Survival, mutagenesis, and host cell reactivation in a Chinese hamster ovary cell ERCC1 knock-out mutant

Positive selection-negative selection gene targeting was usedto disrupt the nucleotide excision repair gene ERCC1 in a Chinesehamster ovary cell line, CHO-K1. Southern and Northern analysisshowed that a cell clone isolated by this targeting approach,CHO-7-27, had an ERCC1 gene structure consistent with targeteddisruption of ERCC1 exon V, and did not express ERCC1 mRNA.CHO-7-27 was further characterized with respect to UV and mitomycinC sensitivities, and was shown to exhibit severe mutagen sensitivityphenotypes consistent with those of other CHO cell ERCC1 mutants.Mutation frequency experiments showed that CHO-7-27 was UV-hypermutableat the hypoxanthine-guanine phosphoribosyltransferase locus.Experiments assessing host cell reactivation of viral DNA synthesisfor UV-irradiated adenovirus showed that CHO7-27 exhibited aseverely deficient HCR phenotype similar to that of UV20 cells.Our results demonstrate that CHOK1 cells are hemizygous forthe ERCC1 gene, and show that the comparatively mild mutagensensitivities and lack of severely deficient HCR phenotypesof conventionally derived CHO-K1 ERCC1 mutants, in contrastto the severe phenotypes of CHO-AA8-derived mutants, are notdue to any intrinsic genetic differences between CHO-K1 andCHO-AA8 parental cell lines. ⁴To whom correspondence should be addressed     

The induction of operational tolerance is not prevented by simultaneous administration of cyclosporin A1

In this study, the effect of combining anti-CD4 monoclonal antibody (mAb) and cyclosporin (CyA) therapy at the time of transplantation was examined. A mouse cardiac allograft model was used. Anti-CD4 mAb administered perioperatively induces long-term survival. The addition of a short course of CyA given subcutaneously in a regimen of either a high-dose treatment or a standard dose treatment to the anti-CD4 mAb treatment protocol did not have a detrimental effect on graft survival. Despite having no significant effect on graft survival, the addition of CyA to the treatment protocol did result in a significant decrease in the level of IL-2 present in the hearts 7 days after transplantation. The decrease in IL-2 production was directly related to the presence of CyA in vivo. When CyA treatment was continued throughout the period during which unresponsiveness to the graft is induced by anti-CD4 mAb therapy, 50 % of the grafted hearts were rejected once the CyA was discontinued. In conclusion, the combined use of anti-CD4 mAb therapy and CyA did not have a negative effect on graft survival in this model when the two agents were used concurrently at the time of transplantation. Received: 2 October 1996 Received after revision: 31 January 1997 Accepted: 5 February 1997     

The Effect of High-Dose Ascorbate Supplementation on Plasma Lipoprotein(a) Levels in Patients With Premature Coronary Heart Disease

Study Objective . To determine the efficacy of high-dose ascorbate supplementation in lowering lipoprotein(a) [Lp(a)] levels in patients with premature coronary heart disease (CHD). Design . Randomized, double-blind, placebo-controlled trial. Setting . Outpatient clinic. Patients . Forty-four patients with documented premature CHD. defined as confirmed myocardial infarction and/or angiographically determined stenosis of 50% or greater in at least one major coronary artery before age 60 years. Interventions . Patients were block randomized on the basis of age, gender, and screening Lp(a) concentrations to receive ascorbate 4.5 g/day or placebo for 12 weeks. Measurements and Main Results . High-dose ascorbate was well tolerated and produced a marked elevation in mean plasma ascorbate levels (+1.2 mg/dl; p<0.001). Multiple linear regression analysis revealed no significant effect of supplementation on postintervention Lp(a) levels (p=0.39) in a model that included treatment group assignment, and baseline Lp(a) levels. Conclusions . Our findings do not support a clinically important lowering effect of high-dose ascorbate on plasma Lp(a) in patients with premature CHD.     

Mixed h2 and h∞ optimal robust control design

This paper introduces a design methodology for a dynamic compensator that simultaneously minimizes the upper bound of a quadratic performance index and the H_∞-norm of a disturbance transfer function matrix of a multiple-input/multiple-output system whose model contains parameter uncertainty in the state and input matrices. The real parameter uncertainty is modelled as additional measurement outputs and as additional weights on the existing noise inputs and measurement outputs of the system. The compensator equations are derived by taking the dual of a system with parameter variation in the state and output matrices, for which the compensator equations have previously been derived, and then taking the dual of the compensator equations. An algorithm for applying this theory is given and an example is shown.     

Pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate concentrations in blood and tissues of mice fed ethanol-containing liquid diets

The effects of chronic ethanol administration on vitamin B-6 metabolism were studied in female Long-Sleep (LS) and Short-Sleep (SS) mice. Animals were fed an ethanol containing liquid diet (AIN-76) for four weeks. Concentration of ethanol in the diet increased from 10 to 25% ethanol-derived calories (EDC) during weeks 1-3 and was maintained at 30% EDC for 1 additional week. We measured concentrations of pyridoxal 5'-phosphate (PLP) in plasma, erythrocytes and whole blood, and liver and brain PLP and pyridoxamine 5'-phosphate (PMP) in ethanol-fed and pair-fed control mice. Chronic ethanol administration significantly increased PMP and total (PLP + PMP) levels in the liver of SS mice. In LS mice ethanol feeding significantly decreased PMP and total (PLP + PMP) levels in the brain, but these values were still within normal limits. These results suggest that both control and ethanol-containing liquid diets are nutritionally adequate with respect to vitamin B-6, and that chronic ethanol administration does not adversely affect vitamin B-6 metabolism in adult mice.     

Are Continuity Clinic Patients Less Satisfied When the Resident Is Postcall?

Due to recent public debate and newly imposed resident work hour restrictions, we decided to investigate the relationship of resident call status to their ambulatory patients' satisfaction. Resident continuity clinic patients were asked to rate their level of satisfaction on a 10-point Likert-type scale. Using multiple regression approaches, these data were then assessed as a function of resident call status. We found that in 646 patient encounters, patient satisfaction scores were significantly less when the resident was postcall, 8.99 ± 1.8, than when not postcall, 9.31 ± 1.3. We herein discuss etiologies and implications of these findings for both patient care and medical education.     

Use of suture-mediated vascular closure devices for the management of femoral vein access after transcatheter procedures.

Groin complications remain the most common complication of cardiac catheterization procedures. While the use of closure devices is increasing for arterial sheaths, venous sheaths tend to be removed and hemostasis achieved with manual compression. We report our experience using Perclose suture-mediated vascular closure device to achieve hemostasis and early mobility in patients who have had venous access as part of their procedure. There were a total of 42 patients (21 males; average age, 63.5 years) studied. The majority of the patients had 7 Fr sheaths (24), with access sites of sheaths up to 14 Fr being closed with this technique. Two patients developed complications at the access site: one patient requiring rehospitalization for intravenous antibiotics because of late access site infection, and one patient with deep venous thrombosis and pulmonary emboli. We conclude that the use of the Perclose suture-mediated closure device for closure of femoral venous access sites is feasible and should be considered especially in patients with larger venous sheaths and those at increased risk of groin complications.