Genetic analysis of the yeast NUD1 endo-exonuclease: a role in the repair of DNA double-strand breaks

The deoxyribonucleases (DNases) have been shown genetically to be important in the vital processes of DNA repair and recombination. The NUD1 gene, which codes for an endo-exonuclease of Saccharomyces cerevisiae, was analyzed for its role in the DNA double-strand break (DSB) repair processes. While the nud1 strain is only slightly sensitive to ionizing radiation, expression of the HO-endonuclease to introduce a DSB at the MAT locus in that strain results in cell death. Cell survival is inversely proportional to the duration of HO-endonuclease expression. Analysis of the surviving colonies from the nud1 strain indicated that many of the survivors are sterile and that the proportion of these sterile survivors increases with the time of HO-endonuclease expression. On the other hand, the surviving colonies from the isogenic NUD1 strain are mating-proficient. Interestingly, double mutants of nud1 rad52 are more resistant to ionizing irradiation than the rad52 strain and have a cell-survival fraction of 32% for rad52-1 nud1 and 9% for rad52::URA3 nud1 following prolonged HO-endonuclease expression, indicating that nud1 has a suppressor effect on the DSB-induced lethality in rad52. Polymerase chain reaction analysis showed that many of the nud1 survivors contained small alterations within the MAT locus, suggesting that the survivors arose through the process of non-homologous end-joining. These results suggest that the endo-exonuclease acts at a DSB to promote DNA repair via the homologous recombination pathway. Received: 20 July / 20 September 1998     

Acetate metabolism and bicarbonate generation during hemodialysis: 10 years of observation

The capacity of chronically hemodialyzed patients to metabolize acetate during conventional hemodialysis was evaluated using a retrospective study in 219 patients dialyzed for up to ten years under similar dialysis conditions. For each patient, and using all available data, a regression line relating the changes of plasma total CO2 during dialysis as a function of the pre-dialysis value was calculated. The intercept of this function indicates the plasma concentration where the losses of bicarbonate in the dialysate is matched by the generation of bicarbonate arising from the metabolism of acetate. This value therefore represents an individual index of the capacity of each patient to metabolize acetate. A value for this index smaller than 18.0 mM was considered abnormal. It was shown that around 10% of chronically hemodialyzed patients are clearly unable to metabolize acetate optimally. This defect is not related to the duration of dialysis, body weight or quality of hemodialysis treatments but is strongly related to sex, 19 of the 22 "acetate intolerant" patients being women. In a prospective study, all the 60 patients of the same population undergoing active dialysis were studied, and this index identified 12 abnormal (11 women, 1 man) patients and 48 normal patients. Plasma acetate measured at the end their dialysis treatments were significantly higher in abnormal than in normal patients. It is concluded: that this index is useful to identify the patients unable to metabolize acetate optimally; that only around 10% of hemodialyzed patients present a severe problem when dialyzed against acetate and should be dialyzed against bicarbonate; that dialysis against acetate does not fully correct the metabolic acidosis even in "normal" patients.     

Acute Alcohol Use and Injury Patterns in Young Adult Prehospital Patients

The objective was to determine if acute alcohol consumption is associated with differences in injury pattern among young adult patients with traumatic injuries presenting to emergency medical services (EMS). A cross-sectional, retrospective review of prehospital patient care reports (PCRs) was conducted evaluating injured patients who presented to a collegiate EMS agency from January 1, 2011 to December 31, 2012. Included patients were age 18–24 y and sustained an injury within the previous 24 h. PCRs were reviewed independently by two abstractors to determine if the patient was documented to have acutely consumed alcohol proximate to his/her injury. Primary and secondary sites of regional body injury were recorded. Injury severity was recorded using the Revised Trauma Score (RTS). The association between primary injury site and acute alcohol use was assessed using a chi-square test. Multiple logistic regression was used to control for sex in predicting injury type. Of 440 injured patients, 135 (30.6%) had documented alcohol use prior to injury. Acute alcohol consumption altered the overall pattern of regional injury (p < 0.001). Alcohol users were more likely to present with injury secondary to assault, fall/trip, and unknown mechanism of injury (p < 0.001, all comparisons). RTS scores were statistically lower in the alcohol group (p < 0.001), although the clinical significance of this is unclear. Controlling for sex, acute alcohol consumption predicted increased risk of head/neck injury 5.59-fold (p < 0.001). Acute alcohol use in collegiate EMS patients appears to alter injury patterns in young adults and increases risk of head/neck injury. EMS providers in similar agencies should consider these trends when assessing and treating injured college-aged patients.     

