Survival of Mammary Stem Cells in Suspension Culture: Implications for Stem Cell Biology and Neoplasia

There is increasing evidence that a variety of neoplasms including breast cancer may result from transformation of normal stem and progenitor cells. In the past, isolation and characterization of mammary stem cells has been limited by the lack of suitable culture systems able to maintain these cells in an undifferentiated state in vitro. We have recently described a culture system in which human mammary stem and progenitor cells are able to survive in suspension and produce spherical colonies composed of both stem and progenitor cells. Recent observation that adult stem cells from other tissues may also retain the capacity for growth under anchorage independent conditions suggests a common underlying mechanism. We propose that this mechanism involves the interaction between the canonical Wnt signal pathway and E-cadherin. The Wnt pathway has been implicated in normal stem cell self-renewal in vivo. Furthermore, there is evidence that deregulation of this pathway in the mammary gland and other organs may play a key role in carcinogenesis. Thus, the development of in vitro suspension culture systems not only provides an important new tool for the study of mammary cell biology, but also may have important implications for understanding key molecular pathways in both normal and neoplastic stem cells.     

Interleukin-10 gene polymorphism in Parkinson's disease patients

BACKGROUND: The etiology of sporadic Parkinson's disease (PD) is not well established. Recent studies revealed that inflammatory processes might also play an important role in the pathogenesis of PD. We hypothesized that genetically determined differences in the immune response, especially in anti-inflammatory cytokines production, might influence the risk of sporadic PD development and/or onset. To prove this hypothesis, two DNA polymorphisms at IL-10 promoter (-1082 and -519) were examined in sporadic PD patients. METHODS: The study enrolled 341 patients with diagnosed idiopathic PD. All cases of secondary parkinsonism were excluded from the study. For the purpose of this study the patients were also divided into two subgroups: group 1: patients with onset of Parkinson's disease, i.e., <50 years of age (early onset) included 60 patients, as well as group 2: patients with onset of Parkinson's disease >50 years of age (late onset) comprising 281 subjects. Control samples were from 315 randomly selected healthy individuals from the same geographical region who were free from signs of parkinsonism as evaluated by consultant neurologists. PCR-RFLP methods were used for genotyping. RESULTS: No statistically significant differences between PD patients and controls were found in the frequency of a single locus (-1082, -519) of IL-10 promoter. Likewise, haplotype analysis did not demonstrate any significant differences between evaluated groups. The frequency of the evaluated IL-10 genotypes was also similar in EOPD and LOPD patients. CONCLUSIONS: Results from our study revealed that the IL-10 (-1082G>A, -592C>A) polymorphism is not a risk factor of sporadic Parkinson's disease in a Polish population.     

Antidepressant-like effect of Tagetes lucida Cav. extract in rats: involvement of the serotonergic system

It has been demonstrated that the decoction of the aerial parts of Tagetes lucida Cav. produces an antidepressant effect during the forced swimming test (FST) in rats. The aim of this study was to evaluate the effect of different organic extracts and one aqueous extract of the aerial parts of T. lucida on the FST. In addition, the possible involvement of the serotonergic system in the antidepressant-like effect of T. lucida in the FST was evaluated, as was its potential toxicological effect. The different extracts of T. lucida (methanol, hexane, dichloromethane and aqueous, 10 and 50 mg/kg), as well as fluoxetine (FLX, 5 mg/kg), were administered per os (p.o.) to rats for 14 days. All animals were subjected to the FST. Only the aqueous extract of T. lucida at a dose of 50 mg/kg significantly reduced immobility behavior and increased swimming in the FST, similar to FLX. Later, the aqueous extract of T. lucida (50mg/kg) was administered for 1, 7 and 14 days. An antidepressant effect was observed after 7 days of treatment. To evaluate the participation of the serotoninergic system, the animals were pretreated with PCPA, an inhibitor of serotonin synthesis (100 mg/kg/day for 4 consecutive days). The animals were treated with the aqueous extract of T. lucida (50 mg/kg) and FLX (5 mg/kg) 24 h after the final injection and were then subjected to the FST. Pretreatment with PCPA inhibited the antidepressant effect of both T. lucida and FLX. Finally, T. lucida was administered p.o. and intraperitoneal route to evaluate its acute toxicological effect. The aqueous extract of T. lucida, administered p.o., did not produce lethality or any significant changes in behavior. In conclusion, the aqueous extract of T. lucida manifested an antidepressant-like effect in the FST mediated by the serotonergic system, with no adverse effects when administered p.o.     

Further insight on the hypoglycemic and nonhypoglycemic effects of troglitazone 400 or 600 mg/d: effects on the very-low-density and high-density lipoprotein particle distribution.

