Prediction of heparin binding sites in bone morphogenetic proteins (BMPs)

Heparin is a glycosaminoglycan known to bind bone morphogenetic proteins (BMPs) and the growth and differentiation factors (GDFs) and has strong and variable effects on BMP osteogenic activity. In this paper we report our predictions of the likely heparin binding sites for BMP-2 and 14. The N-terminal sequences upstream of TGF-β-type cysteine-knot domains in BMP-2, 7 and 14 contain the basic residues arginine and lysine, which are key components of the heparin/HS-binding sites, with these residues being highly non-conserved. Importantly, evolutionary conserved surfaces on the beta sheets are required for interactions with receptors and antagonists. Furthermore, BMP-2 has electropositive surfaces on two sides compared to BMP-7 and BMP-14. Molecular docking simulations suggest the presence of high and low affinity binding sites in dimeric BMP-2. Histidines were found to play a role in the interactions of BMP-2 with heparin; however, a pKa analysis suggests that histidines are likely not protonated. This is indicative that interactions of BMP-2 with heparin do not require acidic pH. Taken together, non-conserved amino acid residues in the N-terminus and residues protruding from the beta sheet (not overlapping with the receptor binding sites and the dimeric interface) and not C-terminal are found to be important for heparin–BMP interactions.

Effect of surface conditions on flow of a micropolar fluid driven by a porous stretching sheet

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Flow of a micropolar fluid bounded by a stretching sheet

We consider boundary layer flow of a micropolar fluid driven by a porous stretching sheet. A similarity solution is defined, and numerical solutions using Runge-Kutta and quasilinearisation schemes are obtained. A perturbation analysis is also used to derive analytic solutions to first order in the perturbing parameter. The resulting closed form solutions involve relatively complex expressions, and the analysis is made more tractable by a combination of offline and online work using a computational algebra system (CAS). For this combined numerical and analytic approach, the perturbation analysis yields a number of benefits with regard to the numerical work. The existence of a closed form solution helps to discriminate between acceptable and spurious numerical solutions. Also, the expressions obtained from the perturbation work can provide an accurate description of the solution for ranges of parameters where the numerical approaches considered here prove computationally more difficult.

Vitronectin modulates human mesenchymal stem cell response to insulin-like growth factor-I and transforming growth factor beta 1 in a serum-free environment

The use of animal sera for the culture of therapeutically important cells impedes the clinical use of the cells. We sought to characterize the functional response of human mesenchymal stem cells (hMSCs) to specific proteins known to exist in bone tissue with a view to eliminating the requirement of animal sera. Insulin-like growth factor-I (IGF-I), via IGF binding protein-3 or -5 (IGFBP-3 or -5) and transforming growth factor-beta 1 (TGF-beta(1)) are known to associate with the extracellular matrix (ECM) protein vitronectin (VN) and elicit functional responses in a range of cell types in vitro. We found that specific combinations of VN, IGFBP-3 or -5, and IGF-I or TGF-beta(1) could stimulate initial functional responses in hMSCs and that IGF-I or TGF-beta(1) induced hMSC aggregation, but VN concentration modulated this effect. We speculated that the aggregation effect may be due to endogenous protease activity, although we found that neither IGF-I nor TGF-beta(1) affected the functional expression of matrix metalloprotease-2 or -9, two common proteases expressed by hMSCs. In summary, combinations of the ECM and growth factors described herein may form the basis of defined cell culture media supplements, although the effect of endogenous protease expression on the function of such proteins requires investigation.

Influence of smectite suspension structure on sheet orientation in dry sediments : XRD and AFM applications

