Lack of biologically active estrogens in commercial cow milk.

Recently, many studies have investigated potential estrogenic compounds in the human diet. Several of these investigations have studied cow milk, a mainstay of the diets of both young and old. In vitro studies have determined that estrogens can be found in milk, and that the concentration of estrogen may be correlated to the fat content in the milk. Regardless, the majority of these studies have concluded that the levels of estrogens in milk are too low to have a physiological effect. However, a recent study found that commercial 1% cow milk was uterotrophic in rats, suggesting that it contained biologically significant levels of estrogen. Using the rat model, we tested milk samples from commercial sources and with varying fat content for estrogenic activity. Ovariectomized female rats were given milk ad libitum for a period of 2 wk. After 12 d of treatment, rats were tested sequentially in an open field and an elevated plus maze to determine any effect of milk on anxiety levels. Upon completion of the behavior testing, uterine weights were examined. Regardless of milk type, no difference was observed in daily volume of milk consumed. Contrary to previous publications, no differences existed in either the behavior or the uterine weights between animals that consumed any milk type and the negative controls. These results demonstrated that none of the commercial milk types that we tested contained biologically significant estrogenic activity.

Relationship of the lumbar lordosis angle to the abdominal aortic bifurcation and inferior vena cava confluence levels.

The objective of this study is to determine the relationship of the variations of the lumbar lordosis angle (LLA) to the aortic bifurcation level and inferior vena cava (IVC) confluence level using CT angiography. A retrospective study was conducted using the data available on abdominopelvic CT angiography scans. The LLA, the level and angle of bifurcation of the aorta, the level and angle of confluence of the IVC were identified using multiplanar and 3D reconstruction. Linear regression models were fitted to the data. We interpreted 181 scans for 181 individuals having a mean age of 55 years (18-89). The most common site of aortic bifurcation was at L4-L5 disc space (34.8%) and that of vena confluence was at the upper of L5 (29.3%). The mean LLA was 34.65° (13°-77°). The mean aortic bifurcation angle was 47.43° (17°-100°) and the mean IVC confluence angle was 71.86° (30°-120°). The positions of the aortic bifurcation and venous confluence levels showed a proximal shift with an increasing LLA P < 0.001. This study showed that the level of bifurcation of the aorta and the level of confluence of the IVC may vary with the variations of the LLA.

Adsorption of short-chain thiols and disulfides onto gold under defined mass transport conditions: coverage, kinetics, and mechanism.

The adsorption of water-soluble alkane thiols and their corresponding disulfides onto gold was followed in real time using highly sensitive surface conductivity measurements. Particular attention was paid to producing clean surfaces and to the purity of the adsorbates. The rate of mass transport to the surface was constant, controlled, and measured, over the whole time course of the experiment (1-10(4) s), by convective diffusion. An adsorption rate equation derived for coupled steady state convective-diffusion mass transport and Langmuir kinetics shows that systems limited by mass transport must also be slowed by Langmuir kinetics. Thiols and disulfides adsorbed at the same rate, limited mainly by mass transport. The distinct slowdown in adsorption rate for longer alkanethiols, attributed to conformational transitions (lying down → standing up), was less evident for the neutral thiols/disulfides. The slower rate of charged thiol adsorption is thought to stem from steric interactions of large, hydrated tail groups, although calcium as a counterion accelerated monolayer formation. The adsorption kinetics of a charged thiol were almost the same under screened (by extra added salt) or unscreened conditions. Therefore, long-range electrostatic interactions appear to be less important than short-range steric ones in limiting adsorption rates at surfaces.

Electrochemically addressed cross-links in polyelectrolyte multilayers: cyclic duravoltammetry.

In situ nanoindentation was performed on a multilayer of poly(acrylic acid) and a high molecular weight, pendant chain polyviologen under controlled electrochemical potential. The modulus of the thin film of polyelectrolyte complex was reversibly modulated, by about an order of magnitude, upon changing the state of charge within the material using the electrochemically active and addressable viologen repeat units. The applied potential, under aqueous conditions, is believed to control the extent of cross-link formation. Simultaneous quartz crystal microbalance measurements revealed the flux of ions into or out of the multilayer during redox cycling. Apparent film modulus also depends on the identity of the last layer.

