Continuous maintenance of transformed fibroblasts under reduced serum conditions: utility as a model system for investigating growth factor-specific effects in nonquiescent cells

We report the continuous growth maintenance of untransformed and chemically transformed fibroblasts (AKR-2B, AKR-MCA cells) in low concentrations of serum (0.1% FBS). The cell lines established (AKR-0.1F, MCA-0.1F) proliferated at rates comparable to cells maintained under high serum conditions (10% FBS). Complete removal of serum from the cells did not induce quiescence. The MCA-0.1F cells were more similar to the untransformed AKR-2B fibroblasts in their morphology, saturation density, inability to form colonies under anchorage-independent conditions, steady-state level of c-myc expression, and kinetics of induction of c-myc in response to specific growth factors. This report demonstrates the utility of this cell line as a nonquiescent model system for investigating growth factor-specific effects in serum-free, cycling cells. Addition of transforming growth factor-beta (TGF-beta) (5 ng/ml) to proliferating MCA-0.1F cells, in the absence of any serum, induced a multilayered growth pattern at confluency, similar to that of AKR-MCA cells maintained in 10% FBS. Other growth factors tested did not elicit this effect. The induction of this growth pattern by TGF-beta was associated with a sustained induction of the c-myc proto-oncogene at confluency, but not with a restoration of anchorage-independent growth. The data suggest that TGF-beta may play a role in the up-regulation of c-myc at confluency previously described for AKR-MCA cells maintained in 10% serum.     

The Microflora of Liquid Whole Egg Made from Incubator Reject Eggs

S ummary . The microflora of liquid whole egg made by centrifuging incubator reject shell eggs was investigated by examining a selection of colonies growing on different selective culture media. As a result of the method of manufacture micro-organisms were present and of the 200 isolates characterized, most belonged to the Bacillaceae, Enterobacteriaceae, Lactobacillaceae, Micrococcaceae and Pseudomonadaceae. A pasteurization treatment showed the presence of a thermoresistant flora. Some representatives of the Bacillaceae and the Lactobacillaceae showed resistance to the heat treatment given.     

Dosage effect of a gene with a regulating effect on anthocyanin synthesis in a trisomic Petunia hybrida

A Petunia hybrida inbred line (W 28) has white flowers with red spots on the corolla. These spots are the result of back mutations of an unstable allele of the gene Anl for anthocyanin synthesis. Among the progeny of a population of selfed plants a primary trisomic with red-spotted white flowers was found. The reversion frequency was more than twice as high as compared with disomic plants of the same family.It was found that the chromosome in triplicate was not the chromosome on which the gene Anl is localized. It can be concluded that there is an independently segregating factor which influences the frequency of back mutations of the Anl locus. Twin spots were found among the flowers of the trisomic. They consisted of two adjacent sectors, one with a spot frequency equal to that of the flowers of disomic plants, and the other with a spot frequency more than twice as high as that of the trisomic. Probably an irregular distribution of the extra chromosome resulted in one sector with the normal diploid number of chromosomes, and an adjacent sector with two extra chromosomes. The reversion frequencies in the sector suggest that the factor which affects the reversion frequency of the unstable alleles of Anl exhibits a dosage effect.     

A medium for the detection of bacteria causing green discolouration of cooked cured meat products

Summary A cooked haemoglobin medium was developed for the detection of hydrogen-peroxide-producing bacteria. The composition and preparation of this medium are described. The medium has proved to be an adequate medium for diagnosis of the greening of cooked cured meat products caused by hydrogen-peroxide-producing lactic acid bacteria. Offprint requests to: F. K. Stekelenburg     

Influence of macrolide maintenance therapy and bacterial colonisation on exacerbation frequency and progression of COPD (COLUMBUS): Study protocol for a randomised controlled trial

