Conductometric and voltammetric studies on the bis(triphenyl phosphine) ruthenium(II) complex, cis-[RuCl2(L)(PPh3)2], where L: 2-(2′-pyridyl)quinoxaline

Conductometric investigation on the bis(triphenyl phosphine) ruthenium(II) complex, cis-[RuCl₂(L)(PPh₃)₂] (A) (where PPh₃: triphenyl phosphine and L: 2-(2′-pyridyl)quinoxaline, C₁₃N₃H₉), in dimethylsulfoxide (DMSO) was performed at temperatures ranging from 25 to 50 °C. In addition, cyclic voltammograms of A were recorded on platinum working electrode in dichloromethane (DCM) and dimethylsulfoxide (DMSO) using n-tetrabutylammonium hexafluorophosphate (NBu₄PF₆) as supporting electrolyte at 25 °C. The molar conductivities (Λ) demonstrate that A behaves as uni-univalent electrolyte in DMSO over the whole temperature range. This behavior can be explained in terms of the replacement upon dissolution of chlorine and PPh₃ ligands by DMSO molecules, and consequently, the formation of the ion-pair [RuCl(L)(PPh₃)(DMSO)₂]Cl [B⁺Cl⁻] which is dissociated in some extent. The Λ values were analyzed by means of the Lee-Wheaton conductivity equation in order to estimate the limiting molar conductivities (Λ^o) and the ion-pair association constants (K_A) of [B⁺Cl⁻]. The limiting ion conductivities for the B⁺ ion were evaluated using n-tetrabutylammonium chloride (NBu₄Cl) as “reference electrolyte”. The thermodynamic functions related with the ion association, such as Gibbs free energy , enthalpy , and entropy , were evaluated as well. The mobility of B⁺ was found to increase linearly with rising temperature and the consequent decrease of the viscosity (η) of DMSO. The K_A and values indicate that the association of [B⁺Cl⁻] increases to some extent with the rise of the temperature followed by the decrease of the dielectric constant (ε) of DMSO. The voltammetric experiments indicated that the couple Ru^3+/2+ is reversible and diffusion controlled in DCM and completely irreversible in DMSO.     

Preparation of poly(ethylene glycol) protected nanoparticles with variable bioconjugate ligand density

Maleimide-functional poly(ethylene glycol)-b-poly(epsilon-caprolactone) nanoparticles (NPs) were prepared via the Flash NanoPrecipitation technique. Subsequent reaction with a model ligand, bovine serum albumin (BSA), was conducted using thiol-maleimide conjugation. Reaction of up to 22% of NP surface maleimide-PEG tethers was obtained, with the percent conversion being essentially independent of the ratio of maleimide-PEG to methyl-PEG over the range 30-100%, respectively. At the highest surface coverage, BSA is calculated to essentially cover the NP surface area. Reaction parameters (reaction order and docking constant) describing the extent of ligand conjugation were determined. The reaction order is applicable to the conjugation of ligands presenting free thiol functionalities, while the value of the docking constant is ligand-dependent and accounts for physical and dynamic properties of the ligand-PEG interaction. Jointly, the particle formation process, using block copolymer-directed kinetically controlled assembly and surface functionalization represent a versatile new platform for the preparation of bioconjugated NPs with accurate control of ligand density and minimal processing steps.     

The mode of lymphoblastoid cell death in response to gas phase cigarette smoke is dose-dependent

Background

Human embryonic stem cells (hESC) have the capacity to differentiate in vivo and in vitro into cells from all three germ lineages. The aim of the present study was to investigate the effect of specific culture conditions on the differentiation of hESC into lung epithelial cells.

Methods

Undifferentiated hESC, grown on a porous membrane in hESC medium for four days, were switched to a differentiation medium for four days; this was followed by culture in air-liquid interface conditions during another 20 days. Expression of several lung markers was measured by immunohistochemistry and by quantitative real-time RT-PCR at four different time points throughout the differentiation and compared to appropriate controls.