The structure-building phenomena within clay aggregates are governed by forces acting between clay particles. Measurements of such forces are important to understand in order to manipulate the aggregate structure for applications such as dewatering of mineral processing tailings. A parallel particle orientation is required when conducting XRD investigation on the oriented samples and conduct force measurements acting between basal planes of clay mineral platelets using at. force microscopy (AFM). To investigate how smectite clay platelets were oriented on silicon wafer substrate when dried from suspension range of methods like SEM, XRD and AFM were employed. From these investigations, we conclude that high clay concns. and larger particle diams. (up to 5 μm) in suspension result in random orientation of platelets in the substrate. The best possible laminar orientation in the clay dry film, represented in the XRD 0 0 1/0 2 0 intensity ratio of 47 was obtained by drying thin layers from 0.02 wt.% clay suspensions of the natural pH. Conducted AFM investigations show that smectite studied in water based electrolytes show very long-range repulsive forces lower in strength than electrostatic forces from double-layer repulsion. It was suggested that these forces may have structural nature. Smectite surface layers rehydrate in water environment forms surface gel with spongy and cellular texture which cushion approaching AFM probe. This structural effect can be measured in distances larger than 1000 nm from substrate surface and when probe penetrate this gel layer, structural linkages are forming between substrate and clay covered probe. These linkages prevent subsequently smooth detachments of AFM probe on way back when retrieval. This effect of tearing new formed structure apart involves larger adhesion-like forces measured in retrieval. It is also suggested that these effect may be enhanced by the nano-clay particles interaction.

Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts

In this study, cell sheets comprising multilayered porcine bone marrow stromal cells (BMSC) were assembled with fully interconnected scaffolds made from medical-grade polycaprolactone–calcium phosphate (mPCL–CaP), for the engineering of structural and functional bone grafts. The BMSC sheets were harvested from culture flasks and wrapped around pre-seeded composite scaffolds. The layered cell sheets integrated well with the scaffold/cell construct and remained viable, with mineralized nodules visible both inside and outside the scaffold for up to 8 weeks culture. Cells within the constructs underwent classical in vitro osteogenic differentiation with the associated elevation of alkaline phosphatase activity and bone-related protein expression. In vivo, two sets of cell-sheet-scaffold/cell constructs were transplanted under the skin of nude rats. The first set of constructs (554mm3) were assembled with BMSC sheets and cultured for 8 weeks before implantation. The second set of constructs (10104mm3) was implanted immediately after assembly with BMSC sheets, with no further in vitro culture. For both groups, neo cortical and well-vascularised cancellous bone were formed within the constructs with up to 40% bone volume. Histological and immunohistochemical examination revealed that neo bone tissue formed from the pool of seeded BMSC and the bone formation followed predominantly an endochondral pathway, with woven bone matrix subsequently maturing into fully mineralized compact bone; exhibiting the histological markers of native bone. These findings demonstrate that large bone tissues similar to native bone can be regenerated utilizing BMSC sheet techniques in conjunction with composite scaffolds whose structures are optimized from a mechanical, nutrient transport and vascularization perspective.

A functional screen identifies lateral transfer of beta-glucuronidase (gus) from bacteria to fungi

Lateral gene transfer (LGT) from prokaryotes to microbial eukaryotes is usually detected by chance through genome-sequencing projects. Here, we explore a different, hypothesis-driven approach. We show that the fitness advantage associated with the transferred gene, typically invoked only in retrospect, can be used to design a functional screen capable of identifying postulated LGT cases. We hypothesized that beta-glucuronidase (gus) genes may be prone to LGT from bacteria to fungi (thought to lack gus) because this would enable fungi to utilize glucuronides in vertebrate urine as a carbon source. Using an enrichment procedure based on a glucose-releasing glucuronide analog (cellobiouronic acid), we isolated two gus(+) ascomycete fungi from soils (Penicillium canescens and Scopulariopsis sp.). A phylogenetic analysis suggested that their gus genes, as well as the gus genes identified in genomic sequences of the ascomycetes Aspergillus nidulans and Gibberella zeae, had been introgressed laterally from high-GC gram(+) bacteria. Two such bacteria (Arthrobacter spp.), isolated together with the gus(+) fungi, appeared to be the descendants of a bacterial donor organism from which gus had been transferred to fungi. This scenario was independently supported by similar substrate affinities of the encoded beta-glucuronidases, the absence of introns from fungal gus genes, and the similarity between the signal peptide-encoding 5' extensions of some fungal gus genes and the Arthrobacter sequences upstream of gus. Differences in the sequences of the fungal 5' extensions suggested at least two separate introgression events after the divergence of the two main Euascomycete classes. We suggest that deposition of glucuronides on soils as a result of the colonization of land by vertebrates may have favored LGT of gus from bacteria to fungi in soils.