Correlating the compliance and permeability of photo-cross-linked polyelectrolyte multilayers.

Photo-cross-linkable polyelectrolyte multilayers were made from poly(allylamine) (PAH) and poly(acrylic acid) (PAA) modified with a photosensitive benzophenone. Nanoindentation, using atomic force microscopy (AFM) of these and unmodified PAH/PAA multilayers, was used to assess their mechanical properties in situ under an aqueous buffer. Under the conditions employed (and a 20 nm radius AFM tip), reliable nanoindentations that appeared to be decoupled from the properties of the silicon substrate were obtained for films greater than 150 nm in thickness. A strong difference in the apparent modulus was observed for films terminated with positive as compared to negative polyelectrolytes. Films terminated with PAA were more glassy, suggesting better charge matching of polyelectrolytes. Multilayers irradiated for up to 100 min showed a smooth, controlled increase in the modulus with little change in the water contact angle. The permeability to iodide ion, measured electrochemically, also decreased in a controlled fashion.

Smooth muscle cell phenotype modulation and contraction on native and cross-linked polyelectrolyte multilayers.

Smooth muscle cells convert between a motile, proliferative "synthetic" phenotype and a sessile, "contractile" phenotype. The ability to manipulate the phenotype of aortic smooth muscle cells with thin biocompatible polyelectrolyte multilayers (PEMUs) with common surface chemical characteristics but varying stiffness was investigated. The stiffness of (PAH/PAA) PEMUs was varied by heating to form covalent amide bond cross-links between the layers. Atomic force microscopy (AFM) showed that cross-linked PEMUs were thinner than those that were not cross-linked. AFM nanoindentation demonstrated that the Young's modulus ranged from 6 MPa for hydrated native PEMUs to more than 8 GPa for maximally cross-linked PEMUs. Rat aortic A7r5 smooth muscle cells cultured on native PEMUs exhibited morphology and motility of synthetic cells and expression of the synthetic phenotype markers vimentin, tropomyosin 4, and nonmuscle myosin heavy chain IIB (nmMHCIIB). In comparison, cells cultured on maximally cross-linked PEMUs exhibited the phenotype markers calponin, smooth muscle myosin heavy chain (smMHC), myocardin, transgelin, and smooth muscle alpha-actin (smActin) that are characteristic of the smooth muscle "contractile" phenotype. Consistent with those cells being "contractile", A7r5 cells grown on cross-linked PEMUs produced contractile force when stimulated with a Ca(2+) ionophore.

Human coronary artery smooth muscle cell responses to bioactive polyelectrolyte multilayer interfaces.

Under normal physiological conditions, mature human coronary artery smooth muscle cells (hCASMCs) exhibit a "contractile" phenotype marked by low rates of proliferation and protein synthesis, but these cells possess the remarkable ability to dedifferentiate into a "synthetic" phenotype when stimulated by conditions of pathologic stress. A variety of polyelectrolyte multilayer (PEMU) films are shown here to exhibit bioactive properties that induce distinct responses from cultured hCASMCs. Surfaces terminated with Nafion or poly(styrenesulfonic acid) (PSS) induce changes in the expression and organization of intracellular proteins, while a hydrophilic, zwitterionic copolymer of acrylic acid and 3-[2-(acrylamido)-ethyl dimethylammonio] propane sulfonate (PAA-co-PAEDAPS) is resistant to cell attachment and suppresses the formation of key cytoskeletal components. Differential expression of heat shock protein 90 and actin is observed, in terms of both their magnitude and cellular localization, and distinct cytoplasmic patterns of vimentin are seen. The ionophore A23187 induces contraction in confluent hCASMC cultures on Nafion-terminated surfaces. These results demonstrate that PEMU coatings exert direct effects on the cytoskeletal organization of attaching hCASMCs, impeding growth in some cases, inducing changes consistent with phenotypic modulation in others, and suggesting potential utility for PEMU surfaces as a coating for coronary artery stents and other implantable medical devices.

