Monolayer cultures of rat hepatocytes were treated with increasing concentrations of saponin (prepared from Gypsophila plants) for 30 min at 6 degrees C. Differential permeabilization of the intracellular membranes could be demonstrated: at 0.040 mg of saponin/ml the plasma membrane was permeabilized, as assessed by the release of 50% of the total cellular amount of lactate dehydrogenase, and at 0.20 mg/ml the endoplasmic reticulum was permeabilized, as measured by the release of 50% of pulse-35S-labelled albumin. The Golgi complex was permeabilized at an intermediate saponin concentration, as indicated by the release of homogeneously 35S-labelled albumin; about half the intracellular albumin is located in this organelle. At 1.0 up to 5.0 mg of saponin/ml 90-95% of the radioactively labelled albumin was released. Even at 5.0 mg/ml less than 10% of the membrane of the endoplasmic reticulum was solubilized, as judged by the degree of release of a membrane-bound enzyme specific for this organelle. These results demonstrate the usefulness of saponin as a tool for investigating the interior of different intracellular compartments.

Gamete recognition has been studied extensively in the mouse. In this system, it is generally believed that sperm bind to a class of O-linked oligosaccharides on the zona pellucida glycoprotein, ZP3. The best characterized sperm receptor for ZP3 is β1,4-galactosyltransferase (GalT), which functions in a lectin-like capacity by binding to N-terminal N-acetylglucosamine residues on ZP3 oligosaccharides. Multivalent oligosaccharides on ZP3, as well as synthetic polymers terminating in N-acetylglucosamine aggregate GalT, leading to activation of a heterotrimeric G protein cascade and culminating in the acrosome reaction. Following fertilization, cortical granules release N-acetylglucosaminidase, which removes the binding site for sperm GalT and facilitates the zona block to polyspermic binding. Genetic manipulation of GalT expression has confirmed its function as a ZP3 receptor. Overexpressing GalT on sperm leads to increased binding of ZP3, increased G protein activation, and precocious acrosome reactions. In contrast, sperm from mice made null for GalT by homologous recombination are refractory to ZP3, in that they are unable to bind soluble ZP3 and fail to undergo the acrosome reaction in response to zona glycoproteins. Surprisingly, GalT null sperm still bind to the zona and achieve low rates of fertilization in vitro. This then suggests that sperm-egg binding involves receptor-ligand interactions independent of GalT and ZP3. The current model suggests that GalT functions as the ZP3 receptor that is responsible for inducing the acrosome reaction, whereas initial sperm-zona binding is dictated by other sperm surface receptors. Consistent with this, at least three other zona pellucida monosaccharides have been implicated in sperm binding, and novel sperm surface glycoproteins have been suggested to function in gamete binding. A large scaffolding protein has been identified that associates with the GalT cytoplasmic domain and may be responsible for orchestrating its signal transduction capacities that lead to the acrosome reaction.

Objective MicroRNA-133a (miR-133a) and insulin-like growth factor-1 (IGF-1) are two different molecules known to regulate cardiovascular cell proliferation. This study tested whether miR-133a affects expression of IGF-1 receptor (IGF-1R) and proliferation of IGF-1-stimulated vascular smooth muscle cells (VSMC) in a murine model of atherosclerosis. Methods and Results Expression of IGF-1R was analyzed by immuno-fluorescence and immuno-blotting, and miR-133a by qRT-PCR in the aortas of wild-type C57BL/6J (WT) and apolipoprotein-E deficient (ApoE−/−) mice. Compared to those in WT aortas, the IGF-1R and miR-133a levels were lower in ApoE−/− aortas. ApoE−/− VSMC grew slower than WT cells in the cultures with IGF-1-containing medium. MiR-133a-specific inhibitor decreased miR-133a, IGF-1R expression, IGF-1-stimulated VSMC growth in lipoprotein-deficient media. By contrast, miR-133a precursor increased IGF-1R levels and promoted IGF-1-induced VSMC proliferation. In the luciferase-IGF-1R 3’UTR reporter system, the reporter luciferase activity was not inhibited in VSMC with miR-133a overexpression. IGF-1R mRNA half-life in ApoE−/− VSMC was shorter than that in WT VSMC. MiR-133a inhibitor reduced but precursor increased the mRNA half-life, although the effects appeared less striking in ApoE−/− VSMC than in WT cells. Conclusion MiR-133a serves as a stimulatory factor for IGF-1R expression through prolonging IGF-1R mRNA half-life. In atherosclerosis induced by ApoE deficiency, reduced miR-133a expression is associated with lower IGF-1R levels and suppressive VSMC growth. Administration of miR-133a precursor may potentiate IGF-1 stimulated VSMC survival and growth.

Embryonic stem (ES) cells are pluripotent cells that are capable of differentiating all the somatic cell lineages, including those in the liver tissue. We describe the generation of functional hepatic-like cells from mouse ES (mES) cells using a biodegradable polymer scaffold and a rotating bioreactor that allows simulated microgravity. Cells derived from ES cells cultured in the three-dimensional (3D) culture system with exogenous growth factors and hormones can differentiate into hepatic-like cells with morphologic characteristics of typical mature hepatocytes. Reverse-transcription polymerase chain-reaction testing, Western blot testing, immunostaining, and flow cytometric analysis show that these cells express hepatic-specific genes and proteins during differentiation. Differentiated cells on scaffolds further exhibit morphologic traits and biomarkers characteristic of liver cells, including albumin production, cytochrome P450 activity, and low-density lipoprotein uptake. When these stem cell-bearing scaffolds are transplanted into severe combined immunodeficient mice, the 3D constructs remained viable, undergoing further differentiation and maturation of hepatic-like cells in vivo. In conclusion, the growth and differentiation of ES cells in a biodegradable polymer scaffold and a rotating microgravity bioreactor can yield functional and organizational hepatocytes useful for research involving bioartificial liver and engineered liver tissue.

