High efficiency electrotransformation of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> spp. <Emphasis Type="Italic">lactis</Emphasis>cells pretreated with lithium acetate and dithiothreitol

Background  

A goal for the food industry has always been to improve strains of Lactococcus lactis and stabilize beneficial traits. Genetic engineering is used extensively for manipulating this lactic acid bacterium, while electropolation is the most widely used technique for introducing foreign DNA into cells. The efficiency of electrotransformation depends on the level of electropermealization and pretreatment with chemicals which alter cell wall permeability, resulting in improved transformation efficiencies is rather common practice in bacteria as in yeasts and fungi. In the present study, treatment with lithium acetate (LiAc) and dithiothreitol (DTT) in various combinations was applied to L. lactis spp. lactis cells of the early-log phase prior to electroporation with plasmid pTRKH3 (a 7.8 kb shuttle vector, suitable for cloning into L. lactis). Two strains of L. lactis spp. lactis were used, L. lactis spp. lactis LM0230 and ATCC 11454. To the best of our knowledge these agents have never been used before with L. lactis or other bacteria.     

Serum osteoprotegerin and RANKL are not specifically altered in women with postmenopausal osteoporosis treated with teriparatide or risedronate: a randomized, controlled trial.

Risedronate and teriparatide have opposite actions on the osteoblast-osteoclast dipole and are expected to influence the RANK/RANKL/osteoprotegerin (OPG) system. We aimed to evaluate changes in serum OPG and RANKL after risedronate or teriparatide administration in postmenopausal osteoporotic women. Seventy-four postmenopausal Caucasian women (age 64.1+/-1.0 years) were studied. Women with osteopenia served as controls (group 1, n=30). Women with osteoporosis were randomly assigned to either risedronate 35 mg once weekly (group 2, n=21) or teriparatide 20 microg once daily (group 3, n=23) for six months. Blood samples for serum RANKL, OPG, N-terminal propeptide of type 1 collagen (P1NP), and C-terminal telopeptide of type 1 collagen (CTx) were obtained before treatment and three and six months after treatment. P1NP and CTx levels remained unchanged in group 1, decreased in group 2 (p<0.001), and increased in group 3 women (p<0.001) throughout the treatment. OPG levels remained unchanged while RANKL decreased gradually in all groups (p<0.001). There was no correlation between OPG or RANKL and P1NP or CTx. Our data suggest that neither antiresorptive nor osteoanabolic therapy causes specific alterations of serum OPG/RANKL levels; therefore, these cytokines cannot substitute for the established markers of bone turnover in the monitoring of response to osteoporosis treatment.     

Seasonal water quality of shallow and eutrophic Lake Pamvotis, Greece: implications for restoration

Lake Pamvotis is a moderately sized (22 km²) shallow (z _avg=4 m) lake with a polymictic stratification regime located in northwest Greece. The lake has undergone cultural eutrophication over the past 40 years and is currently eutrophic (annual averages of FRP=0.07 mg P l^-1, TP=0.11 mg P l^-1, NH₄ ⁺=0.25 mg N l^-1, NO₃ ^–=0.56 mg N l^-1). FRP and NH₄ ⁺ levels are correlated to external loading from streams during the winter and spring, and to internal loading during multi-day periods of summer stratification. Algal blooms occurred in summer (July–August green algae, August–September blue-green algae), autumn (October blue-green algae and diatoms), and winter (February diatoms), but not in the spring (March–June). The phytoplankton underwent brief periods of N- and P-limitation, though persistent low transparency (secchi depth of 60–80 cm) also suggests periods of light limitation. Rotifers counts were highest from mid-summer to early autumn whereas copepods were high in the spring and cladocerans were low in the summer. Removal of industrial and sewage point sources a decade ago resulted in a decrease in FRP. A phosphorus mass balance identified further reductions in external loading from the predominately agricultural catchment will decrease FRP levels further. The commercial fishery and lake hatchery also provides opportunities to control algal biomass through biomanipulation measures.     

Abdominal muscle fatigue after maximal ventilation in humans

Kyroussis, Dimitris, Gary H. Mills, Michael I. Polkey,Carl-Hugo Hamnegard, Nicholaos Koulouris, Malcolm Green, and John Moxham. Abdominal muscle fatigue after maximal ventilation inhumans. J. Appl. Physiol. 81(4):1477-1483, 1996.Abdominal muscles are the principal muscles ofactive expiration. To investigate the possibility of abdominal musclelow-frequency fatigue after maximal ventilation in humans, westimulated the nerve roots supplying the abdominal muscles. We used amagnetic stimulator (Magstim 200) powering a 90-mm circular coil andstudied six normal subjects. To assess the optimum level of stimulationand posture, we stimulated at each intervertebral level betweenT₇ andL₁ in the prone, supine, andseated positions. At T₁₀, we usedincreasing power outputs to assess the pressure-power relationship.Care was taken to avoid muscle potentiation. Twitch gastric pressure(Pga) was recorded with a balloon-tipped catheter. Mean (±SD)baseline twitch Pga measured with the subjects in the prone position atT₁₀ was 23.5 ± 5.4 cmH₂O. Within-occasion mean twitchPga coefficient of variation was 4.6 ± 1.1%. Twitch Pga wasmeasured with the subjects in the prone position with stimulation overT₁₀ before and after 2 min ofmaximal isocapnic ventilation (MIV). Twenty minutes after MIV, meantwitch Pga fell by 17 ± 9.1%(P = 0.03) and remained low 90 minafter MIV. We conclude that after maximal ventilation in humans thereis a reduction of twitch Pga and, therefore, of low-frequency fatiguein abdominal muscles.