Results

Expression of CC16 and NKX2.1 showed a 1,000- and 10,000- fold increase at day 10 of differentiation. Other lung markers such as SP-C and Aquaporin 5 had the highest expression after twenty days of culture, as well as two markers for ciliated cells, FOXJ1 and β-tubulin IV. The results from qRT-PCR were confirmed by immunohistochemistry on paraffin-embedded samples. Antibodies against CC16, SP-A and SP-C were chosen as specific markers for Clara Cells and alveolar type II cells. The functionality was tested by measuring the secretion of CC16 in the medium using an enzyme immunoassay.

Conclusion

These results suggest that by using our novel culture protocol hESC can be differentiated into the major cell types of lung epithelial tissue.     

Hydroxyindole-O-Methyltransferase in Y-79 Human Retinoblastoma Cells: Effect of Cell Attachment

Hydroxyindole-O-methyltransferase (HIOMT), the enzyme in the final step of melatonin synthesis, is present in the Y-79 human retinoblastoma cell line. Using electroblot immunolabellings, a single band corresponding to HIOMT was observed. Immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and quantification of enzyme activity all revealed dramatic increases in HIOMT in cells attached to substrate compared to cells in suspension culture.     

Size at maturity of Mediterranean marine fishes

In this review we collected data on the length at maturity (L_m) and maximum reported total length (L_max) of 565 Mediterranean marine fish stocks, representing 150 species, 68 families, 24 orders and 3 classes. Overall, L_m ranged from 2 cm, for the males of the toothcarp Aphanius fasciatus, to 350 cm, for the females of the bluntnose sixgill shark Hexanchus griseus. L_m was positively linearly related with L_max for Actinopterygii (logL_m = ?0.123 + 0.92 × logL_max; r ² = 0.87, n = 471, P < 0.001) and Elasmobranchii (logL_m = ?0.008 + 0.922 × logL_max; r ² = 0.90, n = 92, P < 0.001) with the two slopes being significantly different (ANCOVA: F = 2,904, P < 0.001). The reproductive load (L_m/L_max) ranged between 0.23 (sand steenbras Lithognathus mormyrus) and 0.94 (angular roughshark Oxynotus centrina and thornback ray Raja clavata). The mean L_m/L_max was significantly (ANOVA, F = 34.14, P < 0.001) lower for Actinopterygii (mean = 0.59, SD = 0.122, n = 471) compared to Elasmobranchii (mean = 0.70, SD = 0.132, n = 92) and Holocephali (mean = 0.77, SD = 0.077, n = 2). The L_m/L_max was significantly (ANOVA, F = 43.80, P < 0.001) higher for species providing some form of parental care, i.e. guarders, bearers, nesters (mean L_m/L_max ± SD = 0.68 ± 0.141, n = 111) compared to non-guarders (mean L_m/L_max ± SD = 0.59 ± 0.123, n = 454). The mean L_m/L_max displayed a remarkable constancy with longitude (northern and southern Mediterranean coastline: ANOVA, F = 0.01, P = 0.93), latitude (western, central and eastern regions: ANOVA, F = 1.25, P = 0.29) and habitat (ANOVA, F = 0.85, P = 0.51).     

A new example of a tetranuclear iron(III) cluster containing the [Fe4O2] core: preparation, X-ray crystal structure, magnetochemistry and Mössbauer study of [Fe4O2(O2CMe)6(N3)2(phen)2]

Two synthetic procedures have been employed that allow access to the new tetranuclear cluster [Fe₄O₂(O₂CMe)₆(N₃)₂(phen)₂] (1), where phen is 1,10-phenanthroline. Complex 1 · 3MeCN displays an unusual structural asymmetry (observed for the second time) in its [Fe₄O₂]⁸⁺ core that can be considered as a hybrid of the bent (butterfly) and planar dispositions of four metal ions seen previously in such compounds with transition metals. Complex 1 has been characterized by variable-temperature magnetic susceptibility studies, and by IR and variable-temperature ⁵⁷Fe Mössbauer spectroscopies. Magnetochemical data reveal a diamagnetic ground state (S=0) with antiferromagnetic body-body and body-wingtip interactions between the iron(III) ions of the butterfly core (J_bb=−11 cm⁻¹, J_wb=−70 cm⁻¹). Magnetochemical and Mössbauer studies on 1 show that its structural asymmetry has practically no influence on these properties compared with the more symmetric types.     