Coexpression of rat glutathione S-transferase A3 and human cytidine deaminase by a bicistronic retroviral vector confers in vitro resistance to nitrogen mustards and cytosine arabinoside in murine fibroblasts

The transfer of drug resistance genes into hematopoietic cells is an experimental approach to protect patients from drug-induced myelosuppression. Because anti-cancer drugs are often administered in combination to increase their clinical efficacy, vectors that express two drug resistance genes are being developed to broaden the spectrum of chemoprotection. We have constructed a bicistronic vector, MFG/GST-IRES-CD (MFG/GIC) coexpressing rat glutathione S-transferase (GST) A3 isoform (rGST Yc1) and human cytidine deaminase (CD). Murine NIH 3T3 fibroblast cells transduced with this vector were evaluated for their resistance to nitrogen mustards and cytosine nucleoside analogs. GIC-transduced polyclonal cell populations (GIC cells) demonstrated marked increases in selenium-independent glutathione peroxidase (peroxidase) and CD activities, as well as increased resistance to melphalan (2.3-fold), chlorambucil (3.4-fold), and cytosine arabinoside (Ara-C) (8.1-fold). After selection with Ara-C, the peroxidase and CD activities of GIC cells were augmented 2.6- and 2.9-fold, respectively, in comparison with unselected cells, and the resistance to melphalan, chlorambucil, and Ara-C was further increased to 3.7-, 5.9-, and 53-fold, respectively. Melphalan selection of GIC cells likewise augmented their peroxidase (2.3-fold) and CD (1.9-fold) activities. GIC cells proliferated in the simultaneous presence of melphalan and Ara-C at drug concentrations that completely inhibited the growth of untransduced cells. The growth rate of unselected GIC cells exposed to the drug combination averaged 18% that of drug-free cultures. The growth rate of GIC cells exposed to the drug combination increased to 30% of controls after Ara-C selection and to 50% after melphalan selection. Our results suggest that retroviral transfer of MFG/GIC may be useful for chemoprotection against the toxicities of nitrogen mustards and cytosine nucleoside analogs.     

A prospective, randomized trial comparing a transparent dressing and a dry gauze on the exit site of long term central venous catheters of hemodialysis patients

The objective of this study was to assess the risk of bacteremia, estimate the cost and evaluate the quality of life by using a transparent dressing (TD) versus (vs) a dry gauze (DG) on the exit site of long term central I.V. catheters (LTCC) of hemodialysis patients. This 6-months preliminary study was conducted on 58 patients (pts) randomized to receive DG replaced 3 times/week (29 pts) or TD replaced every 7 days (29 pts). Data on patients, conditions of the exit site, local infection, bacteremia, quality of life and cost related to each type of dressing were collected. Two pts in the DG group experienced bacteremia related to their LTCC vs 1 pt in the group TD. A total of 7 (DG) vs 13 (TD) pts experienced skin condition changes at the catheter exit site. Some skin reactions, erythema and pruritus, did occur initially in the group TD and was due in part to insufficient drying time of the skin preparation solution. The estimated individual, weekly costs for using the DG was $7.60 vs $4.72 Canadian dollars for the TD. The SF-36trade mark scores did not show a significant difference between the 2 groups during the study (3.8 (PCS), 6.4 (MCS) at study end). Although this study was statistically underpowered, it suggests that the incidence of bacteremia was not increased with the use of a TD. Moreover, the use of a TD allowed fewer dressing changes, lowered total treatment costs, with no observed unfavorable impact on the quality of life and without significant local complications of the exit site. Based on the positive results observed in this pilot study, further study is warranted to examine the cost effectiveness of long-term use of TD dressings on dialysis catheter exit sites.     

