Human osteoblast-like cells in three-dimensional culture with fluid flow

In a previous study, we showed that the combination of appropriately designed three-dimensional (3D) microcarrier scaffolds and fluid flow through and around the scaffolds during high aspect ratio vessel (HARV) rotation enhances the elaboration of mineralized bone matrix by osteoblast-like cells. In this study, we describe the ongoing characterization of our 3D culture system, including the investigation of interior fluid flow within the scaffolds and early stage integrin expression during hydrodynamic culture. Using theoretical and experimental methods, we have estimated that cells cultured on the interior of microcarrier scaffolds experience an interior nutrient flow velocity between 1 x 10(-3) and 1 x 10(-2) cm/s and maximum shear stress of 0.03 N/m(2). Under these conditions, osteoblast-like cells grew extensively in the interior regions of the scaffold and retained their osteoblastic phenotype as measured by alkaline phosphatase. In addition, flow cytometric analysis of the overall cell population showed that cells constitutively expressed integrin alpha3beta1 during 3D hydrodynamic culture.     

Conditioned defeat in male and female Syrian hamsters

A brief exposure to social defeat in male Syrian hamsters (Mesocricetus auratus) leads to profound changes in the subsequent agonistic behavior exhibited by the defeated animals. Following defeat in the home cage of an aggressive conspecific, male hamsters will subsequently fail to defend their home territory even if the intruder is a smaller, nonaggressive male. This phenomenon has been called conditioned defeat. In Experiment 1, we examined the duration of conditioned defeat by repeatedly testing (every 3-5 days) defeated hamsters with a nonaggressive intruder. We found that conditioned defeat occurs in all defeated male hamsters and persists for a prolonged period of time (at least 33 days) in the majority of male hamsters tested despite the fact that these animals are never attacked by the nonaggressive intruders. In Experiment 2, we examined whether conditioned defeat could be induced in female Syrian hamsters. While conditioned defeat occurred in some females, they displayed only low levels of submissive/defensive behavior and, in contrast to males, the conditioned defeat response did not persist beyond the first test. These results suggest that in male hamsters conditioned defeat is a profound, persistent behavioral change characterized by a total absence of territorial aggression and by the frequent display of submissive and defensive behaviors. Conversely, social defeat in female hamsters does not appear to induce long-term behavioral changes. Finally, in Experiment 3, we determined that plasma adrenocorticotropin-like immunoreactivity increases in females following social defeat in a manner similar to that seen in males, suggesting that the disparate behavioral reactions of males and females are not due to sex differences in the release of, or response to, plasma adrenocorticotropin.     

Apical EGF receptors regulate epithelial barrier to gastric acid: endogenous TGF-alpha is an essential facilitator

In previous studies, we found that apical and basolateral EGF receptors (EGFR) on primary canine gastric monolayers decreased paracellular permeability, evident by increased transepithelial electrical resistance (TER) and decreased flux of [(3)H]mannitol (MF). After studying monolayers in Ussing chambers, we now report that treatment with apical, but not basolateral, EGF enhanced tolerance to apical H(+), evident by a slower decay in TER and an attenuated rise in MF. Enhanced tolerance to apical acid was evident within 10 min of treatment with apical EGF. Immunoneutralization of endogenous transforming growth factor (TGF)-alpha accelerated the drop in TER and the rise in MF in response to apical acidification; apical EGF reversed these effects. Study of monolayers cultured in Transwell inserts showed that immunoblockade of basolateral, but not apical, EGFR also impaired the resistance to apical acidification and enhanced MF. We conclude that apical EGFR regulates the barrier to apical acidification via effects on paracellular resistance. Although exogenous basolateral EGF has a less apparent effect on the barrier to acid, endogenous ligand active at basolateral EGFR plays an important role in maintaining the barrier to apical acid. Our data implicate a role for an apical EGFR ligand, which may be EGF or another member of the EGF family.     

Secretin regulates paracellular permeability in canine gastric monolayers by a Src kinase-dependent pathway

Previous studies found that epidermal growth factor (EGF) decreased paracellular permeability in gastric mucosa, but the other physiological regulators and the molecular mechanisms mediating these responses remain undefined. We investigated the role of secretin and Src in regulating paracellular permeability because secretin regulates gastric chief cell function and Src mediates events involving the cytoskeletal-membrane interface, respectively. Confluent monolayers were formed from canine gastric epithelial cells in short-term culture on Transwell filter inserts. Resistance was monitored in the presence of secretin with or without specific kinase inhibitors. Tyrosine phosphorylation of Src at Tyr(416) was measured with a site-specific phosphotyrosine antibody. Basolateral, but not apical, secretin at concentrations from 1 to 100 nM dose dependently increased resistance; this response was rapid and sustained over hours. PP2 (10 microM), a selective Src tyrosine kinase inhibitor, but not the inactive isomer PP3, abolished the increase in resistance by secretin but only modestly attenuated apical EGF effects. AG-1478 (100 nM), a specific EGF receptor tyrosine kinase inhibitor, attenuated the resistance increase to EGF but not secretin. Secretin, but not EGF, induced tyrosine phosphorylation of Src at Tyr(416) in a dose-dependent fashion, with the maximal response observed at 1 min. PP2, but not PP3, dramatically inhibited this tyrosine phosphorylation. Secretin increases paracellular resistance in gastric mucosa through a Src-mediated pathway, while the effect of EGF is Src independent. Src appears to mediate the physiological effects of this G(s)-coupled receptor in primary epithelial cells.     

