Am improved procedure for the isolation of chloroplast DNA fromChlamydomonas reinhardtii

The isolation of chloroplast DNA fromChlamydomonas reinhardtii requires the efficient separation of this AT-rich genome from the GC-rich nuclear genome by density-gradient centrifugation. We describe a simple and efficient method for separating these DNA fractions by using a sodium iodide gradient in combination with the DNA-binding dye, bisbenzimide. The yield of chloroplast DNA is close to the theoretical maximum and the DNA is suitable for restriction enzyme analysis and cloning. This method is applicable to the isolation of AT-rich plastid genomes from other organisms and may be appropriate as a general method for separating species of DNA that differ in their AT/GC ratios. An erratum to this article is available at .     

Characterization of two new facultative methanotrophs

Two new facultative methane-oxidizing bacteria have been isolated from lake water enrichments. The organisms have been characterized in terms of colony types, growth characteristics, the guanine plus cytosine content of their deoxyribonucleic acid, thin sections, oxidation rates, and carbon assimilation pathways. Methane-grown cells of both organisms contained intracytoplasmic membranes similar to those described as type II in other methanotrophic bacteria. Neither organism had such membranes when grown heterotrophically. Both organisms assimilated methane by way of the isocitrate lyase-negative serine pathway for formaldehyde incorporation. The enzymes of this pathway were high in specific activity in cells grown on methane and were at low levels in cells grown either on heterotrophic substrates or on heterotrophic substrates plus methane. It is proposed that both organisms be classified in the genus Methylobacterium as two new species, Methylobacterium ethanolicum and Methylobacterium hypolimneticum.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Exercise training in childhood-onset systemic lupus erythematosus: a controlled randomized trial

Introduction

Exercise training has emerged as a promising therapeutic strategy to counteract physical dysfunction in adult systemic lupus erythematosus. However, no longitudinal studies have evaluated the effects of an exercise training program in childhood-onset systemic lupus erythematosus (C-SLE) patients. The objective was to evaluate the safety and the efficacy of a supervised aerobic training program in improving the cardiorespiratory capacity in C-SLE patients.

Methods

Nineteen physically inactive C-SLE patients were randomly assigned into two groups: trained (TR, n = 10, supervised moderate-intensity aerobic exercise program) and non-trained (NT, n = 9). Gender-, body mass index (BMI)- and age-matched healthy children were recruited as controls (C, n = 10) for baseline (PRE) measurements only. C-SLE patients were assessed at PRE and after 12 weeks of training (POST). Main measurements included exercise tolerance and cardiorespiratory measurements in response to a maximal exercise (that is, peak VO₂, chronotropic reserve (CR), and the heart rate recovery (ΔHRR) (that is, the difference between HR at peak exercise and at both the first (ΔHRR1) and second (ΔHRR2) minutes of recovery after exercise).

Results

The C-SLE NT patients did not present changes in any of the cardiorespiratory parameters at POST (P > 0.05). In contrast, the exercise training program was effective in promoting significant increases in time-to-exhaustion (P = 0.01; ES = 1.07), peak speed (P = 0.01; ES = 1.08), peak VO₂ (P = 0.04; ES = 0.86), CR (P = 0.06; ES = 0.83), and in ΔHRR1 and ΔHRR2 (P = 0.003; ES = 1.29 and P = 0.0008; ES = 1.36, respectively) in the C-SLE TR when compared with the NT group. Moreover, cardiorespiratory parameters were comparable between C-SLE TR patients and C subjects after the exercise training intervention, as evidenced by the ANOVA analysis (P > 0.05, TR vs. C). SLEDAI-2K scores remained stable throughout the study.

Conclusion

A 3-month aerobic exercise training was safe and capable of ameliorating the cardiorespiratory capacity and the autonomic function in C-SLE patients.

Trial registration

NCT01515163.     

Survival Rate and Enzyme Activities ofLactobacillus acidophilusFollowing Frozen Storage

The ability of two strains of Lactobacillus acidophilus, CRL 640 and CRL 800, to survive and retain their biological activities under frozen storage was determined. Freezing and thawing, as well as frozen storage, damaged the cell membrane, rendering the microorganisms sensitive to sodium chloride and bile salts. Both lactic acid production and proteolytic activity were depressed after 21 days at -20 degreesC, whereas beta-galactosidase activity per cell unit was increased. Cell injury was partially overcome after repair in a salt-rich medium. Copyright 1998 Academic Press.     

