Passage of 17 kDa calmodulin through gap junctions of three vertebrate species

Gap junctions of some vertebrates are capable of passing the elongate molecule, calmodulin, with a molecular weight 8-17 times greater than the previously recognized size limits. Fluorescently labeled calmodulin (FCaM) (17.34 kDa) microinjected into oocytes of ovarian follicles from an amphibian, Xenopus laevis, and from two species of teleost fish, Danio rerio (Zebrafish) and Oryzias latipes (Medaka), is shown to transit their gap junctions and enter the surrounding epithelial cells. Passage of FCaM was terminated when follicles were first treated with 1 mM octanol, a molecule known to down-regulate gap junctions. There was no FCaM detected in the surrounding medium, nor did epithelial cells become fluorescent when follicles were incubated in medium containing dye. Calmodulin is well known to modulate many cytoplasmic reactions; thus, its passage through gap junctions opens possibilities of additional means by which cells may be supplied with this signaling molecule, and by which their supply may be regulated.     

A phenomenological mixture model for biosynthesis and linking of cartilage extracellular matrix in scaffolds seeded with chondrocytes

A phenomenological mixture model is presented for interactions between biosynthesis of extracellular matrix (ECM) constituents and ECM linking in a scaffold seeded with chondrocytes. A system of three ordinary differential equations for average apparent densities of unlinked ECM, linked ECM and scaffold is developed along with associated initial conditions for scaffold material properties. Equations for unlinked ECM synthesis and ECM linking include an inhibitory mechanism where associated rates decrease as unlinked ECM concentration in the interstitial fluid increases. Linking rates are proposed to depend on average porosity in the evolving tissue construct. The resulting initial value problem contains nine independent parameters that account for scaffold biomaterial properties and interacting mechanisms in the engineered system. Effects of parameter variations on model variables are analyzed relative to a baseline case with emphasis on the evolution of solid phase apparent density, which is often correlated with the compressive elastic modulus of the tissue construct. The new model provides an additional quantitative framework for assessing and optimizing the design of engineered cell-scaffold systems and guiding strategies for articular cartilage tissue engineering.     

Conical tomography of a ribbon synapse: structural evidence for vesicle fusion

To characterize the sites of synaptic vesicle fusion in photoreceptors, we evaluated the three-dimensional structure of rod spherules from mice exposed to steady bright light or dark-adapted for periods ranging from 3 to 180 minutes using conical electron tomography. Conical tilt series from mice retinas were reconstructed using the weighted back projection algorithm, refined by projection matching and analyzed using semiautomatic density segmentation. In the light, rod spherules contained ～470 vesicles that were hemi-fused and ～187 vesicles that were fully fused (omega figures) with the plasma membrane. Active zones, defined by the presence of fully fused vesicles, extended along the entire area of contact between the rod spherule and the horizontal cell ending, and included the base of the ribbon, the slope of the synaptic ridge and ribbon-free regions apposed to horizontal cell axonal endings. There were transient changes of the rod spherules during dark adaptation. At early periods in the dark (3-15 minutes), there was a) an increase in the number of fully fused synaptic vesicles, b) a decrease in rod spherule volume, and c) an increase in the surface area of the contact between the rod spherule and horizontal cell endings. These changes partially compensate for the increase in the rod spherule plasma membrane following vesicle fusion. After 30 minutes of dark-adaptation, the rod spherules returned to dimensions similar to those measured in the light. These findings show that vesicle fusion occurs at both ribbon-associated and ribbon-free regions, and that transient changes in rod spherules and horizontal cell endings occur shortly after dark onset.     

Cell sourcing for bone tissue engineering: amniotic fluid stem cells have a delayed, robust differentiation compared to mesenchymal stem cells

Cell based therapies for bone regeneration are an exciting emerging technology, but the availability of osteogenic cells is limited and an ideal cell source has not been identified. Amniotic fluid-derived stem cells (AFS) and bone-marrow derived mesenchymal stem cells (MSCs) were compared to determine their osteogenic differentiation capacity in both 2D and 3D environments. In 2D culture, the AFS cells produced more mineralized matrix but delayed peaks in osteogenic markers. Cells were also cultured on 3D scaffolds constructed of poly-ε-caprolactone for 15 weeks. MSCs differentiated more quickly than AFS cells on 3D scaffolds, but mineralized matrix production slowed considerably after 5 weeks. In contrast, the rate of AFS cell mineralization continued to increase out to 15 weeks, at which time AFS constructs contained 5-fold more mineralized matrix than MSC constructs. Therefore, cell source should be taken into consideration when used for cell therapy, as the MSCs would be a good choice for immediate matrix production, but the AFS cells would continue robust mineralization for an extended period of time. This study demonstrates that stem cell source can dramatically influence the magnitude and rate of osteogenic differentiation in vitro.     

