Formation of intracellular vesicles within the Gram+ Lactococcus lactis induced by the overexpression of Caveolin-1ß.

BACKGROUND: Caveolae are invaginated plasma membrane domains of 50-100 nm in diameter involved in many important physiological functions in eukaryotic cells. They are composed of different proteins, including the membrane-embedded caveolins and the peripheric cavins. Caveolin-1 has already been expressed in various expression systems (E. coli, insect cells, Toxoplasma gondii, cell-free system), generating intracellular caveolin-enriched vesicles in E. coli, insect cells and T. gondii. These systems helped to understand the protein insertion within the membrane and its oligomerization. There is still need for fundamental insights into the formation of specific domains on membrane, the deformation of a biological membrane driven by caveolin-1, the organization of a caveolar coat, and the requirement of specific lipids and proteins during the process. The aim of this study was to test whether the heterologously expressed caveolin-1ß was able to induce the formation of intracellular vesicles within a Gram+ bacterium, Lactococcus lactis, since it displays a specific lipid composition different from E. coli and appears to emerge as a good alternative to E. coli for efficient overexpression of various membrane proteins. RESULTS: Recombinant bacteria transformed with the plasmid pNZ-HTC coding for the canine isoform of caveolin-1ß were shown to produce caveolin-1ß, in its functional oligomeric form, at a high expression level unexpected for an eukaryotic membrane protein. Electron microscopy revealed several intracellular vesicles from 30 to 60 nm, a size comparable to E. coli h-caveolae, beneath the plasma membrane of the overexpressing bacteria, showing that caveolin-1ß is sufficient to induce membrane vesiculation. Immunolabelling studies showed antibodies on such neo-formed intracellular vesicles, but none on plasma membrane. Density gradient fractionation allowed the correlation between detection of oligomers on Western blot and appearance of vesicles measurable by DLS, showing the requirement of caveolin-1ß oligomerization for vesicle formation. CONCLUSIONS: Lactococcus lactis cells can heterologously overexpress caveolin-1ß, generating caveolin-1ß enriched intracellular neo-formed vesicles. These vesicles might be useful for potential co-expression of membrane proteins of pharmaceutical interest for their simplified functional characterization.

Protective effects of the phytogenic feed additive "comfort" on growth performance via modulation of hypothalamic feeding- and drinking-related neuropeptides in cyclic heat-stressed broilers.

Identification of alternatives to antibiotics in livestock and poultry is necessary. Fueled by consumer preferences, phytogenic feed additives are increasingly used in the food system; however, their mode of action is not well defined. Here, we used broiler chickens, in which appetite and feeding behavior regulation are controlled by complex mechanisms, to determine the effect of the phytogenic feed additive "comfort" (PFA-C) as well as its underlying molecular mechanisms on growth performance in heat-stressed broiler chickens. Heat stress significantly increased birds' core body temperature, water intake, and the hypothalamic expression of heat shock protein (HSP) 70, whereas it decreased feed intake, BW, and woody breast incidence. Phytogenic feed additive "comfort" supplementation downregulated the hypothalamic expression of HSP70, reduced core body temperature, increased feed and water intake, and improved BW in HS broilers. At molecular levels, the effect of PFA-C on growth performance seemed to be mediated by modulation of hypothalamic expression of melanocortin receptor 2, arginine vasopressin, aquaporin 2, and sodium and potassium-transporting ATPase subunit beta 1 polypeptides. In summary, PFA-C supplementation ameliorates heat stress productivity losses via a potential cytoprotective effect, reduction of hypothalamic intracellular stress, and modulation of hypothalamic feeding- and drinking-related polypeptide expression.

Divergent selection for relative breast yield at 4 D posthatch and the effect on embryonic and early posthatch development.