     

Determination of bacteriocin activity with bioassays carried out on solid and liquid substrates: assessing the factor "indicator microorganism"

Background  

Successful application of growth inhibition techniques for quantitative determination of bacteriocins relies on the sensitivity of the applied indicator microorganism to the bacteriocin to which is exposed. However, information on indicator microorganisms' performance and comparisons in bacteriocin determination with bioassays is almost non-existing in the literature. The aim of the present work was to evaluate the parameter "indicator microorganism" in bioassays carried out on solid -agar diffusion assay- and liquid -turbidometric assay- substrates, applied in the quantification of the most studied bacteriocin nisin.     

Engineering the central pathways in Lactococcus lactis: functional expression of the phosphofructokinase (pfk) and alternative oxidase (aox1) genes from Aspergillus niger in Lactococcus lactis facilitates improved carbon conversion rates under oxidizing conditions

The present work describes a novel central pathway engineering method that has been designed with the aim to increase the carbon conversion rates under oxidizing conditions in L. lactis fermentations. The nisin producer L. lactis ATCC11454 strain has been genetically engineered by cloning a truncated version of the phosphofructokinase gene (pfk13), along with the pkaC, encoding for the catalytic subunit of cAMP-dependent protein kinase, and the alternative oxidase (aox1) genes of A. niger. Functional expression of the above genes resulted in enhanced PFK activity and the introduction of AOX activity and alternative respiration in the presence of a source of heme in the substrate, under fully aerobic growth conditions. The constructed strain is capable of fermenting high concentrations of glucose as was demonstrated in a series of glucostat fed-batch fermentations with glucose levels maintained at 55, 138 and 277 mM. The high maximum specific uptake rate of glucose of 1.8 mMs(-1)gCDW(-1) at 277 mM glucose is characteristic of the improved ability of the microorganism to handle elevated glucose concentrations under conditions otherwise causing severe reduction of PFK activity. The increased carbon flow through glycolysis led to increased protein synthesis that was reflected in increased biomass and nisin levels. The pfk 13-pkaC-aox1-transformant strain's fermentation at 277 mM glucose gave a final biomass concentration of 7.5 g/l and nisin activity of 14,000 IU/ml which is, compared to the parental strain's production levels at its optimal 55 mM glucose, increased by a factor of 2.34 for biomass and 4.37 for nisin.     

Glycolysis and the regulation of glucose transport in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> spp. <Emphasis Type="Italic">lactis</Emphasis> in batch and fed-batch culture

Background  

Despite the fact that many reports deal with glycolysis in Lactococcus lactis, there is not much information on the regulation of uptake of glucose itself. The aim of the present work was to investigate the effect of the glucose level on its specific uptake rate.     

Metabolism-mediated neurotoxicity: the significance of genetically engineered cell lines and new three-dimensional cell cultures

Until now, no in vitro methods for determining neurotoxic effects, on Phase I and Phase II biotransformation-driven metabolite formation or for the evaluation of the metabolism-mediated hazard of a chemical, have been validated. The current test guidelines are based on studies in vivo, involving animals exposed to the test substance. Novel in vitro testing instead of animal testing is required by Directive 86/609/EEC. In the EU White Paper on a Strategy for a Future Chemicals Policy, which may result in up to 20,000 chemicals being screened for toxicity, the use of non-animal test methods is seen as essential and is encouraged. The aim of the present work was to demonstrate the significance of novel technologies, including the use of genetically engineered cell lines and three-dimensional cell culture techniques for direct application in the regulatory hazard-assessment process. Furthermore, attempts were made to make in vitro toxicity tests for specific applications more-readily available for inclusion in the chemical hazard-assessment process, by exploiting advances made in the life sciences.     

Lactococcus lactis as a cell factory: a twofold increase in phosphofructokinase activity results in a proportional increase in specific rates of glucose uptake and lactate formation

Despite the fact that the area of glycolysis in Lactococcus lactis has been intensively studied, only a limited number of studies have been focused on the regulation of uptake of glucose itself. Using the tool of the glucostat fed-batch mode of culture, it was demonstrated in our earlier work that the concentration of glucose regulates its uptake rate and that the control of the glycolytic flux resides to a large extent in processes outside the pathway itself, like glucose transport and the ATP consuming reactions, while allosteric properties of key enzymes like phosphofructokinase (PFK) have a significant influence on the control. Extending our work, we report here the results of fermentations with engineered L. lactis strains with altered PFK activity in which the pfkA gene from Aspergillus niger, and its truncated version pfk13 that encodes a shorter PFK1 fragment were cloned. The results in this study suggest that, under the optimum for the microorganism applied microaerobic conditions, the glycolytic capacity of L. lactis was significantly increased in engineered strains with increased PFK activity. The transformant strain in which the truncated pfk13 gene of A. niger was expressed performed more efficiently as it was able to grow successfully in glucostat cultures with 277 mM glucose - while the optimum glucose concentration for the parental strain was 55 mM. The present work demonstrates the direct effect of PFK activity on the glycolytic flux in L. lactis since a twofold increase in specific PFK activity (from 7.1 to 14.5 U/OD₆₀₀) resulted in a proportional increase of the maximum specific rates of glucose uptake (from 0.8 to 1.7 μM s⁻¹ g CDW⁻¹) and lactate formation (from 15 to 22.8 g lactate (g CDW)⁻¹ h⁻¹).