N-isopropylacrylamide-based thermoresponsive polyelectrolyte multilayer films for human mesenchymal stem cell expansion.

Human mesenchymal stem cells (hMSCs) are colony-forming unit fibroblasts (CFU-F) derived from adult bone marrow and have significant potential for many cell-based tissue-engineering applications. Their therapeutic potential, however, is restricted by their diminishing plasticity as they are expanded in culture. In this study, we used N-isopropylacrylamide (NIPAM)-based thermoresponsive polyelectrolyte multilayer (N-PEMU) films as culture substrates to support hMSC expansion and evaluated their effects on cell properties. The N-PEMU films were made via layer-by-layer adsorption of thermoresponsive monomers copolymerized with charged monomers, positively charged allylamine hydrochloride (PAH), or negatively charged styrene sulfonic acid (PSS) and compared to fetal bovine serum (FBS) coated surfaces. Surface charges were shown to alter the extracellular matrix (ECM) structure and subsequently regulate hMSC responses including adhesion, proliferation, integrin expression, detachment, and colony forming ability. The positively charged thermal responsive surfaces improved cell adhesion and growth in a range comparable to control surfaces while maintaining significantly higher CFU-F forming ability. Immunostaining and Western blot results indicate that the improved cell adhesion and growth on the positively charged surfaces resulted from the elevated adhesion of ECM proteins such as fibronectin on the positively charge surfaces. These results demonstrate that the layer-by-layer approach is an efficient way to form PNIPAM-based thermal responsive surfaces for hMSC growth and removal without enzymatic treatment. The results also show that surface charge regulates ECM adhesion, which in turn influences not only cell adhesion but also CFU-forming ability and their multi-lineage differentiation potential.

Hydrogen bonding versus ion pairing in polyelectrolyte multilayers with homopolynucleotides.

Homopolynucleotides--poly(adenylic acid), poly(A), and poly(uridylic acid), poly(U)--were assembled, layer-by-layer, into thin films with poly(ethylenimine), PEI. Various combinations and sequences of polynucleotide and PEI were used to highlight contributions of electrostatic versus hydrogen bonding as driving forces for multilayer build-up. Assembly of alternating poly(A) and poly(U) failed to yield growing films, due to excessively strong interactions between these complimentary strands. The surface morphology of multilayers depended on the deposition order and whether films had been annealed by salt. Films assembled from preformed A/U duplexes (having high persistence lengths) were very smooth. Individual adsorption steps, followed by optical waveguide light-mode spectroscopy, showed that only complementary polynucleotides adsorb by H-bonding to the surface of a growing multilayer. In contrast to behavior usually observed for polyelectrolyte multilayer build-up, the films decreased in thickness with increasing salt concentration.

Fibronectin and cell attachment to cell and protein resistant polyelectrolyte surfaces.

Culture of A7r5 smooth muscle cells on a polyelectrolyte multilayer film (PEMU) can influence various cell properties, including adhesion, motility, and cytoskeletal organization, that are modulated by the extracellular matrix (ECM) in vivo. ECM contribution to cell behavior on PEMUs was investigated by determining the amount of fibronectin (FN) bound to charged and hydrophobic PEMUs by optical waveguide lightmode spectroscopy and immunofluorescence microscopy. FN bound best to poly(allylamine hydrochloride) (PAH)-terminated and Nafion-terminated PEMUs. FN bound poorly to PEMUs terminated with a copolymer of poly(acrylic acid) (PAA) and 3-[2-(acrylamido)-ethyl dimethylammonio] propane sulfonate (PAA-co-AEDAPS). Cells adhered and spread well on the Nafion-terminated PEMU surfaces. In contrast, cells spread less and migrated more on both FN-coated and uncoated PAH-terminated PEMU surfaces. Both cells and FN interacted much better with Nafion than with PAA-co-PAEDAPS in a micropatterned PEMU. These results indicate that A7r5 cell adhesion, spreading, and motility on PEMUs can be independent of FN binding to the surfaces.