Early stimulation of ATP turnover induced by growth factors : Synergistic effect of EGF and insulin and correlation with DNA synthesis

Addition of a mixture of EGF + insulin to quiescent cell cultures synergistically stimulates the cells to reinitiate DNA synthesis and cell division. We have previously demonstrated that this mixture rapidly increases ATP turnover in quiescent cells. The present work shows that each of the two growth factors, EGF and insulin, when added separately to quiescent cells was able to stimulate the phosphorylation of the organic acid-soluble compounds (Po) pool and ATP turnover. The stimulation of ATP turnover was closely correlated with the increase in phosphorylation of the Po pool which suggests that Po labelling reflects the ATP turnover. In many experiments, the synergy between the two growth factors on the early increase in phosphorylation of the Po pool was clearly shown. Doubling the concentration of EGF (12-24 ng/ml) or insulin (50-100 ng/ml) did not increase early stimulation of phosphorylation of the Po pool, whereas simultaneous addition of the two growth factors induced a greater stimulation than that of each growth factor separately added. The augmentation in Po labelling after addition of EGF or insulin alone was transient. The synergistic effect of the two growth factors was more significant when determined 150 or 300 min after growth-factor addition. In our experimental conditions, each of the two growth factors, EGF and insulin, was able to induce a stimulation of DNA synthesis. However, the best stimulatory effect was observed with the mixture of the two which synergistically increased DNA synthesis determined between 6 and 24 h after growth-factor addition. The comparison between DNA replication and Po labelling suggests a correlation between the increase in DNA replication and in the total ATP synthesized in the first 5 h after cell stimulation by growth factors added separately or in combination.     

Early stimulation of ATP turnover by EGF + insulin. Relation to external pH and Na+/H+ exchange system

We previously shown a rapid increase in ATP turnover after addition of epidermal growth factor and insulin to quiescent 3T3 cell cultures. Here, the relationship between this increase in ATP turnover and the activation by growth factors of Na+/H+ and Na+/K+ exchange systems was studied. Our results show that alkalinization of the medium enhances ATP turnover but they do not support the assumption that stimulation by growth factors of the Na+/H+ exchange induces an increase in ATP turnover since this increase was not inhibited by amiloride. Conversely, when ATP synthesis was abolished, the increase, in intracellular pH, by growth factors, was significantly decreased.     

Impacto del ejercicio físico en variables relacionadas con el bienestar emocional y funcional en adultos mayores

Background and aimThe benefits of the physical exercise in aging, and specially in frailty, have been associated with reduced risk of mortality, chronic disease, and cognitive and functional impairments. Multi-component training, which combines strength, endurance, balance, and gait training, represents the most beneficial kind of physical exercise in older adults.MethodsGiven the effectiveness of the multi-component training, a physical exercise program «Actívate» (based on the methodology Vivifrail), with the focus on «active aging», was conducted in the present study. Forty-nine older adults over 60 years participated in this program.ResultsThe physical exercise intervention led to a reduction in diastolic blood pressure, pain threshold and sleep disturbances (e. g. hypersomnia) (t ≥ 2.72, p < 0.01), as well as an increase of walking speed (t = 7.84, p ≤ 0.001). Further, quality of life factors (GENCAT scale), like emotional well-being, personal development, physical well-being, self-determination, and social inclusion, were greater after intervention (t ≥ ?2.06, p < 0.05).ConclusionsThese findings underline the benefits of multi-component training in functionality of older adults, and further, provide relevant aspects about the modulation of pain perception, sleep disturbances, social factors and physical and emotional well-being. Physical exercise programs such as «Actívate» should be promoted, in order to encourage healthy lifestyle habits, in the older adults’ population.     

Europium Nanoparticle-Based High Performing Immunoassay for the Screening of Treponemal Antibodies

Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p = 0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a screening test for treponemal antibodies.     

Shear Viscosity Investigation on Mango Juice with High Frequency Longitudinal Ultrasonic Waves and Rotational Viscosimetry

Ultrasonic velocity and attenuation measurements were performed on mango juices at 25 MHz in order to estimate longitudinal viscosity. Juices were extracted from fruits, removed periodically from fruit batches undergoing ripening for 3 weeks under controlled conditions. The correlation between longitudinal viscosity and apparent dynamic shear viscosity, obtained from flow tests, showed that up to 12–13 wt.% of Soluble Solids Content (SSC), the juices presented a Newtonian behavior. In this case the relation between longitudinal viscosity measured by ultrasound and shear viscosity measured by flow tests was very simple leading to the conclusion that ultrasound could replace rotating viscosimeters for specific applications. Over this limit, the results were also clearly correlated but the correlation depended on the shear rate because of the shear thinning behavior of the juices certainly due to soluble pectins. The use of longitudinal ultrasonic waves as a tool for viscosity determination on large batches of samples is discussed at the end of this communication.     