Tyrosine Phosphorylation of Wiskott-Aldrich Syndrome Protein (WASP) by Hck Regulates Macrophage Function

We have shown previously that tyrosine phosphorylation of Wiskott-Aldrich syndrome protein (WASP) is important for diverse macrophage functions including phagocytosis, chemotaxis, podosome dynamics, and matrix degradation. However, the specific tyrosine kinase mediating WASP phosphorylation is still unclear. Here, we provide evidence that Hck, which is predominantly expressed in leukocytes, can tyrosine phosphorylate WASP and regulates WASP-mediated macrophage functions. We demonstrate that tyrosine phosphorylation of WASP in response to stimulation with CX3CL1 or via Fcγ receptor ligation were severely reduced in Hck^−/− bone marrow-derived macrophages (BMMs) or in RAW/LR5 macrophages in which Hck expression was silenced using RNA-mediated interference (Hck shRNA). Consistent with reduced WASP tyrosine phosphorylation, phagocytosis, chemotaxis, and matrix degradation are reduced in Hck^−/− BMMs or Hck shRNA cells. In particular, WASP phosphorylation was primarily mediated by the p61 isoform of Hck. Our studies also show that Hck and WASP are required for passage through a dense three-dimensional matrix and transendothelial migration, suggesting that tyrosine phosphorylation of WASP by Hck may play a role in tissue infiltration of macrophages. Consistent with a role for this pathway in invasion, WASP^−/− BMMs do not invade into tumor spheroids with the same efficiency as WT BMMs and cells expressing phospho-deficient WASP have reduced ability to promote carcinoma cell invasion. Altogether, our results indicate that tyrosine phosphorylation of WASP by Hck is required for proper macrophage functions.     

Fluidization, resolidification, and reorientation of the endothelial cell in response to slow tidal stretches

Mechanical stretch plays an important role in regulating shape and orientation of the vascular endothelial cell. This morphological response to stretch is basic to angiogenesis, neovascularization, and vascular homeostasis, but mechanism remains unclear. To elucidate mechanisms, we used cell mapping rheometry to measure traction forces in primary human umbilical vein endothelial cells subjected to periodic uniaxial stretches. Onset of periodic stretch of 10% strain amplitude caused a fluidization response typified by attenuation of traction forces almost to zero. As periodic stretch continued, the prompt fluidization response was followed by a slow resolidification response typified by recovery of the traction forces, but now aligned along the axis perpendicular to the imposed stretch. Reorientation of the cell body lagged reorientation of the traction forces, however. Together, these observations demonstrate that cellular reorientation in response to periodic stretch is preceded by traction attenuation by means of cytoskeletal fluidization and subsequent traction recovery transverse to the stretch direction by means of cytoskeletal resolidification.     

Insulin secretion dynamics of free and alginate-encapsulated insulinoma cells

This study investigates the effect of alginate/poly-l-lysine/alginate (APA) encapsulation on the insulin secretion dynamics exhibited by an encapsulated cell system. Experiments were performed with the aid of a home-built perfusion apparatus providing a 1 min temporal resolution. Insulin profiles were measured from: (i) murine insulinoma βTC3 cells encapsulated in calcium alginate/poly-l-lysine/alginate (APA) beads generated with high guluronic (G) or high mannuoric (M) content alginate, and (ii) murine insulinoma βTC-tet cells encapsulated in high M APA beads and propagated in the presence and absence of tetracycline. Results show that encapsulation in APA beads did not affect the insulin secretion profile shortly post-encapsulation. However, remodeling of the beads due to cell proliferation affected the insulin secretion profiles; and inhibiting remodeling by suppressing cell growth preserved the secretion profile. The implications of these findings regarding the in vivo function of encapsulated insulin secreting cells are discussed.