (AGT). This protein is called Mgmt (or MGMT) in mammals and Mgt1 in the yeast Saccharomyces cerevisiae. AGT proteins remove methyl and other alkyl groups from an alkylated O^6 in guanine by transferring the adduct to an active-site cysteine residue. The resulting S-alkyl-Cys of AGT is not restored back to Cys, so repair proteins of this kind can act only once. We report here that S. cerevisiae Mgt1 is cotargeted for degradation, through a degron near its N terminus, by 2 ubiquitin-mediated proteolytic systems, the Ubr1/Rad6-dependent N-end rule pathway and the Ufd4/Ubc4-dependent ubiquitin fusion degradation (UFD) pathway. The cotargeting of Mgt1 by these pathways is synergistic, in that it increases not only the yield of polyubiquitylated Mgt1, but also the processivity of polyubiquitylation. The N-end rule and UFD pathways comediate both the constitutive and MNNG-accelerated degradation of Mgt1. Yeast cells lacking the Ubr1 and Ufd4 ubiquitin ligases were hyperresistant to MNNG but hypersensitive to the toxicity of overexpressed Mgt1. We consider ramifications of this discovery for the control of DNA repair and mechanisms of substrate targeting by the ubiquitin system

Peer role-play and standardised patients in communication training: a comparative study on the student perspective on acceptability, realism, and perceived effect

<p>Abstract</p> <p>Background</p> <p>To assess the student perspective on acceptability, realism, and perceived effect of communication training with peer role play (RP) and standardised patients (SP).</p> <p>Methods</p> <p>69 prefinal year students from a large German medical faculty were randomly assigned to one of two groups receiving communication training with RP (N = 34) or SP (N = 35) in the course of their paediatric rotation. In both groups, training addressed major medical and communication problems encountered in the exploration and counselling of parents of sick children. Acceptability and realism of the training as well as perceived effects and applicability for future parent-physician encounters were assessed using six-point Likert scales.</p> <p>Results</p> <p>Both forms of training were highly accepted (RP 5.32 ± .41, SP 5.51 ± .44, n.s.; 6 = very good, 1 = very poor) and perceived to be highly realistic (RP 5.60 ± .38, SP 5.53 ± .36, n.s.; 6 = highly realistic, 1 = unrealistic). Regarding perceived effects, participation was seen to be significantly more worthwhile in the SP group (RP 5.17 ± .37, SP 5.50 ± .43; p < .003; 6 = totally agree, 1 = don't agree at all). Both training methods were perceived as useful for training communication skills (RP 5.01 ± .68, SP 5.34 ± .47; 6 = totally agree; 1 = don't agree at all) and were considered to be moderately applicable for future parent-physician encounters (RP 4.29 ± 1.08, SP 5.00 ± .89; 6 = well prepared, 1 = unprepared), with usefulness and applicability both being rated higher in the SP group (p < .032 and p < .009).</p> <p>Conclusions</p> <p>RP and SP represent comparably valuable tools for the training of specific communication skills from the student perspective. Both provide highly realistic training scenarios and warrant inclusion in medical curricula. Given the expense of SP, deciding which method to employ should be carefully weighed up. From the perspective of the students in our study, SP were seen as a more useful and more applicable tool than RP. We discuss the potential of RP to foster a greater empathic appreciation of the patient perspective.</p

DNA glycosylases: in DNA repair and beyond

The base excision repair machinery protects DNA in cells from the damaging effects of oxidation, alkylation, and deamination; it is specialized to fix single-base damage in the form of small chemical modifications. Base modifications can be mutagenic and/or cytotoxic, depending on how they interfere with the template function of the DNA during replication and transcription. DNA glycosylases play a key role in the elimination of such DNA lesions; they recognize and excise damaged bases, thereby initiating a repair process that restores the regular DNA structure with high accuracy. All glycosylases share a common mode of action for damage recognition; they flip bases out of the DNA helix into a selective active site pocket, the architecture of which permits a sensitive detection of even minor base irregularities. Within the past few years, it has become clear that nature has exploited this ability to read the chemical structure of DNA bases for purposes other than canonical DNA repair. DNA glycosylases have been brought into context with molecular processes relating to innate and adaptive immunity as well as to the control of DNA methylation and epigenetic stability. Here, we summarize the key structural and mechanistic features of DNA glycosylases with a special focus on the mammalian enzymes, and then review the evidence for the newly emerging biological functions beyond the protection of genome integrity

Inhibition of Stearoyl-CoA Desaturase 1 (SCD1) Dissociates Insulin Resistance and Obesity From Atherosclerosis

Background—Stearoyl-CoA Desaturase 1 (SCD1) is a well-known enhancer of the metabolic syndrome. The purpose of the present study was to investigate the role of SCD1 in lipoprotein metabolism and atherosclerosis progression. Methods and Results—Antisense oligonucleotides were used to inhibit SCD1 in a mouse model of hyperlipidemia and atherosclerosis (LDLr-/-Apob100/100). In agreement with previous reports, inhibition of SCD1 protected against diet-induced obesity, insulin resistance, and hepatic steatosis. However, unexpectedly SCD1 inhibition strongly promoted aortic atherosclerosis, which could not be reversed by dietary oleate. Further analyses revealed that SCD1 inhibition promoted accumulation of saturated fatty acids in plasma and tissues, reduced plasma triglyceride, yet had little impact on LDL cholesterol. Since dietary SFAs have been shown to promote inflammation through toll-like receptor 4 (TLR4), we examined macrophage TLR4 function. Interestingly, SCD1 inhibition resulted in alterations in macrophage membrane lipid composition and marked hypersensitivity to TLR4 agonists. Conclusions—This study demonstrates that atherosclerosis can occur independently of obesity and insulin resistance, and argues against SCD1 inhibition as a safe therapeutic target for the metabolic syndrome