The effects of troglitazone 400 or 600 mg/d on the glycemic control, very-low-density lipoprotein (VLDL), and high-density lipoprotein (HDL) subclass concentrations and plasminogen-activator inhibitor 1 (PAI-1) levels were assessed in patients with type 2 diabetes that had not been controlled with dietary treatment. This was a multicenter, open-label, parallel-groups study. It included a run-in 4-week diet period and a 24-week randomized treatment. Fifty one patients received 400 mg/d and 55 patients 600 mg. The mean HbA(1c) concentration at the end of the study was similar for both doses. Troglitazone, regardless of dose, significantly improved insulin sensitivity assessed by the homeostasis model (HOMA). PAI-1 levels were significantly decreased in both groups by 13%. Higher HDL cholesterol concentrations and lower triglycerides levels were observed at the end of treatment. Triglyceride contents were reduced only in the lighter VLDL1. The change in HDL cholesterol concentration resulted from a combination of increased HDL3 cholesterol and lower HDL2 cholesterol levels. No differences were found in the effects of both treatment groups on the evaluated parameters. Our data provide new information about the actions of the drug on the lipid profile. Troglitazone reduces triglyceride levels by lowering the triglycerides content of the VLDL1 particles and increases HDL cholesterol concentrations by increasing HDL3 cholesterol levels.     

A single injection of pregabalin induces short- and long-term beneficial effects on fear memory and anxiety-like behavior in rats with experimental type-1 diabetes mellitus

Metabolic Brain Disease - Anxiety Disorders and Posttraumatic Stress Disorders (PTSD) associated with type-1 diabetes mellitus (T1DM) are increasingly common comorbidities and the treatment is...     

The effect of alcohols as the third component on diffusion in mixtures of aromatics and ketones

With laboratory and numerical work, we demonstrate that one of the main diffusion coefficients and the smaller eigenvalue of the Fick diffusion matrix are invariant to the number of methylene groups of the alcohol in ternary mixtures composed of an aromatic (benzene), a ketone (acetone) and one of three different alcohols (methanol, ethanol or 2-propanol). A critical analysis of the relationship between the kinetic and thermodynamic contributions to the diffusion coefficients allows us to explain this intriguing behaviour of this class of mixture. These findings are reflected by the diffusive behaviour of the according binary subsystems. Our approach provides a promising systematic framework for future investigations into the important and challenging problem of transport diffusion in multicomponent liquids.

The Fick diffusion coefficient matrix of three ternary mixtures composed of an aromatic (benzene), a ketone (acetone) and one of three different alcohols (methanol, ethanol or 2-propanol) is investigated with laboratory and numerical work.

