Postnatal maturation of the intestinal epithelial barrier in prairie voles.

Intestinal epithelium develops during gestation and continues to mature post-natally into a selective barrier that will protect the individual while still allowing passage of nutrients. Until fully mature, the risk of translocation of microorganisms, toxins or antigens into the sub-epithelial tissue is high and could result in pathologies with life-altering consequences, or even premature death. Because of their monogamous mating system, prairie voles are an emerging model for studying the role of the intestinal microbiota in modulating social behavior via the microbiota-gut-brain-behavior axis. However, knowledge about the voles' intestinal barrier maturation is lacking. Understanding the maturation of the intestine epithelial barrier can complement the extensive behavioral literature for future studies involving the vole gut-brain axis. In this study, we characterized intestinal barrier function by demonstrating that two-week-old prairie voles have high paracellular absorption of FITC-dextran molecules prior to markedly decreased permeability at three weeks of age. In light of the fundamental role of tight junctions in maintaining epithelial integrity regulating intestinal permeability, we examined tight junction gene expression profiles. Transmission electron microscopy was used to visualize tight junction structure. Our results provide a timeline for intestinal barrier maturation and point to tight junction proteins involved in this process in prairie voles.

Combined micro CT and histopathology for evaluation of skeletal metastasis in live animals.

Bone is a favored site for solid tumor metastasis, especially among patients with breast, lung or prostate carcinomas. Micro CT is a powerful and inexpensive tool that can be used to investigate tumor progression in xenograft models of human disease. Many previous studies have relied on terminal analysis of harvested bones to document metastatic tumor activity. The current protocol uses live animals and combines sequential micro CT evaluation of lesion development with matched histopathology at the end of the study. The approach allows for both rapid detection and evaluation of bone lesion progression in live animals. Bone resident tumors are established either by direct (intraosseous) or arterial (intracardiac) injection, and lesion development is evaluated for up to eight weeks. This protocol provides a clinically relevant method for investigating bone metastasis progression and the development of osteotropic therapeutic strategies for the treatment of bone metastases.

A flagellar A-kinase anchoring protein with two amphipathic helices forms a structural scaffold in the radial spoke complex.

A-kinase anchoring proteins (AKAPs) contain an amphipathic helix (AH) that binds the dimerization and docking (D/D) domain, RIIa, in cAMP-dependent protein kinase A (PKA). Many AKAPs were discovered solely based on the AH-RIIa interaction in vitro. An RIIa or a similar Dpy-30 domain is also present in numerous diverged molecules that are implicated in critical processes as diverse as flagellar beating, membrane trafficking, histone methylation, and stem cell differentiation, yet these molecules remain poorly characterized. Here we demonstrate that an AKAP, RSP3, forms a dimeric structural scaffold in the flagellar radial spoke complex, anchoring through two distinct AHs, the RIIa and Dpy-30 domains, in four non-PKA spoke proteins involved in the assembly and modulation of the complex. Interestingly, one AH can bind both RIIa and Dpy-30 domains in vitro. Thus, AHs and D/D domains constitute a versatile yet potentially promiscuous system for localizing various effector mechanisms. These results greatly expand the current concept about anchoring mechanisms and AKAPs.

Regulation of flagellar length in Chlamydomonas.

Chlamydomonas reinhardtii has two apically localized flagella that are maintained at an equal and appropriate length. Assembly and maintenance of flagella requires a microtubule-based transport system known as intraflagellar transport (IFT). During IFT, proteins destined for incorporation into or removal from a flagellum are carried along doublet microtubules via IFT particles. Regulation of IFT activity therefore is pivotal in determining the length of a flagellum. Reviewed is our current understanding of the role of IFT and signal transduction pathways in the regulation of flagellar length.

Medium calcium concentration determines keratin intermediate filament density and distribution in immortalized cultured thymic epithelial cells (TECs).

Isolation and culture of thymic epithelial cells (TECs) using conventional primary tissue culture techniques under conditions employing supplemented low calcium medium yielded an immortalized cell line derived from the LDA rat (Lewis [Rt1l] cross DA [Rt1a]) that could be manipulated in vitro. Thymi were harvested from 4-5-day-old neonates, enzymically digested using collagenase (1 mg/ml, 37 degrees C, 1 h) and cultured in low calcium WAJC404A medium containing cholera toxin (20 ng/ml), dexamethasone (10 nM), epidermal growth factor (10 ng/ml), insulin (10 mug/ml), transferrin (10 mug/ml), 2% calf serum, 2.5% Dulbecco's Modified Eagle's Medium (DMEM), and 1% antibiotic/antimycotic. TECs cultured in low calcium displayed round to spindle-shaped morphology, distinct intercellular spaces (even at confluence), and dense reticular-like keratin patterns. In high calcium (0.188 mM), TECs formed cobblestone-like confluent monolayers that were resistant to trypsinization (0.05%) and displayed keratin intermediate filaments concentrated at desmosomal junctions between contiguous cells. Changes in cultured TEC morphology were quantified by an analysis of desmosome/membrane relationships in high and low calcium media. Desmosomes were significantly increased in the high calcium medium. These studies may have value when considering the growth conditions of cultured primary cell lines like TECs.

Increased intercellular communication through gap junctions may contribute to progression of osteoarthritis.

Our aim was to support the hypothesis of a specific association between gap junctions in synovial tissue and the presence of osteoarthritis, as evidenced by differences between osteoarthritis and non-osteoarthritis synovia in the number of gap junctions, the amount of gap-junction protein, and the amount of enzymatic activity produced through a pathway mediated by gap-junction intercellular communication. An average of 4.41 gap junctions were found per 100 cells counted in the osteoarthritis synovia, compared with 1.00 in the controls. The amount of the gap-junction protein connexin 43 in synovial lining cells was approximately 50% greater in patients with osteoarthritis. Synovial lining cells from patients with osteoarthritis produced matrix metalloproteinases constitutively and, at higher levels, in response to stimulation by interleukin-1 beta. In both cases, intercellular communication through gap junctions was shown to be critical to the ability of the cells to secrete matrix metalloproteinases. Overall, the results indicated that gap junctions between synovial lining cells were altered significantly in patients with osteoarthritis, as a consequence of the disease process or as part of the causal chain. In either case, gap junctions seem to be a rational therapeutic target.