Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.     

Disruption of the GDNF binding site in NCAM dissociates ligand binding and homophilic cell adhesion

Most plasma membrane proteins are capable of sensing multiple cell-cell and cell-ligand interactions, but the extent to which this functional versatility is founded on their modular design is less clear. We have identified the third immunoglobulin domain of the Neural Cell Adhesion Molecule (NCAM) as the necessary and sufficient determinant for its interaction with Glial Cell Line-derived Neurotrophic Factor (GDNF). Four charged contacts were identified by molecular modeling as the main contributors to binding energy. Their mutation abolished GDNF binding to NCAM but left intact the ability of NCAM to mediate cell adhesion, indicating that the two functions are genetically separable. The GDNF-NCAM interface allows complex formation with the GDNF family receptor alpha1, shedding light on the molecular architecture of a multicomponent GDNF receptor.     

The use of environmental DNA metabarcoding and quantitative PCR for molecular detection of marine invasive non-native species associated with artificial structures

Biological Invasions - Artificial coastal structures associated with coastal defences, energy generation, ports, marinas and other developments, are known to support lower levels of biodiversity...     

Wood addition in the hatchery and river environments affects post‐release performance of overwintering brown trout

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Quantitative detection in the attomole range for immunochromatographic tests by means of a flatbed scanner

This work describes the use of the combination of carbon black as an antibody label, a membrane-based immunochromatographic device, and a flatbed scanner as a quantitative test system. The scanner detected 0.4-345 ng carbon black/mm(2) on a nitrocellulose membrane (0.2-170 amol carbon black/mm(2)) with an imprecision (coefficient of variation, CV) lower than 2% for the carbon black determination and a detection limit of 0.04 ng carbon black/mm(2) (0.02 amol/mm(2)). The detection ability was compared to that obtained with alkaline phosphatase (ALP) using a substrate yielding a chemiluminescent signal (0.02 amol ALP/well), beta-galactosidase using a substrate yielding a fluorescent signal (0.3 amol beta-galactosidase/well), and horseradish peroxidase (HRP) using a substrate yielding a colored signal (5 amol HRP/microtiter well). The carbon black immunochromatographic test for immunoglobulin E (IgE) showed a detection limit of 0.13 pM IgE (0.01 kU/L) after a testing time of 10 min. The scanner detection imprecision for the IgE determination was 0.6% CV in the range 1-10 kU IgE/L when 2.3 mm(2) was used for detection and 1% CV when 0.19 mm(2) was used. A flatbed scanner is an inexpensive instrument with multiple uses, which now also includes the sensitive evaluation of immunoassays.     

Characterization of a molten globule state of bovine carbonic anhydrase III: loss of asymmetrical environment of the aromatic residues has a profound effect on both the near- and far-UV CD spectrum

Bovine muscle carbonic anhydrase (isoenzyme III; BCAIII) exhibited a three-state unfolding process at equilibrium upon denaturation in guanidine hydrochloride (GuHCl). The stable folding intermediate appeared to be of molten globule type. The stability towards GuHCl in terms of mid-point concentrations of denaturation were very similar for BCAIII and human CAII (HCAII). It was further demonstrated that the aromatic amino acid residues contributed significantly to the circular dichroism (CD) spectrum in the far-UV wavelength region during the native-->molten globule state transition. Thus, the ellipiticity change at 218 nm was shown to monitor the loss of tertiary interactions of aromatic side chains at the first unfolding transition as well as the rupture of secondary structure at the second unfolding transition. Similar aromatic contributions to the far-UV CD spectrum, but with varying magnitudes, were also noted for BCAII and HCAII, further emphasizing that interference of aromatic residues should not be neglected at wavelengths that normally are assigned to secondary structural changes.     