Multicomponent diffusion plays a crucial role in various natural and industrial processes involving mass transfer.^1–3 Liquids appearing in nature and technical applications are essentially multicomponent. However, only data on binary diffusion coefficients are relatively abundant because the diffusion behavior of ternary and higher mixtures is much more complex.^4,5 Describing the isothermal–isobaric diffusion of a ternary mixture by Fick’s law requires four different diffusion coefficients that are composition dependent. The presence of cross diffusion coefficients aggravates the interpretation and data processing in experimental work, resulting in large uncertainties.^6,7 Thus, efforts are being made to develop new methods for analysis of multicomponent diffusion explicitly addressing various degrees of complexity.^8–10 Predictive equations for multicomponent diffusion of liquids mostly rely on extensions of the Darken relation,^11–13 which is only valid for ideal mixtures.¹⁴ The underlying physical phenomena in non-ideal mixtures are not well understood and the lack of experimental data impedes the development and verification of new predictive equations.The objective of this study was not only to measure and predict the Fick diffusion coefficient matrix for a series of ternary liquid mixtures, rather, the emphasis lied on understanding common features and whether they can be related to the behavior of the pure components and binary subsystems. Three ternary mixtures that are composed of organic compounds were selected, i.e. an aromatic, a ketone and an alcohol. Throughout, the first two components were benzene (1) and acetone (2) and the third component was one of the alcohols, methanol, ethanol or 2-propanol. For each mixture, nine state points along a composition path with a constant content of benzene, x₁ = 0.33 mol mol⁻¹, were studied under ambient conditions (298.15 K and 0.1 MPa). Seven of the state points were ternary mixtures and two were binary subsystems. To obtain reliable results for the Fick diffusion coefficient matrix, two complementary approaches were used, i.e. experiments and predictive molecular simulations. This combination allows for a critical analysis and leads to a deeper understanding of the underlying phenomena.^14,15The Taylor dispersion technique was utilized for the experiments.^16,17 In this method, a small quantity of mixture with a slightly different composition is injected into a laminar stream. It disperses due to convection and diffusion while flowing through a capillary tube and the refractive index is measured at its end to sample the concentration distribution. We have used the same apparatus as in previous works.^6,7 The Fick diffusion matrix is obtained by fitting working equations to the measured signal, i.e. the Taylor peak. The mathematical model of the Taylor dispersion technique was originally developed on the basis of Fick’s law in the volume reference frame. In a ternary mixture, two molar fluxes J^v_i relative to a volume averaged velocity are related to gradients of molar concentration ∇C_i with four diffusion coefficients D^v_ij. Alternatively, fluxes expressed in the molar reference frame J_i are relative to a molar averaged velocity and the mole fraction gradients ∇x_i act as a driving force1with molar density ρ. The fluxes of all three components are constrained by ΣJ_i = 0. The main diffusion coefficients D₁₁ and D₂₂ relate the flux of one component to its own mole fraction gradient and the cross diffusion coefficients D₁₂ and D₂₁ describe the coupling of the flux of one component with the gradient of the other. The third component does not appear in eqn (1) explicitly, but in general it affects all four diffusion coefficients. The transformation of experimental data from the volume to the molar reference frame (D^v_ij to D_ij) could be done here on the basis of the pure component volumes (see the ESI^†).Equilibrium molecular dynamics (MD) simulations were employed in this work, allowing for examination at the microscopic scale. The underlying molecular models were rigid, non-polarizable force fields of united atom type, consisting of a varying number of Lennard–Jones, point charge, dipole and quadrupole sites (see the ESI^†). Note that the force field parameters were adjusted to pure fluid properties only so that all simulation results for the mixtures are strictly predictive. Diffusion coefficients were sampled with the Green–Kubo formalism, based on integrated correlation functions of net velocities of the contained species.^11,15 Thereby, phenomenological coefficients Δ_ij were obtained, associating the diffusive fluxes with the chemical potential gradients ∇μ_i2with gas constant R and temperature T. Fluxes J_i correspond to the molar reference frame as in eqn (1).The diffusion coefficients from experiment and simulation are related to different driving forces so that the chemical potential gradients have to be transformed to the mole fraction gradients for their comparison.¹⁸ This transformation is contained in the thermodynamic factor matrix Γ3with the activity coefficient of species i being γ_i, which expresses the non-ideality of a mixture with respect to the composition. This relationship shows that the Fick diffusion coefficients are actually the product of two contributions, a kinetic Δ_ij and a thermodynamic Γ_ij. The separate observation of these two contributions promotes understanding of the underlying physical phenomena. In the present study, the thermodynamic factor was calculated using the Wilson excess Gibbs energy (g^E) model, using parameters fitted to experimental vapor–liquid equilibrium data of the binary subsystems (see the ESI^†). This combination of MD simulation results with a g^E model was successfully used in previous work to predict Fick diffusion coefficients, including several binary subsystems of the ternary mixtures studied here.¹⁹The four elements of the Fick diffusion coefficient matrix were determined for the three ternary mixtures, benzene + acetone + methanol/ethanol/2-propanol, for nine different compositions, each at ambient temperature and pressure.Results for the first main element of the diffusion matrix D₁₁, which relates the flux of benzene to its own mole fraction gradient, are shown in Fig. 1(a). The experimental data agree quantitatively with the molecular simulation data. D₁₁ increases with the acetone content in the ternary mixture. Since mixtures with a constant mole fraction of benzene (x₁ = 0.33 mol mol⁻¹) were studied throughout, the left edge of Fig. 1(a) corresponds to the binary limit of benzene + alcohol, while the right edge corresponds to that of benzene + acetone. Analysis of the ternary diffusive fluxes implies the following asymptotic behavior of the diffusion coefficients towards the binary limits:⁷ (i) at the infinite dilution limit, x₂ → 0, the ternary coefficient D₁₁ tends to the binary Fick diffusion coefficient of benzene + alcohol; (ii) at the other limit, x₃ → 0, D₁₁ − D₁₂ = D₂₂ − D₂₁ → D_bin (benzene + acetone) should hold. The present experimental and simulation results for D₁₁ are consistent with these asymptotic limits.Open in a separate windowFig. 1Top: The main Fick diffusion coefficient (molar reference frame) of benzene D₁₁ in the three ternary mixtures benzene (1) + acetone (2) + alcohol (3) at a constant benzene mole fraction x₁ = 0.33 mol mol⁻¹ from experiment (triangles) and MD simulation combined with the Wilson g^E model (circles). Both data sets were sampled at the same compositions, but are slightly shifted in the plot for visibility reasons. The symbols at the edges of this plot are the binary diffusion coefficients of benzene + alcohol (x₂ → 0) and of benzene + acetone (x₃ → 0). Bottom: The binary Fick diffusion coefficient of the subsystems benzene + alcohol and benzene + acetone. Most of the binary experimental data were taken from the literature.^20–27An inspection of Fig. 1(a) provides an unexpected finding: the main element D₁₁ is almost identical for all three mixtures along the examined composition path, i.e. it is independent of the contained type of alcohol. To explain this intriguing behavior of D₁₁, the properties of the pure components are considered first (see