Genetic selections for growth promotion in poultry have been highly successful in improving growth, yield, and feed conversion in the modern broiler. These selections have focused on the use of hypertrophy, the increase of muscle fiber size to improve growth. Muscle growth however is not limited solely to hypertrophy but is largely attributable to both hypertrophy and hyperplasia, the increase in muscle fiber number. As muscle fiber size has been theorized to reach an eventual physiological limit, it was determined to develop a novel method of selection focusing on hyperplasia. Divergent selection for 4-day relative breast yield (BY4) was chosen as it is believed to occur at point at which muscle cell number per gram is maximized and satellite cell activity is higher than later in life. Using a random bred control population, divergent selection was undergone for BY4. The 2 broiler lines divergently selected for BY4 are noted as the high and low BY4 lines, respectively (high 4-day breast yield and low 4-day breast yield). Heritability estimates for selection of 4-day breast percentage in the upward and downward directions were 0.63 and 0.44, respectively. Divergent selection resulted in clear divergence in BY4 and shows promise in utilizing BY4 to promote broiler growth and body composition.

The association of cognitive distortions and the type of gambling in problematic and disordered gambling.

BACKGROUND: Previous studies have shown that particular types of gambling are related to the development of gambling-related problems. Further, gambling-related cognitive distortions contribute to the development of disordered gambling. The aim of the present study is to compare different gambling types with respect to cognitive distortions and the development of disordered gambling. METHODS: Based on a proactively screened sample of vocational school students (N = 6718), 309 students were selected to undergo an in-depth interview. We assessed the Gamblers-Belief-Questionnaire (GBQ) to measure gambling-related cognitive distortions and the Stinchfield questionnaire for assessing gambling-related problems. Associations between cognitive distortions, gambling-related symptoms, and types of gambling were analysed using logistic regression analyses. RESULTS: Higher scores on the GBQ subscale "belief in luck/perseverance" led to a significantly higher chance to be classified as a person with Gambling Disorder (Conditional Odds Ratio (COR) = 1.05, Confidence Interval (CI) = 1.02-1.08) as well as problematic gambling (COR = 1.04, CI = 1.01-1.06). Higher scores on the subscale "illusion of control" were also associated with problematic gambling (COR = 1.04, CI = 1.00-1.08). The multivariate analyses of the gambling types identified only sports betting as a predictor for problematic gambling (COR = 1.91, CI = 1.05-3.49). When controlling for cognitive distortions, sports betting was not significant anymore. With respect to disordered gambling, gambling on electronic gambling machines (EGMs) turned out to be a risk factor besides cognitive distortions (COR = 2.59, CI = 1.04-6.49). DISCUSSION: The present study confirmed the high relevance of cognitive distortions for problematic and disordered gambling especially for sports betting and gambling on EGMs. Preventive measures and psychotherapy should take these relationships into account.

Processing evaluation of random bred broiler populations and a common ancestor at 55 days under chronic heat stress conditions.

As a result of genetic selection, the modern broiler is more efficient, higher yielding, and faster growing than the bird of the 1950s. Unfortunately, as a result of improvement in growth rate, the modern broiler has the potential to struggle under heat stress conditions. The present study evaluates 3 different random bred populations and a common ancestor under both a thermal neutral and heat stress conditions after a 54-D grow-out period. The lines used in this study included the Athens Canadian Random Bred (ACRB), a 1995 Random Bred (95RAN), a 2015 Random Bred (MRB), and a Junglefowl (JF). Male chicks (n = 150/line) were placed by line in environmentally controlled chambers. An 8-h daily cyclic heat stress (36°C) was applied to half of the chambers beginning on day 28 (HS) and lasting until processing at day 55, while the remaining chambers remained thermal neutral (TN) at 26°C. Dock weights and carcass weights were lower in the HS-95RAN and HS-MRB, compared to their TN counterparts, while the ACRB and JF had no difference in dock and carcass weights regardless of environmental condition. The MRB line had the highest breast yield (27.79%) while the JF (12.79%) and ACRB (12.42%) had the lowest. The 95RAN line had the highest abdominal fat percentage (2.83%) while the MRB line had the lowest moisture uptake during chill. The HS exposure lowered overall breast yield and breast pH at 15 min and 4 h postmortem but did not have an impact on color (L∗) or 24 h breast drip loss. The MRB was scored for both woody breast and white striping. The TN-MRB group had a higher incidence of moderate and severe woody breast and white striping than the HS-MRB group. Based on the results of this study, it appears that HS has a greater negative impact on the higher yielding lines (MRB and 95RAN) than the ACRB and JF and that clear line differences exist between the random bred lines and their common ancestor.