AP-3 directs the intracellular trafficking of HIV-1 Gag and plays a key role in particle assembly

Gag proteins direct the process of retroviral particle assembly and form the major protein constituents of the viral core. The matrix region of the HIV-1 Gag polyprotein plays a critical role in the transport of Gag to the plasma membrane assembly site. Recent evidence indicates that Gag trafficking to late endosomal compartments, including multivesicular bodies, occurs prior to viral particle budding from the plasma membrane. Here we demonstrate that the matrix region of HIV-1 Gag interacts directly with the delta subunit of the AP-3 complex, and that this interaction plays an important functional role in particle assembly. Disruption of this interaction eliminated Gag trafficking to multivesicular bodies and diminished HIV particle formation. These studies illuminate an early step in retroviral particle assembly and provide evidence that the trafficking of Gag to late endosomes is part of a productive particle assembly pathway.     

Protein expression profiling of the drosophila fragile X mutant brain reveals up-regulation of monoamine synthesis

Fragile X syndrome is the most common form of inherited mental retardation, associated with both cognitive and behavioral anomalies. The disease is caused by silencing of the fragile X mental retardation 1 (fmr1) gene, which encodes the mRNA-binding, translational regulator FMRP. Previously we established a disease model through mutation of Drosophila fmr1 (dfmr1) and showed that loss of dFMRP causes defects in neuronal structure, function, and behavioral output similar to the human disease state. To uncover molecular targets of dFMRP in the brain, we use here a proteomic approach involving two-dimensional difference gel electrophoresis analyses followed by mass spectrometry identification of proteins with significantly altered expression in dfmr1 null mutants. We then focus on two misregulated enzymes, phenylalanine hydroxylase (Henna) and GTP cyclohydrolase (Punch), both of which mediate in concert the synthetic pathways of two key monoamine neuromodulators, dopamine and serotonin. Brain enzymatic assays show a nearly 2-fold elevation of Punch activity in dfmr1 null mutants. Consistently brain neurochemical assays show that both dopamine and serotonin are significantly increased in dfmr1 null mutants. At a cellular level, dfmr1 null mutant neurons display a highly significant elevation of the dense core vesicles that package these monoamine neuromodulators for secretion. Taken together, these data indicate that dFMRP normally down-regulates the monoamine pathway, which is consequently up-regulated in the mutant condition. Elevated brain levels of dopamine and serotonin provide a plausible mechanistic explanation for aspects of cognitive and behavioral deficits in human patients.     

Deleterious genomic mutation rate for viability in Drosophila melanogaster using concomitant sibling controls

New deleterious mutations may reduce health and fitness and are involved in the evolution and maintenance of numerous biological processes. Hence, it is important to estimate the deleterious genomic mutation rate (U) in representative higher organisms. However, these estimated rates vary widely, mainly because of inadequate experimental controls. Here we describe an experimental design (the Binscy assay) with concomitant sibling controls and estimate U for viability in Drosophila melanogaster to be 0.31. This estimate, like most published studies, focuses on viability mutations and the overall deleterious genomic mutation rate would therefore be higher.     

Structural basis for a functional antagonist in the transforming growth factor beta superfamily

Within the transforming growth factor beta superfamily, the agonist-antagonist relationship between activin and inhibin is unique and critical to integrated reproductive function. Activin acts in the pituitary to stimulate follicle-stimulating hormone, and is antagonized by endocrine acting, gonadally derived inhibin. We have undertaken a mutational analysis of the activin betaA subunit to determine the precise structural aspects that contribute to inhibin antagonism of activin. By substituting specific amino acid residues in the activin betaA subunit with similarly aligned amino acids from the alpha subunit, we have pinpointed the residues required for activin receptor binding and activity, as well as for inhibin antagonism of activin through its receptors. Additionally, we have identified an activin mutant with a higher affinity for the activin type I receptor that provides structural evidence for the evolution of ligand-receptor interactions within the transforming growth factor beta superfamily.