Endogenous tissue engineering: PTH therapy for skeletal repair

Based on its proven anabolic effects on bone in osteoporosis patients, recombinant parathyroid hormone (PTH_1-34) has been evaluated as a potential therapy for skeletal repair. In animals, the effect of PTH_1-34 has been investigated in various skeletal repair models such as fractures, allografting, spinal arthrodesis and distraction osteogenesis. These studies have demonstrated that intermittent PTH_1-34 treatment enhances and accelerates the skeletal repair process via a number of mechanisms, which include effects on mesenchymal stem cells, angiogenesis, chondrogenesis, bone formation and resorption. Furthermore, PTH_1-34 has been shown to enhance bone repair in challenged animal models of aging, inflammatory arthritis and glucocorticoid-induced bone loss. This pre-clinical success has led to off-label clinical use and a number of case reports documenting PTH_1-34 treatment of delayed-unions and non-unions have been published. Although a recently completed phase 2 clinical trial of PTH_1-34 treatment of patients with radius fracture has failed to achieve its primary outcome, largely because of effective healing in the placebo group, several secondary outcomes are statistically significant, highlighting important issues concerning the appropriate patient population for PTH_1-34 therapy in skeletal repair. Here, we review our current knowledge of the effects of PTH_1-34 therapy for bone healing, enumerate several critical unresolved issues (e.g., appropriate dosing regimen and indications) and discuss the long-term potential of this drug as an adjuvant for endogenous tissue engineering.     

Hight performance gel permeation chromatography of proteins

Proteins in the molecular weight range of 10 000–170 000 were separated by high performance gel permeation chromatography. Silica particles with 30 nm or 50 nm pores were derivatized with glycidoxy-propyltrimethoxysilane and used as support. The proteins were eluted with 50% formic acid. A protein fraction which induces endodermal and mesodermal tissues in amphibian gastrula ectoderm was purified by this method.     

Molecular mechanisms of ceramide-mediated CD95 clustering.

Receptor clustering has been suggested as a crucial mechanism to initiate receptor signaling. Here we show that ceramide in sphingolipid-rich membrane rafts mediates clustering of CD95. Neutralization of surface ceramide or inhibition of its endogenous generation prevented CD95 clustering. Furthermore, application of ceramide at the cell surface triggered clustering of active but not inactive CD95. Apoptosis was inhibited by neutralization of surface ceramide or inhibition of ceramide release in vitro and in vivo. Thus, we conclude that surface ceramide mediates CD95 clustering, which is required for initiation of apoptosis, at least in some cell types.     

Views,landmarks, and routes: how do desert ants negotiate an obstacle course?

The Australian desert ant Melophorus bagoti often follows stereotypical routes through a cluttered landscape containing both distant panoramic views and obstacles (plants) to navigate around. We created an artificial obstacle course for the ants between a feeder and their nest. Landmarks comprised natural objects in the landscape such as logs, branches, and tussocks. Many ants travelled stereotypical routes home through the obstacle course in training, threading repeatedly the same gaps in the landmarks. Manipulations altering the relations between the landmarks and the surrounding panorama, however, affected the routes in two major ways. Both interchanging the positions of landmarks (transpositions) and displacing the entire landmark set along with the starting position of the ants (translations) (1) reduced the stereotypicality of the route, and (2) increased turns and meanders during travel. The ants might have used the entire panorama in view-based travel, or the distal panorama might prime the identification and use of landmarks en route. Despite the large data set, both options (not mutually exclusive) remain viable.     

Seminiferous tubule transfection in vitro to define post-meiotic gene regulation

Background  

Post-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. This article reports the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation.     

The signal sequence suffices to direct export of outer membrane protein OmpA of Escherichia coli K-12.

We studied whether information required for export is present within the mature form of the Escherichia coli 325-residue outer membrane protein OmpA. We had previously analyzed overlapping internal deletions in the ompA gene, and the results allowed us to conclude that if such information exists it must be present repeatedly within the membrane part of the protein encompassing amino acid residues 1 to 177 (R. Freudl, H. Schwarz, M. Klose, N. R. Movva, and U. Henning, EMBO J. 4:3593-3598, 1985). A deletion which removed the codons for amino acid residues 1 to 229 of the OmpA protein was constructed. In this construct the signal sequence was fused to the periplasmic part of the protein. The resulting protein, designated Pro-OmpA delta 1-229, was processed, and the mature 95-residue protein accumulated in the periplasm. Hence, information required for export does not exist within the OmpA protein.