Interaction of macrocyclic lactones with the multidrug transporters: the bases of the pharmacokinetics of lipid-like drugs.

Like most drugs, macrocyclic lactone endectocides (MLs) exert their antiparasitic effects within the defined target tissues where parasites are located, and whose drug concentrations correlate with those in the plasma compartment. The process of drug distribution to the active site constitutes the link in the pharmacokinetic/pharmacodynamic relationship. In the past few years it has become evident that transporter proteins play a major role in regulating the distribution, elimination and metabolism of the antiparasitic macrocyclic lactones. The efflux transporter P-glycoprotein (P-gp) has received the most attention with regards to its strong interaction with ivermectin and other MLs. P-gp has been reported to be involved in restricting the absorption of these drugs, in enhancing their intestinal elimination, in the protection against their neurotoxicity and in the ML resistance mechanisms in parasites. This review focuses on the interaction of MLs with P-glycoprotein and with other multidrug resistance transporters. Given the structural and physicochemical diversity of these drugs, they constitute models of interest to study the major molecular determinants for the interaction with transporters. We will discuss the consequences of such interactions on ML pharmacokinetics and the possibility of benefiting from of drug/drug interaction to reverse multidrug resistance in several therapeutic fights such as against parasites and tumors.

Differential expression of water channel- and noncoding RNA biogenesis-related genes in three lines of chickens under a short-term water restriction.

Genetic selection for high growth rate has resulted in tremendous changes not only in feed efficiency, but also in water consumption between modern broilers and their ancestor jungle fowl (JF). However molecular mechanisms involved in water homeostasis are still not well defined. This study aimed, therefore, to determine the effect of short-term water restriction on the expression of water channel- and noncoding RNA biogenesis-related genes in the kidney and whole blood of JF, broiler population from the 1990s (RB1995), and modern broiler population developed in 2015 (ARB2015). Body weight-matched birds from each population were subjected to water restriction (WR) for 3 h or had ad libitum access to water in a 3 × 2 factorial design. The expression of target genes was determined by real-time quantitative PCR. WR significantly reduced body weight in RB1995, but not in JF or ARB2015. In the kidney, WR up-regulated the expression of AQP2 in all chicken populations, AQP3 in the RB1995, and ATP1B1 in JF and ARB2015. However, it down-regulated the expression of AQP4 in ARB2015 but had no effect on AVP expression. The expression of RNase III family enzymes also was altered by WR in a population-dependent manner, with DICER1 being down-regulated in JF and RB1995, Drosha was decreased in RB1995, and ARG2 was up-regulated in ARB2015. The expression of DGCR8 and TRBP1 was not affected by WR in any population; however, DGCR8 mRNA levels were significantly lower in RB1995 and ARB2015 compared to JF under both conditions. TRBP1 gene expression was significantly lower in RB1995 and ARB2015 compared to JF under WR conditions. In the blood, the expression of these genes also was altered by WR, but with different patterns than the kidney. The mRNA abundances of AQP, AVP, DICER1, DGCR8, AGO2, and TRBP1 were significantly decreased by WR in RB1995. However, the expression of AQP2, AVP, DGCR8, and TRBP1 was increased in WR-ARB2015 compared to the control. In the JF, there was no difference in the expression of these genes except for a significant up-regulation of TRBP1 in WR compared to the control group. To the best of our knowledge, this is the first report showing that water channels and the RNase III enzymes are differentially regulated by WR in a population-dependent manner, which may be due to differential postnatal growth and maturation. Their expression in the circulation could open new vistas for identification of new molecular signatures involved in adaptation to water-deprivation stress.

