Lingulid shell mediation in clay formation

Organophosphatic shells of the brachiopod Lingula squarniformis , collected from Scottish Lower Carboniferous shales and mudstones of intertidal to sublittoral provenance, have been studied to ascertain chemico-structural changes resulting from fossilization. Enough original shell has been preserved at ultrastructural and molecular levels to confirm that Carboniferous and Recent integuments are homologous with stratiform successions of apatitic to organic laminae forming rhythmic sets. One of the main organic constituents, the acidic, hydrophilic gel glycosaminoglycans (GAGs), is the dominant component towards the tops of rhythms. During fossilization of the Carboniferous shells, GAGs degraded incrementally without disturbing apatitic ultrastructures, and the spaces so created became partly filled with sheets of recrystal-lized apatite with some kaolinite or with books and plates of kaolinite. The kaolinite in the shells contrasts with the illite of the entombing sediments and suggests that degrading acidic GAGs mediated in clay formation in situ . The sediments also contain framboidal pyrite, which is virtually absent from the shells themselves but is usually even more abundant, with a greater range of trace metals, in the sedimentary fills of complete shells. This imbalance suggests mediation by another gel, the glycocalyx, secreted by the inner epithelium of the brachiopod mantle. The glycocalyx would have lined the shell interior and could have served as a sorption film for dissolved metals precipitated as compounds on decomposition of body tissue.     

Synthesis of stromal glycosaminoglycans in response to injury

Our goal is to examine the synthesis and deposition of corneal glycosaminoglycans (GAGs) in response to a wound created by the insertion of porous discs into stromal interlamellar pockets. The disc and the surrounding stromal tissue were assayed and compared to contralateral control stroma and to sham operated corneas at 14,42, and 84 days. The tissue and/or discs were removed and labeled with ³⁵S-sulfate for 18 h; GAGs were extracted with 4 M guanidine–HCl. Extracts were chromatographed on Q-Sepharose columns, bound proteoglycans were eluted with a linear salt gradient, and radioactive fractions were analyzed. Total GAG content was determined colorimetrically, using dimethylmethylene blue. Specific GAGs were determined using enzymatic digestion with selective polysaccharide lyases and protein cores were examined using SDS–PAGE. The nonbound fractions from the chromatography were assayed for TGF-β using Western blot analysis and for hyaluronic acid using an ¹²⁵I-radiometric assay. Specific GAGs were localized 42 days after the disc had been implanted in the stroma. The placement of the discs into the stroma resulted in a decrease in the total amount of GAG. However, the ratio of dermatan-chondroitin sulfate and heparan sulfate to keratan sulfate increased in the surrounding tissue and disc. Hyaluronic acid was elevated at day 14 in the surrounding tissue, and not until day 84 in the disc. Western blot analysis of surrounding tissue extracts revealed forms of TGF-β that migrated with an apparent molecular mass of 63 and 43 kDa. The results indicate that the insertion of discs into interlamellar pockets causes changes in the sulfation and proportion of the glycosaminoglycans in the surrounding tissue and the disc. These changes are coincident with the appearance of TGF-β. After 84 days, the population of glycosaminoglycans in the disc begins to resemble the surrounding stroma. This model will allow us to examine further the synthesis and deposition of proteins following an extensive wound in which cells must migrate to the wound site and then undergo extensive remodeling. © 1995 Wiley-Liss, Inc.     

A comprehensive biochemical characterization of settlement stage leptocephalus larvae of bonefish (Albula vulpes)

Little is known about early development of the near-threatened bonefish (Albula vulpes), a member of superorder Elopomorpha. Members of Elopomorpha are partially defined by their synapomorphic leptocephalus larval stage, for which the nutritional requirements are not well understood. Characterizing the nutritional profile, including major nutrients (such as lipids) used for energetic processes, can help to gain a better understanding of the nutritional requirements for leptocephalus larvae. A total of 24 settlement stage A. vulpes leptocephalus larvae were collected at Long Caye Island, Belize. Samples were used to determine various biochemical characteristics including lipid class, fatty acid and glycosaminoglycan compositions. Each of these biochemical components plays a role in early developmental processes such as cellular membrane formation and is crucial for healthy development. Biochemical characteristics of settlement stage A. vulpes leptocephalus are presented in this study for the first time. The dominant lipid classes and fatty acids detected in these samples were consistent with prior studies using closely related species like the Japanese eel, indicating possible similarities in diets at this stage. In the future, similar analyses can be applied to other species that share the leptocephalus life stage to determine if nutritional requirements at this stage of development are unique to this species. The findings in this study will also help to facilitate the establishment of adequate aquaculture systems for captive bonefish, ultimately leading to improved management strategies for wild bonefish habitats.     

Enhancement of thermal stability of chondroitinase ABC I by site-directed mutagenesis: An insight from Ramachandran plot

The application of chondroitinase ABC I (cABC I) in damaged nervous tissue is believed to prune glycosaminoglycan chains of proteoglycans, thereby facilitates axon regeneration. However, the utilization of cABC I as therapeutics is notably restricted due to its thermal instability. In the present study, we have explored the possibility of thermostabilization of cABC I through release of its conformational strain using Ramachandran plot information. In this regard, Gln140 with non-optimal φ and ψ values were replaced with Gly, Ala and Asn. The results indicated that Q140G and Q140A mutants were able to improve both activity and thermal stability of the enzyme while Q140N variant reduced the enzyme activity and destabilized it. Moreover, the two former variants displayed a remarkable resistance to trypsin degradation. Structural analysis of all mutants showed an increase in intrinsic fluorescence intensity and secondary structure content of Q140G and Q140A compared to the wild type which indicated more compact structure upon mutation. This investigation demonstrated that relief of conformational tension can be considered as a possible approach to increase the stability of the protein.     