Stat3 beta inhibits gamma-globin gene expression in erythroid cells

We demonstrated previously gamma-globin gene inhibition in K562 cells and primary erythroid progenitors treated with interleukin-6. Although several cis-acting elements have been identified in the globin promoters, the precise mechanism for cytokine-mediated globin gene regulation remains to be elucidated. In this report we demonstrate inhibitors of Stat3 phosphorylation abrogate interleukin-6-mediated gamma gene silencing in erythroid cells. DNA-protein binding studies established Stat3 interaction in the 5'-untranslated gamma-globin promoter region. Furthermore, co-transfection experiments with Stat3 beta demonstrate gamma promoter inhibition in a concentration-dependent manner, which was significantly reversed when the cognate Stat3-binding site in the 5'-untranslated region was mutated. These studies establish a novel mechanism for gamma gene silencing through the STAT signal transduction pathway.     

In vitro selection of external guide sequences for directing RNase P-mediated inhibition of viral gene expression

External guide sequences (EGSs) are small RNA molecules that bind to a target mRNA, form a complex resembling the structure of a tRNA, and render the mRNA susceptible to hydrolysis by RNase P, a tRNA processing enzyme. An in vitro selection procedure was used to select EGSs that direct human RNase P to cleave the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1. One of the selected EGSs, TK17, was at least 35 times more active in directing RNase P in cleaving TK mRNA in vitro than the EGS derived from a natural tRNA sequence. TK17, when in complex with the TK mRNA sequence, resembles a portion of tRNA structure and exhibits an enhanced binding affinity to the target mRNA. Moreover, a reduction of 95 and 50% in the TK expression was found in herpes simplex virus 1-infected cells that expressed the selected EGS and the EGS derived from the natural tRNA sequence, respectively. Our study provides direct evidence that EGS molecules isolated by the selection procedure are effective in tissue culture. These results also demonstrate the potential for using the selection procedure as a general approach for the generation of highly effective EGSs for gene-targeting application.     

Priming-induced localization of G(ialpha2) in high density membrane microdomains

Subcellular fractionation of human neutrophils on linear sucrose density gradients was utilized to test the hypothesis that priming regulates the subcellular and sub-plasma membrane distribution of neutrophil G-protein subunits, G(ialpha2) and G(ialpha3), N-formyl peptide receptor, Lyn kinase and phospholipase C(beta2). G(ialpha2), but not G(ialpha3), moved from a lighter to a higher density plasma membrane fraction. Unoccupied N-formyl peptide receptors were found throughout the plasma membrane fractions and this distribution did not change with priming. In unprimed cells G(ialpha2) and its effector, phospholipase C(beta2), were segregated in different membrane compartments; priming caused G(ialpha2) to move to the compartment in which phospholipase C(beta2) resided. Thus, an important component of the mechanism of priming may involve regulation of the location of G-proteins and effector molecules in plasma membrane compartments where their abilities to couple may be enhanced.     

The intracellular domain of the Drosophila cholinesterase-like neural adhesion protein, gliotactin, is natively unfolded

Drosophila gliotactin (Gli) is a 109-kDa transmembrane, cholinesterase-like adhesion molecule (CLAM), expressed in peripheral glia, that is crucial for formation of the blood-nerve barrier. The intracellular portion (Gli-cyt) was cloned and expressed in the cytosolic fraction of Escherichia coli BLR(DE3) at 45 mg/L and purified by Ni-NTA (nitrilotriacetic acid) chromatography. Although migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), under denaturing conditions, was unusually slow, molecular weight determination by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) confirmed that the product was consistent with its theoretical size. Gel filtration chromatography yielded an anomalously large Stokes radius, suggesting a fully unfolded conformation. Circular dichroism (CD) spectroscopy demonstrated that Gli-cyt was >50% unfolded, further suggesting a nonglobular conformation. Finally, 1D-(1)H NMR conclusively demonstrated that Gli-cyt possesses an extended unfolded structure. In addition, Gli-cyt was shown to possess charge and hydrophobic properties characteristic of natively unfolded proteins (i.e., proteins that, when purified, are intrinsically disordered under physiologic conditions in vitro).