Cell electropermeabilization: a new tool for biochemical and pharmacological studies.

Cell electropermeabilization is the transient permeabilization of the plasma membrane by means of short and intense electric pulses. Under optimized conditions, electropermeabilization is compatible with cell survival. It provides a direct access into the cytosol to ions, small molecules, exogenous drugs and macromolecules. As cells remain functional, a large variety of cell biology questions can be addressed. Such 'in situ biochemistry' opens new possibilities beside the more classical studies dealing with unpermeabilized cells or subcellular extracts. Electropermeabilization also allows pharmacological studies with cells, cultured monolayers and in vivo tissues as well as the design of drug controlled-release systems.

Electropermeabilization of cells in tissues assessed by the qualitative and quantitative electroloading of bleomycin.

Using cells in suspension, electropermeabilization is a technique extensively used to transfect living cells or to introduce a variety of compounds inside the cells. Here we demonstrate the reality of the tissue electropermeabilization using qualitative and quantitative determinations of the electroloading of bleomycin considered as an nonpermeant molecule that serves as an indicator of the permeabilization. In tissues, cell electropermeabilization is achieved for electric field intensities lower than those necessary to permeabilize the same cells in suspension. We also emphasize the importance of the geometry of the electric field lines defined by the electrodes for permeabilizing a whole tissue, for example a tumor.

Strontium binding to sarcoplasmic reticulum Ca(2+)-ATPase. Spectroscopic differentiation of the substeps involved.

We investigated the consequences of Sr2+ binding to the transport sites of sarcoplasmic reticulum (SR) Ca(2+)-ATPase for two fluorescent conformational probes located in different regions of the ATPase. Using SR vesicles in which Lys-515 in the ATPase had been previously labeled with fluorescein 5'-isothiocyanate (FITC), we found that the Sr(2+)-induced a drop in the fluorescein fluorescence of this FITC-labeled ATPase shifted toward lower Sr2+ concentrations than the Sr(2+)-induced rise in Trp fluorescence for the same FITC-labeled ATPase. The curve describing the Sr(2+)-dependent rise in Trp fluorescence had a characteristic asymmetric shape, and the changes in Trp fluorescence occurred in parallel with the activation by Sr2+ of pNPP hydrolysis by the ATPase. Analysis of these results in terms of the simplest scheme describing the sequential binding of the two Sr2+ ions suggests that under the conditions of these experiments, i.e. at neutral pH in the presence of potassium, the Sr(2+)-induced rise in the Trp fluorescence mainly reflected the formation of ATPase with two ions bound to the transport sites, whereas the binding of a single Sr2+ ion was virtually sufficient to reduce the fluorescence of bound FITC to its minimal level.

Electrochemotherapy potentiation of antitumour effect of bleomycin by local electric pulses.

In cell culture the cytotoxicity of some anticancer drugs, especially bleomycin, can be greatly enhanced by exposing cells to non-cytotoxic electric pulses. Nude or conventional mice bearing subcutaneous transplanted tumours were treated with intramuscular doses of bleomycin followed by local delivery of electric pulses similar to those used in vitro. Tumors were reduced and even eradicated after this electrochemotherapy. Thus the antitumour effects of bleomycin in mice can be considerably potentiated by local electric pulses.

Electrochemotherapy of spontaneous mammary tumours in mice.

Electrochemotherapy delivers external electric pulses to the tumour site to induce local potentiation of the antitumour activity of intramuscular injections of bleomycin. C3H/Bi mice with spontaneous mammary carcinomas received weekly injections of 50 micrograms bleomycin followed by electric pulses 30 min later. All the 38 tumours treated exhibited at least a partial regression. 23 complete remissions were observed, 3 of which were cures. One difficulty in assessing the cure rate in this model is that frequent parallel or sequential tumours cause early death. Electrochemotherapy appears similarly efficient in spontaneous tumours as in previously studied transplanted tumours.

Very high cytotoxicity of bleomycin introduced into the cytosol of cells in culture.

