Production of cloned mice by nuclear transfer of cumulus cells

Over the past several years, mammals have been successfully cloned by either the splitting of an early stage embryo or nuclear transfer of adult somatic cells [NT] into oocytes. Although it has been 15 years since the generation of the first cloned mammals from somatic cells by NT, the success rate for producing live offspring by this technique is low regardless of the cell type and animal species used. However, these techniques have the potential to be important tools for future research in basic biology. In the present study, we described our experiences in producing successfully cloned mouse using NT method and piezo-actuated micromanipulator. B6D2F1 mice, 8-12 weeks old, were superovulated with injections of 5 IU of pregnant mare serum gonadotropin and 5 IU of human chorionic gonadotropin administered 48 hr apart. Enucleation and donor nuclei cumulus cell injection were performed with a piezo-actuated micromanipulator after which activation and trichostatin A treatment were used for reconstructed oocytes. Two-cell stage cloned embryos that developed in the mWM medium were transferred into the oviducts of pseudopregnant NMRI mice. Of 367 oocytes collected, 131 [69%] developed into 2-cell stage embryos. Of these, 5 [1%] live pups were successfully delivered. We used NMRI foster mother to raise the pups by lactation. One adult cloned mouse was mated, after which she delivered and raised normal offspring. For mouse cloning, the present study also successfully tested the capability of somatic cell nuclear transfer SCNT using a piezo unit

Enhanced cytochrome C oxidase activity in WBC of COPD patients

Chronic obstructive pulmonary disease [COPD] is characterized by decreased expiratory flow rates, increased pulmonary resistance and hyperinflation. Cytochrome C Oxidase [COX] as a key oxidative enzyme modulates oxygen uptake and catalyzes the oxidation of reduced cytochrome C by molecular oxygen. In vitro studies indicate that the activity of COX can be directly regulated by the presence of molecular oxygen. Thus, a better understanding of the role of COX in patients with COPD can provide an important link between the availability of oxygen to tissues and the regulation of oxygen uptake and energy production in these patients. We studied 42 COPD patients [36 males, 6 females] with clinically stable conditions and 50 [42 males, 8 females] healthy sedentary volunteers of similar age. Whole blood was collected by venipuncture in sodium citrate tubes and WBCs were separated by Ficoll according to standard protocol and lysed with microtube pestle homogenizer. The homogenates were centrifuged and the supernatants were used as a cell extract for COX activity determination. Aliquots of this were assayed for total protein content and COX activity. Analysis of COX activity was performed using COX assay kit. Absolute specific COX activity was normalized for total protein. Relative activities were determined by dividing absolute specific COX activity on absolute specific citrate synthase activity. Mitochondrial COX activity and specific activity [absolute and relative] significantly increased in WBCs of patients with COPD in comparison with control samples [p< 0.05] These results indicated that the activity of COX was increased in WBCs of patients with COPD but whether this is a primary or secondary change relevant to hypoxic condition in these patients is not clear and needs further investigation.

Alteration in antioxidant capacity in patients with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease [COPD] is a major public health problem that needs greater attention. Variability in the susceptibility to develop COPD is related to both genetic and environmental factors. Oxidative stress and inflammation are the major hallmarks of COPD and antioxidant status can be used as a biomarker to assess the risk of chronic diseases. We used the FRAP [ferric reducing ability of plasma] assay as a simple and powerful test for determination of the total antioxidant capacity of plasma of patients and normal subjects. The patients were selected by cross-sectional method. The mean average age +/- SD of normal subjects and patients was 56 +/- 4 and 60 +/- 2 years respectively. The spectrophotometeric method was used for this assay. The means of the FRAP assays in the patients were higher [about twice] than those of normal subjects. The differences were significant [p < 0.01]. The high levels of antioxidant capacity in the patient group indicated that the antioxidant defense system had been activated due to the oxidative stress and hypoxic condition. A though, FRAP assay can probably be used for demarcation of severity and risk of developing COPD, clinical follow-up and further investigation are required for the assessment of this hypothesis