Rapid response of a disorder to the addition of lithium carbonate: panic resistant to tricyclic antidepressants

There is evidence that panic disorder and major depression might share some common neurobiological factors. This would be consistent with the fact that most antidepressant drugs are effective in preventing panic attacks. This is a case report of a 40 year old woman who was suffering from a panic disorder. Following the discontinuation of a long-term lorazepam treatment, she developed severe depressive symptoms. The depressive syndrome improved rapidly with amitriptyline (150 mg/day), but the panic attacks persisted. Twelve weeks later, amitriptyline was replaced by clomipramine (150 mg/day), the dosage of which was increased to 225 mg/day three weeks later. The patient remained anxious with no resolution of her panic attacks. Two weeks later, lithium carbonate (900 mg/day) was added to clomipramine. Sixty hours later, a dramatic improvement was observed. The lithium plasma level was 0.8 mEq/L. Because of some tremors, lithium was discontinued five days later. Within four days, the anxiety as well as the panic attacks reappeared. Lithium carbonate (600 mg/day) was then resumed. Forty-eight hours later, the patient was showing a clinical improvement similar to that observed following the first addition of lithium. She remained symptom free with the maintenance of the drug combination. To date, several reports have confirmed the beneficial effect of adding lithium to a tricyclic antidepressant drug in resistant major depression. However, we believe that the present case report is the first one documenting the augmentation of a tricyclic antidepressant drug by lithium in a patient suffering from a panic disorder.     

Antibiotic and metal resistance among hospital and outdoor strains of Pseudomonas aeruginosa

Phenotypic analyses of antibiotic and metal resistance of a collection of 130 strains of Pseudomonas aeruginosa from various outdoor (i.e. soil, water, animals) and hospital (environment, patients, individuals with cystic fibrosis) settings were performed. Resistance was scored according to the origin of the strains and their likely exposure to antibiotics and chemicals. Most of the 76 outdoor strains showed a wild-type antibiotic resistance phenotype, i.e. resistance to minocycline and trimethoprim–sulfamethoxazole. Sixty percent of hospital strains showed a multiresistance phenotype (from 3 to 16 antibiotics) and confirmed that frequent exposure to antibiotics favored selection and maintenance of antibiotic resistance in P. aeruginosa. Twelve percent of outdoor strains naturally exposed to antiseptics and hydrocarbons showed significant resistance profiles, suggesting that chemical contaminants could contribute to selection of antibiotic resistance. For metal resistance, outdoor strains were more frequently resistant to zinc and cadmium, whereas hospital strains were more frequently resistant to mercury and copper. Differences in metal resistance between the 130 strains investigated were not related to previously characterized processes such as those implicating czcA, involved in cadmium, zinc, and cobalt resistance, or copA and copB, involved in copper resistance. Regulatory or new processes were likely to have contributed to the observed variations. Strains showing strong resistance to antibiotics were the least resistant to metals, and inversely. The lack of significant correlations between antibiotic and metal resistance suggests involvement of distinct processes that are rarely co-selected. The effects of the P. aeruginosa collection size and multi-factorial selective pressure on data sets are discussed.     

Valproic acid enhances protein l-isoaspartyl methyltransferase expression by stimulating extracellular signal-regulated kinase signaling pathway

Proteins are susceptible to various non-enzymatic post-translational modifications occurring during aging and in certain pathological states. The protein l-isoaspartyl methyltransferase (PIMT) is an enzyme that recognizes and repairs the abnormal l-isoaspartyl residues in proteins. Recently, we reported that PIMT expression was stimulated by the anti-epileptic drug valproic acid and that this was mediated through the glycogen synthase kinase-3 (GSK-3)/β-catenin pathway. In this study, to gain further insights into which of the signaling pathways activated by valproic acid regulate PIMT abundance, astrocytoma U-87 MG and neuroblastoma SH-SY5Y cells were treated with this drug to investigate the possible involvement of the extracellular-regulated kinase (ERK) pathway in PIMT induction. Valproic acid increased ERK1/2 phosphorylation on Thr202/Tyr204 and Thr185/Tyr187, respectively. Pharmacological inhibitors against the kinases Src, c-Raf, MEK1/2 and ERK1/2 abolished the ERK1/2 phosphorylation stimulated by valproic acid, thus preventing PIMT induction by the drug. Furthermore, MEK1/2 inhibition with U0126 blocked the higher phosphorylation of RSK-1 on Thr359/Ser363 and of GSK-3β on Ser9 as well as the increased expression of RSK-1, β-catenin and PIMT upon treatment with valproic acid. RSK-1 knockdown by interfering RNA abrogated the increased expression of RSK-1, β-catenin and PIMT as well as the induced phosphorylation of RSK-1 and GSK-3β due to valproic acid. Thus, our findings demonstrated that PIMT up-regulation by valproic acid required the activation of the ERK signaling pathway including RSK-1 the latter being responsible for inactivating GSK-3 and subsequently leading to β-catenin stabilization.