We observed previously in vitro that the cytotoxicity of bleomycin (BLM), an anticancer drug in current use, was greatly potentiated by exposing cultured cells to appropriately chosen electric pulses. We then showed in vivo, on tumor-bearing mice, that the same electric pulses also potentiated the antitumoral activity of BLM. In the present work, we demonstrate on DC-3F cells in vitro, that this potentiation is closely related to cell electropermeabilization and the consequent direct internalization of BLM molecules in the cytosol. The survival response curve (SRC) of the electropermeabilized (EP) cells exposed to BLM (plotted as logarithm of survival versus external drug concentration) shows a linear pattern usual for the SRCs of intact cells exposed to current cytotoxic drugs, though in the nanomolar range of concentrations. We have succeeded in determining the relation between BLM cytotoxicity on EP cells and the number of electroloaded BLM molecules per cell (that is the average number, per cell, of BLM molecules internalized into the cytosol). We conclude that (1) BLM molecules possess very intense cytotoxic activity which in non-EP cells is drastically limited by the intact plasma membrane; and (2) in these intact cells, the plasma membrane is responsible for the unusual upward concave curvature of the SRC resulting from exposure to BLM.

Transient electropermeabilization of cells in culture. Increase of the cytotoxicity of anticancer drugs.

The electropermeabilization (EPN) of living cells allows the uptake of non-permeant molecules and can reveal their potential activity on cells without the constraints of the plasma membrane crossing. We decided to compare the cytotoxicity of some anticancer drugs on electropermeabilized (EP) and non-permeabilized (NEP) cultured DC-3F cells exposed to the drugs for a short time. After EPN, the increase in cytotoxicity varies between 1 and more than 700 times, depending on the usual cell uptake pathway of a given drug. The most relevant increase of toxicity was observed with molecules such as netropsin (200-fold) and bleomycin (700-fold) which in ordinary conditions weakly diffuse through the plasma membrane. Only a 3-5-fold increase of the cytotoxicity was observed with lipophilic drugs able to rapidly diffuse through the plasma membrane (actinomycin D, NMHE) both in the case of drug-sensitive and resistant cell strains. This increased toxicity is clearly related to a facilitated uptake because, after electropermeabilization, the effects of melphalan (a drug which enters intact cells via leucine transporters) are not modulated by the external leucine concentration. Thus, EPN enables us to reveal the intrinsic toxicity of hydrophilic molecules which have a limited access to their intracellular targets. We propose that EPN can be used as a novel screening procedure of new cytotoxic molecules which could be modified thereafter in order to facilitate their cellular uptake.

Involvement of membrane bleomycin-binding sites in bleomycin cytotoxicity.

The authors have recently shown the existence of bleomycin (BLM)-binding sites at the surface of DC-3F cells. In order to study the involvement of these sites in the sensitivity of the cells to bleomycin several BLM-resistant cell lines from DC-3F cells were analysed. These mutants were obtained by electrotransfection of the Sh ble gene (D/BlmI cells) or the Sh ble-beta Gal fusion gene (D/BlmII cells) and/or by continuous culture in the presence of BLM (D/BlmIR and D/Blm40 cells). The resistance levels of the D/BlmII and D/Blm40 cells were 50- and 22-fold, respectively, determined at the EC50 level. The D/BlmI cells were only 2-fold resistant, whereas D/BlmIR cells were so resistant that almost no cytotoxicity was detected up to 200 microM BLM external concentration. Electropermeabilization was used in an attempt to bypass the plasma membrane of the cells and permit the distinction between internal resistance and membrane resistance. The former was observed when the products of the transfected genes were present. With respect to membrane resistance, differences were detected in the number of BLM-binding sites in several mutant cell lines, which could account for the differences in cell sensitivity to BLM. This suggests that the BLM-binding sites found at the cell surface may play a crucial role in BLM internalization and consequently in its cytotoxicity.

Internalisation of the bleomycin molecules responsible for bleomycin toxicity: a receptor-mediated endocytosis mechanism.