Dynamic iodide trapping by tumor cells expressing the thyroidal sodium iodide symporter

The thyroidal sodium iodide symporter (NIS) in combination with various radioactive isotopes has shown promise as a therapeutic gene in various tumor models. Therapy depends on adequate retention of the isotope in the tumor. We hypothesized that in the absence of iodide organification, isotope trapping is a dynamic process either due to slow efflux or re-uptake of the isotope by cells expressing NIS. Iodide efflux is slower in ARH-77 and K-562 cells expressing NIS compared to a thyroid cell line. Isotope retention half times varied linearly with the number of cells expressing NIS. With sufficient NIS expression, iodide efflux is a zero-order process. Efflux kinetics in the presence or absence of perchlorate also supports the hypothesis that iodide re-uptake occurs and contributes to the retention of the isotope in tumor cells. Iodide organification was insignificant. In vivo studies in tumors composed of mixed cell populations confirmed these observations.     

Evaluation of live-culture-producing lacticin 3147 as a treatment for the control of Listeria monocytogenes on the surface of smear-ripened cheese

AIMS: A live Lactococcus lactis culture, producing the two-component broad spectrum bacteriocin lacticin 3147, was assessed for ability to inhibit the food pathogen Listeria monocytogenes on the surface of smear-ripened cheese. METHODS AND RESULTS: In initial experiments, the addition of Listeria to a lacticin 3147-containing fermentate produced with L. lactis DPC4275 (a transconjugant strain derived from L. lactis DPC3147) resulted in at least a 4 log reduction of the pathogen in 30 min. Two separate trials were performed in order to assess the most suitable method for application of the potential protective culture to smear-ripened cheese. In the initial trial, the L. lactis was sprayed onto the surface of the cheese either before or after Listeria was deliberately applied. Application of the culture following Listeria challenge, yielded up to a 1000-fold reduction of the pathogen in contrast to the pretreatment where Listeria numbers were unaffected. In a further trial, three applications of the live lacticin 3147-producing culture was used on a cheese surface containing Listeria. Listeria numbers were found to be up to 100-fold lower than in the cheese treated with L. lactis DPC4268 (control). CONCLUSION: While application of the live lacticin 3147 producer did not give complete elimination of the pathogen the results nonetheless demonstrate the potential of the bioprotectant for improving the safety of smear-ripened cheeses and particularly those that contain low level contamination with Listeria. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of lacticin 3147 as a live-culture can serve as a bioprotectant for the control of L. monocytogenes on the surface of smear-ripened cheese.     

The effect of copper supplementation on red blood cell oxidizability and plasma antioxidants in middle-aged healthy volunteers

A multicenter European study (FoodCue) was undertaken to provide data on the significance of increased dietary copper as a pro-oxidant or antioxidant in vivo. The present work describes the effect of Cu supplementation on (2,2'-azo-bis(2-amidinopropane) hydrochloride (AAPH)-induced red blood cell oxidation in middle-aged people. Double-blinded copper supplementation was achieved in 26 healthy volunteers (50-70 years) with pills containing 3 mg CuSO(4), 3 mg Cu glycine chelate (CuG) and 6 mg CuG. Each 6 week supplementation period was preceded and followed by 6 weeks of washout (WO) on placebo. The results show significant increases in time necessary to achieve 50% hemolysis (LT(50)) after 3CuSO(4) and 6CuG compared with values after WO periods. Cu supplementation did not increase the levels of (Cu,Zn)SOD activity in red blood cells. Resistance to hemolysis was significantly and positively correlated (r =.30, p <.01) with alpha- and beta-carotene content in the plasma. Together, these data suggest that intake of copper as high as 7 mg/d has no pro-oxidant activity and may rather result in protection of red blood cells against oxidation. The decreased oxidizability of red blood cells did not result from increased (Cu,Zn)SOD activity and may occur through other mechanisms such as changes in membrane antioxidant content.