Isolation,technological characterization and in vitro probiotic evaluation of Lactococcus strains from traditional Turkish skin bag Tulum cheeses

Purpose

The present study was undertaken to evaluate in vitro prerequisite probiotic and technological characteristics of ten Lactococcus strains isolated from traditional goat skin bags of Tulum cheeses from the Central Taurus mountain range in Turkey.

Methods

All isolates were identified based on the nucleotide sequences of the 16S rRNA gene. Eight isolates belonged to Lactococcus lactis and two belonged to Lactococcus garvieae. Probiotic potential was determined from resistance to acid and bile salt, resistance to gastric and pancreatic juices, resistance to antibiotic, auto-aggregation, co-aggregation, diacetyl, hydrogen peroxide and exopolysaccharide productions. Technological properties were verified by alcohol, NaCl and hydrogen peroxide resistance and temperature tests.

Results

L. lactis NTH7 displayed high growth at all alcohol concentrations while L. lactis NTH4 grew very well even at NaCl concentrations of 10%. All strains showed to some extent resistance to acid and bile. Five strains exhibited desirable survival in gastric juice (pH 2.0), while three strains survived in pancreatic juice (pH 8.0). All Lactococcus isolates were sensitive to ampicillin, chloramphenicol, erythromycin, vancomycin, kanamycin, gentamycin and tetracycline. Also, only L. lactis NTH7 from among the isolates showed resistance against penicillin. L. lactis NTH10 and L. lactis NTH7 had higher auto-aggregation values in comparison with all other strains. All the strains demonstrated a co-aggregation ability against model food pathogens, particularly, L. lactis NTH10 which showed a superior ability with L. monocytogenes. All the ten strains produced H₂O₂ and exopolysaccharide (EPS); however, diacetyl production was detected for only four strains including L. lactis NTH10.

Conclusion

These results demonstrate that the L. lactis NTH10 isolate could be regarded as a favorable probiotic candidate for future in vivo studies.

     

Allosteric Dynamic Control of Binding

Proteins have a highly dynamic nature and there is a complex interrelation between their structural dynamics and binding behavior. By assuming various conformational ensembles, they perform both local and global fluctuations to interact with other proteins in a dynamic infrastructure adapted to functional motion. Here, we show that there is a significant association between allosteric mutations, which lead to high-binding-affinity changes, and the hinge positions of global modes, as revealed by a large-scale statistical analysis of data in the Structural Kinetic and Energetic Database of Mutant Protein Interactions (SKEMPI). We further examined the mechanism of allosteric dynamics by conducting studies on human growth hormone (hGH) and pyrin domain (PYD), and the results show how mutations at the hinge regions could allosterically affect the binding-site dynamics or induce alternative binding modes by modifying the ensemble of accessible conformations. The long-range dissemination of perturbations in local chemistry or physical interactions through an impact on global dynamics can restore the allosteric dynamics. Our findings suggest a mechanism for the coupling of structural dynamics to the modulation of protein interactions, which remains a critical phenomenon in understanding the effect of mutations that lead to functional changes in proteins.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation from young and mature explants of thyme (<Emphasis Type="Italic">Thymus vulgaris</Emphasis> and <Emphasis Type="Italic">T. longicaulis</Emphasis>) resulting in genetically stable shoots

An efficient in vitro propagation protocol, applicable both to young and mature explants of two Thymus spp., results in genetically stable plantlets. In vitro-grown shoot tips of Thymus vulgaris L. were exposed to cytokinins (6-benzyladenine, kinetin, and thidiazuron) alone or in combination with auxins, gibberellic acid (GA₃) and/or silver nitrate in order to optimize in vitro shoot proliferation. Optimum shoot proliferation (97% regeneration rate, with 8.6 shoots produced per explant) was obtained when semi-solid Murashige and Skoog (MS) medium was supplemented with 1 mg L⁻¹ kinetin and 0.3 mg L⁻¹ GA₃. Rooting of the shoots was easily obtained on semi-solid MS medium that was either hormone-free or supplemented with auxins. However, the best root apparatus (92.5% rooting rate, with 19 adventitious roots per shoot) developed on MS medium supplemented with 0.05 mg L⁻¹ 2,4-dichlorophenoxyacetic acid. Genetic stability was confirmed in the in vitro-germinated mother plant as well as the shoots that underwent two, four, six, eight, or ten cycles of in vitro subculturing by random amplified polymorphic DNA (RAPD) analysis. When applied to the micropropagation of mature shoot tips of T. longicaulis C. Presl subsp. longicaulis var. subisophyllus (Borbás) Jalas, the optimized in vitro propagation protocol resulted in a 97.5% shoot regeneration rate, with five shoots formed per explant, and 100% rooting. Rooted plantlets of both species were transferred to 250-mL plastic pots and successfully acclimatized by gradually reducing the relative humidity.