Bleomycin (BLM) does not diffuse through the plasma membrane but nevertheless displays cytotoxic activity due to DNA break generation. The aim of the study was to describe the mechanism of BLM internalisation. We previously provided evidence for the existence of BLM-binding sites at the surface of DC-3F Chinese hamster fibroblasts, as well as of their involvement in BLM cytotoxicity on DC-3F cells and related BLM-resistant sublines. Here we report that A253 human cells and their BLM-resistant subline C-10E also possessed a membrane protein of ca. 250 kDa specifically binding BLM. Part of this C-10E cell resistance could be explained by a decrease in the number of BLM-binding sites exposed at the cell surface with respect to A253 cells. The comparison between A253 and DC-3F cells exposing a similar number of BLM-binding sites revealed that the faster the fluid phase endocytosis, the greater the cell sensitivity to BLM. Moreover, the experimental modification of endocytotic vesicle size showed that BLM cytotoxicity was directly correlated with the flux of plasma membrane area engulfed during endocytosis rather than with the fluid phase volume incorporated. Thus, BLM would be internalised by a receptor-mediated endocytosis mechanism which would first require BLM binding to its membrane receptor and then the transfer of the complex into intracellular endocytotic vesicles, followed by BLM entry into the cytosol, probably from a nonacidic compartment.

Electrochemotherapy tumor treatment is improved by interleukin-2 stimulation of the host's defenses.

We have recently described a new antitumor treatment, electrochemotherapy (ECT), based on the large local potentiation of the effects of the chemotherapeutic agent bleomycin (BLM) by electric pulses (EP) delivered at the tumor site. We demonstrate here that the host's immune response participates in cure achievement. We obtain an increase of the rate of completely cured animals by injecting mice with interleukin-2 (IL-2).

Multiple recognition of various amphiphilic molecules by the multidrug resistance P-glycoprotein: molecular mechanisms and pharmacological consequences coming from functional interactions between various drugs.

P-glycoprotein (P-gp) is an active, ATP-dependent plasma membrane transporter which is responsible for the expulsion of various cytotoxic drugs with different chemical structures out of resistant (MDR) cells. It is also capable of transporting a number of other amphiphilic molecules, the so-called MDR-reversing agents, which belong to a very broad variety of chemical families. Moreover, P-gp can also play a role in steroid secretion and cellular detoxification by transporting various other substrates. In this review, we address the problem of the multiple recognition by P-gp of such a large number of amphiphilic molecules. This is both (i) from a basic viewpoint in order to discuss the underlying molecular mechanisms explaining how the general rule of substrate-enzyme specificity can be violated, and (ii) from a more applied pharmacological viewpoint to show in detail how the interaction of various drugs with P-gp leads to important consequences in terms of the relative effects of these drugs in the anticancer chemotherapy context, as well as for their pharmacokinetic distributions in the whole organism, rationalizing possible adverse drug reactions. In particular, we will present evidence that, independently of the technique used, the mutual interactions between P-gp transport substrates cannot always be reduced to simple competitive effects.

Treatment of murine transplanted subcutaneous tumors using systemic drug administration.

In vitro data have shown an increased cytotoxic drug uptake into electropermeabilized cells in suspension, leading to a marked cytotoxicity increase (1). Preclinical experiments were required to demonstrate the in vivo applicability of these observations. Obviously, the most convenient laboratory animal model to test new antitumor treatments is the mouse. Indeed, there exist many tumors of different histological types which can be transplanted in mice, either in immunocompetent mice in the case of syngeneic tumors or in immunodepressed mice in the case of allogeneic or xenogeneic tumors. From a practical point of view, mice have the advantage to be rather cheap and to allow a large number of experiments. Moreover, murine tumors are generally easy to transplant, grow rapidly, and can be conveniently followed for their evolution, at least in the case of subcutaneous tumors. Finally, murine subcutaneous tumors are well adapted to test the antitumor effects of electrochemotherapy since they allow the use of a rather simple material to conveniently apply transcutaneous permeabilizing electric pulses.