Saturation of the endoplasmic reticulum retention machinery reveals anterograde bulk flow

We have studied the possible mechanisms of endoplasmic reticulum (ER) export and retention by using natural residents of the plant ER. Under normal physiological conditions, calreticulin and the lumenal binding protein (BiP) are efficiently retained in the ER. When the ER retention signal is removed, truncated calreticulin is much more rapidly secreted than truncated BiP. Calreticulin carries two glycans of the typical ER high-mannose form. Both glycans are competent for Golgi-based modifications, as determined from treatment with brefeldin A or based on the deletion of the ER retention motif. In contrast to BiP, calreticulin accumulation is strongly dependent on its retention signal, thereby allowing us to test whether saturation of the retention mechanism is possible. Overexpression of calreticulin led to 100-fold higher levels in dilated globular ER cisternae as well as dilated nuclear envelopes and partial secretion of both BiP and calreticulin. This result shows that both molecules are competent for ER export and supports the concept that proteins are secreted by default. This result also supports previous data suggesting that truncated BiP devoid of its retention motif can be retained in the ER by association with calreticulin. Moreover, even under these saturating conditions, cellular calreticulin did not carry significant amounts of complex glycans, in contrast to secreted calreticulin. This result shows that calreticulin is rapidly secreted once complex glycans have been synthesized in the medial/trans Golgi apparatus and that the modified protein does not appear to recycle back to the ER.     

Introduction to galectins

Good evidence suggest roles of galectins in cancer, immunity and inflammation, and development, but a unifying picture of their biological function is lacking. Instead galectins appear to have a particularly diverse, bewildering but intriguing array of activities both inside and outside cells--"clear truths and mysteries are inextricably twined". Fortunately this has not discouraged but rather enthused a large number of good galectin researchers, some of which have contributed to this special issue of Glycoconjugate Journal to provide a personal, critical status of the field. Here we will give a brief introduction to the galectins as a protein family with some comments on nomenclature.     

Evidence for myofibril remodeling as opposed to myofibril damage in human muscles with DOMS: an ultrastructural and immunoelectron microscopic study

The myofibrillar and cytoskeletal alterations observed in delayed onset muscle soreness (DOMS) caused by eccentric exercise are generally considered to represent damage. By contrast our recent immunohistochemical studies suggested that the alterations reflect myofibrillar remodeling (Yu and Thornell 2002; Yu et al. 2003). In the present study the same human muscle biopsies were further analyzed with transmission electron microscopy and immunoelectron microscopy. We show that the ultrastructural hallmarks of DOMS, Z-disc streaming, Z-disc smearing, and Z-disc disruption were present in the biopsies and were significantly more frequent in biopsies taken 2–3 days and 7–8 days after exercise than in those from controls and 1 h after exercise. Four main types of changes were observed: amorphous widened Z-discs, amorphous sarcomeres, double Z-discs, and supernumerary sarcomeres. We confirm by immunoelectron microscopy that the main Z-disc protein alpha-actinin is not present in Z-disc alterations or in the links of electron-dense material between Z-discs in longitudinal register. These alterations were related to an increase of F-actin and desmin, where F-actin was present within the strands of amorphous material. Desmin, on the other hand, was seen in less dense regions of the alterations. Our results strongly support that the myofibrillar and cytoskeletal alterations, considered to be the hallmarks of DOMS, reflect an adaptive remodeling of the myofibrils.     

The Lim homeobox gene Lhx2 is required for olfactory sensory neuron identity

Progenitor cells in the mouse olfactory epithelium generate over a thousand subpopulations of neurons, each expressing a unique odorant receptor (OR) gene. This event is under the control of spatial cues, since neurons in different epithelial regions are restricted to express region-specific subsets of OR genes. We show that progenitors and neurons express the LIM-homeobox gene Lhx2 and that neurons in Lhx2-null mutant embryos do not diversify into subpopulations expressing different OR genes and other region-restricted genes such as Nqo1 and Ncam2. Lhx2-/- embryos have, however, a normal distribution of Mash1-positive and neurogenin 1-positive neuronal progenitors that leave the cell cycle, acquire pan-neuronal traits and form axon bundles. Increased cell death in combination with increased expression of the early differentiation marker Neurod1, as well as reduced expression of late differentiation markers (Galphaolf and Omp), suggests that neuronal differentiation in the absence of Lhx2 is primarily inhibited at, or immediate prior to, onset of OR expression. Aberrant regional expression of early and late differentiation markers, taken together with unaltered region-restricted expression of the Msx1 homeobox gene in the progenitor cell layer of Lhx2-/- embryos, shows that Lhx2 function is not required for all aspects of regional specification of progenitors and neurons. Thus, these results indicate that a cell-autonomous function of Lhx2 is required for differentiation of progenitors into a heterogeneous population of individually and regionally specified mature olfactory sensory neurons.