The potentiation of myeloperoxidase activity by the glycosaminoglycan-dependent binding of myeloperoxidase to proteins of the extracellular matrix

Background

Myeloperoxidase (MPO) is an abundant hemoprotein expressed by neutrophil granulocytes that is recognized to play an important role in the development of vascular diseases. Upon degranulation from circulating neutrophil granulocytes, MPO binds to the surface of endothelial cells in an electrostatic-dependent manner and undergoes transcytotic migration to the underlying extracellular matrix (ECM). However, the mechanisms governing the binding of MPO to subendothelial ECM proteins, and whether this binding modulates its enzymatic functions are not well understood.

Methods

We investigated MPO binding to ECM derived from aortic endothelial cells, aortic smooth muscle cells, and fibroblasts, and to purified ECM proteins, and the modulation of these associations by glycosaminoglycans. The oxidizing and chlorinating potential of MPO upon binding to ECM proteins was tested.

Results

MPO binds to the ECM proteins collagen IV and fibronectin, and this association is enhanced by the pre-incubation of these proteins with glycosaminoglycans. Correspondingly, an excess of glycosaminoglycans in solution during incubation inhibits the binding of MPO to collagen IV and fibronectin. These observations were confirmed with cell-derived ECM. The oxidizing and chlorinating potential of MPO was preserved upon binding to collagen IV and fibronectin; even the potentiation of MPO activity in the presence of collagen IV and fibronectin was observed.

Conclusions

Collectively, the data reveal that MPO binds to ECM proteins on the basis of electrostatic interactions, and MPO chlorinating and oxidizing activity is potentiated upon association with these proteins.

General significance

Our findings provide new insights into the molecular mechanisms underlying the interaction of MPO with ECM proteins.     

Acidolysis-based component mapping of glycosaminoglycans by reversed-phase high-performance liquid chromatography with off-line electrospray ionization–tandem mass spectrometry: Evidence and tags to distinguish different glycosaminoglycans

Diverse monosaccharide analysis methods have been established for a long time, but few methods are available for a complete monosaccharide analysis of glycosaminoglycans (GAGs) and certain acidolysis-resistant components derived from GAGs. In this report, a reversed-phase high-performance liquid chromatography (RP–HPLC) method with pre-column 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization was established for a complete monosaccharide analysis of GAGs. Good separation of glucosamine/mannosamine (GlcN/ManN) and glucuronic acid/iduronic acid (GlcA/IdoA) was achieved. This method can also be applied to analyze the acidolysis-resistant disaccharides derived from GAGs, and the sequences of these disaccharides were confirmed by electrospray ionization–collision-induced dissociation–tandem mass spectrometry (ESI–CID–MS/MS). These unique disaccharides could be used as markers to distinguish heparin/heparan sulfate (HP/HS), chondroitin sulfate/dermatan sulfate (CS/DS), and hyaluronic acid (HA).     

Biotechnological challenges of bioartificial kidney engineering

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed.     

Virus Entry: Looking Back and Moving Forward

Research over a period of more than half a century has provided a reasonably accurate picture of mechanisms involved in animal virus entry into their host cells. Successive steps in entry include binding to receptors, endocytosis, passage through one or more membranes, targeting to specific sites within the cell, and uncoating of the genome. For some viruses, the molecular interactions are known in great detail. However, as more viruses are analyzed, and as the focus shifts from tissue culture to in vivo experiments, it is evident that viruses display considerable redundancy and flexibility in receptor usage, endocytic mechanism, location of penetration, and uncoating mechanism. For many viruses, the picture is still elusive because the interactions that they engage in rely on sophisticated adaptation to complex cellular functions and defense mechanisms.     

Glycomics and Proteomics Approaches to Investigate Early Adenovirus–Host Cell Interactions

Adenoviruses as most viruses rely on glycan and protein interactions to attach to and enter susceptible host cells. The Adenoviridae family comprises more than 80 human types and they differ in their attachment factor and receptor usage, which likely contributes to the diverse tropism of the different types. In the past years, methods to systematically identify glycan and protein interactions have advanced. In particular sensitivity, speed and coverage of mass spectrometric analyses allow for high-throughput identification of glycans and peptides separated by liquid chromatography. Also, developments in glycan microarray technologies have led to targeted, high-throughput screening and identification of glycan-based receptors. The mapping of cell surface interactions of the diverse adenovirus types has implications for cell, tissue, and species tropism as well as drug development. Here we review known adenovirus interactions with glycan- and protein-based receptors, as well as glycomics and proteomics strategies to identify yet elusive virus receptors and attachment factors. We finally discuss challenges, bottlenecks, and future research directions in the field of non-enveloped virus entry into host cells.     

Structural aspects of pathology in Alzheimer's disease

The characteristic lesions of Alzheimer's disease, neurofibrillary tangles and neuritic plaques, are the sites of accumulation of abnormal fibrillar material. The structure of the paired helical filament from tangles has been analysed by electron microscopy and biochemical studies have shown that it contains microtubule associated protein tau as a component. Fibrils of β-amyloid in the neuritic plaque arise by polymerization of a small proteolytic fragment of a much larger precursor protein. It is not yet clear what triggers the events that lead to assembly of the abnormal structures nor why the structures once formed are so resistant to turnover.