In atherosclerosis, the loss of vascular stem cells via apoptosis impairs the capacity of the vascular wall to repair or regenerate the tissue damaged by atherogenic factors. Recruitment of exogenous stem cells to the plaque tissue may repopulate vascular cells and help repair the arterial tissue. Ultrasound-enhanced liposomal targeting may provide a feasible method for stem cell delivery into atheroma. Bifunctional echogenic immunoliposomes (BF-ELIP) were generated by covalently coupling two antibodies to liposomes; the first one specific for CD34 antigens on the surface of stem cells and the second directed against the intercellular adhesion molecule-1 (ICAM-1) antigens on the inflammatory endothelium covering atheroma. CD34+ stem cells from adult bone marrow were incubated on the ICAM-1-expressing endothelium of the aorta of swine fed high cholesterol diets, which was preloaded with BF-ELIP. Significantly increased stem cell adherence and penetration were detected, in particular in the aortic segments treated with 1 MHz low-amplitude continuous wave ultrasound. Fluorescence and scanning electron microscopy confirmed the presence of BF-ELIP-bound CD34+ cells in the intimal compartment of the atheromatous arterial wall. Ultrasound treatment increased the number of endothelial cell progenitors migrating into the intima. Thus, under ultrasound enhancement, BF-ELIP bound CD34+ stem cells selectively bind to the ICAM-1 expressing endothelium of atherosclerotic lesions.

Intrinsically echogenic liposomes (ELIP) can be adapted to encapsulate nitric oxide to facilitate ultrasound-enhanced delivery of therapeutic agents to atherosclerotic plaques. However, the NO loading of targeted ELIP caused a 93% decrease of antibody (Ab) immunoreactivity. The following hypothesis was tested: biotin/avidin-mediated coupling of NO-ELIP and Ab-conjugated ELIP will enable co-delivery of bioactive gases and ELIP that can encapsulate other agents without loss of targeting efficiency. Complex formation was initiated by addition of excess streptavidin to equal proportions of biotinylated Ab-ELIP and NO-ELIP. Fluorescence deconvolution microscopy, Coulter Multisizer 3 analysis and flow cytometry demonstrated that the ELIP coupling procedure formed mixed aggregates of ≥10 liposomes within 1 minute. Intravascular ultrasound imaging and ELISA showed that echogenicity and targeting efficiency were completely and 69-99% retained, respectively. When complexed to NO-ELIP, ELIP bifunctionally targeted to both CD34 and ICAM-1 (BF-ELIP) increased human mononuclear cell migration through human coronary artery endothelial cell monolayers in transwell plates 4-fold relative to a nonspecific IgG-ELIP control and 2-fold relative to BF-ELIP alone. It was concluded that this novel multi-functional conjugation methodology provides a platform technology for site-specific co-delivery of bioactive gases and other agents.

Our earlier studies in mouse have shown that the cystatin-related epididymal spermatogenic (CRES) protein is highly expressed in elongating spermatids in the testis and is present in mouse sperm acrosomes, suggesting specific roles in sperm function, fertilization, or both. However, whether the human CRES gene is similar to that of the mouse and is expressed in germ cells has not yet been determined. Therefore, the present study was undertaken to characterize the human ortholog of mouse CRES: Northern blot and in situ hybridization experiments showed that CRES is highly expressed in the human testis, specifically within clusters of round spermatids. Furthermore, reverse transcription-polymerase chain reaction detected CRES mRNA in the epididymis. Western blot analysis of protein lysates prepared from human testis and ejaculated spermatozoa showed a predominant 19-kDa protein and a minor 14-kDa protein. However, in contrast to the acrosomal localization of CRES protein in mouse spermatozoa, indirect immunofluorescence of human spermatozoa treated with methanol/acetic acid using anti-human CRES antibodies revealed that CRES was strictly localized to the equatorial segment. Furthermore, the same staining was observed in both capacitated and acrosome-reacted spermatozoa. To determine whether CRES was associated with the plasma membrane, live spermatozoa were incubated with CRES antibody after capacitation and acrosome reaction. Only acrosome-reacted spermatozoa showed a weak but specific equatorial staining. Taken together, these studies show that CRES protein is present in the sperm equatorial segment and becomes accessible to the extracellular environment during fertilization.

SummaryMaternal nicotine exposure causes alteration of gene expression and cardiovascular programming. The discovery of nicotine-medicated regulation in cardiogenesis is of major importance for the study of cardiac defects. The present study investigated the effect of nicotine on cardiac gene expression and epigenetic regulation during myocardial differentiation. Persistent nicotine exposure selectively inhibited expression of two cardiac genes, Tbx5 and Gata4, by promoter DNA hypermethylation. The nicotine-induced suppression on cardiac differentiation was restored by general nicotinic acetylcholine receptor inhibition. Consistent results of Tbx5 and Gata4 gene suppression and cardiac function impairment with decreased left ventricular ejection fraction were obtained from in vivo studies in offspring. Our results present a direct repressive effect of nicotine on myocardial differentiation by regulating cardiac gene suppression via promoter DNA hypermethylation, contributing to the etiology of smoking-associated cardiac defects.