ABSTRACT: BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterised by progressive development of airflow limitation that is poorly reversible. Because of a poor understanding of COPD pathogenesis, treatment is mostly symptomatic and new therapeutic strategies are limited. There is a direct relationship between the severity of the disease and the intensity of the inflammatory response. Besides smoking, one of the hypotheses for the persistent airway inflammation is the presence of recurrent infections. Macrolide antibiotics have bacteriostatic as well as anti-inflammatory properties in patients with cystic fibrosis and other inflammatory pulmonary diseases. There is consistent evidence that macrolide therapy reduces infectious exacerbations, decreases the requirement for additional antibiotics and improves nutritional measures. Because of these positive effects we hypothesised that maintenance macrolide therapy may also have beneficial effects in patients with COPD who have recurrent exacerbations. The effects on development of bacterial resistance to macrolides due to this long-term treatment are unknown. Until now, studies investigating macrolide therapy in COPD are limited. The objective of this study is to assess whether maintenance treatment with macrolide antibiotics in COPD patients with three or more exacerbations in the previous year decreases the exacerbation rate in the year of treatment and to establish microbial resistance due to the long-term treatment. Methods/design The study is set up as a prospective randomised double-blind placebo-controlled single-centre trial. A total of 92 patients with COPD who have had at least three exacerbations of COPD in the previous year will be included. Subjects will be randomised to receive either azithromycin 500 mg three times a week or placebo. Our primary endpoint is the reduction in the number of exacerbations of COPD in the year of treatment. DISCUSSION: We investigate whether long-term therapy with macrolide antibiotics can prevent exacerbations in patients with COPD. Additionally, our study aims to assess the effect of long-term use of macrolide on the development of antimicrobial resistance and on inflammatory parameters related to COPD. We believe this study will provide more data on the effects of macrolide treatment in patients in COPD and will add more knowledge on its working mechanisms. Trial registration www.clinicaltrials.gov NCT00985244.     

Radiation inactivation ofSalmonella panama andEscherichia coli K 12 present on deep-frozen broiler carcasses

Summary Low doses of ionizing radiation have been used to extend the shelf life of refrigerated poultry carcasses and to reduce the numbers of Salmonellae present. This report gives results of experiments on irradiation of deep-frozen poultry carcasses which were, before freezing, artificially contaminated withSalmonella panama and with a nalidixic acid-resistantEscherichia coli K 12. The D-values (decimal reduction) obtained with the inoculated carcasses were compared with D-values obtained with carcasses which were slaughtered in the normal way.The D-values forS.panama and forE.coli K 12 were 64.9 krad and 55.9 krad in the dripwater. Under commercial conditions approximately 100 krad were required for one decimal reduction of the Enterobacteriaceae present.The D-values estimated on the skin were higher forS.panama than forE. coli K 12 (128.6 krad vs 57.6 krad). If it is assumed that 1 positive carcass in 10,000 is allowed, the deep-frozen carcasses should be irradiated with doses of at least 700 krad to be sure of the absence of the testedS. panama strain.     

On the mechanism of inhibition of methanol dehydrogenase by cyclopropane-derived inhibitors

Extraction of cyclopropanol-inactivated methanol dehydrogenase (MDH) gave a mixture of two interconverting compounds. The same compounds could be prepared from 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinoline-4,5-dione (PQQ) and cyclopropanol using a metal oxide (e.g. Ag2O) as a catalyst. Structure elucidation revealed that a C5 3-propanal adduct of PQQ is formed which is present in the extract as a diastereoisomeric mixture of the ring-closed form. Cyclopropanone gave an analogous product, while cyclopropylmethanol behaved as a substrate and was oxidized by the enzyme without ring-opening. From the work described, several arguments can be derived to reject the idea that inactivation proceeds via formation of a pair of free radicals. The mechanism probably consists of a concerted proton abstraction, rearrangement of the cyclopropoxy anion to a ring-opened carbanion and attack of the latter on the electrophilic C5 of PQQ. The measured rate of inactivation (3.7 s-1) is in agreement with such a mechanism. The role of the metal oxide and the enzyme in this process is the catalysis of the addition step and possibly a positioning of the reactants. As only a sole type of quinoprotein alcohol dehydrogenase becomes inhibited, the cyclopropane derivatives studied here can be regarded as mechanism-based inhibitors. The modified PQQ in cyclopropanone-inactivated MDH is fluorescent. A fluorescent intermediate was also observed in the catalytic cycle of MDH with methanol as a substrate. Its rate of formation and decay and the strongly decreased level of fluorescence in the presence of activator are in accordance with the view that the fluorescing species is the previously found oxidized-MDH.substrate (MDHox.S) complex. Since the decomposition of this complex requires activator and model studies have failed so far to mimic the enzyme, it seems that the combination of enzyme and activator is essential for the oxidation of the alcohol substrate.