Collagen fibril morphology in developing chick metatarsal tendoms: 1. X-ray diffraction studies

The low angle equatorial X-ray diffraction ($\text{R ? 30 μ}\text{m}{}^{\mathrm{?1}}$) from hydrated embryonic chick metatarsal tendon contains minima and maxima that are not seen in mature tendons. This diffraction derives from the disordered array of parallel, cylindrical fibrils of collagen of small, uniform diameter that comprise the major part of this tissue. Comparison of the positions of the minima and maxima with those expected from an array of cylinders allows estimation of the mean diameter of the cylinders and the average centre-to-centre nearest neighbour separation. It was found that in the age range from 13 to 19 days fetal, the mean diameter increased from ～ 46 to ～ 58 nm, whereas the mean nearest neighbour separation remained constant at ～ 90 nm. Detailed analysis of the X-ray intensity profile of a 17 day fetal tendon indicated the presence of a paucidisperse distribution of fibril diameters with two or more discrete populations of preferred diameters separated by 10 to 12 nm.     

A centrosomal inclusion (striped nebulous body) in pinealocytes of the golden hamster

Summary The postnatal maturation of regions of the epididymis and intragonadal segment of the deferens duct was studied in the rat by light-and transmission electron microscopy. Maturation of the genital duct starts in the distal cauda epididymidis and ductus deferens after one week of life, and one week later, in the more cranial segments of the epididymis. Epithelial principal cells and peritubular contractile cells are structurally mature 35 days after birth. The synchronous changes of these cells indicate that the same factors control their postnatal maturation. The epithelial principal cells obtain an endocytotic apparatus and long stereocilia, whereas peritubular cells acquire contractile features. These changes are associated with a progressive increase in the immunoreaction for smooth muscle actin in both cell types. Smooth muscle myosin is detected in the apical region of the epithelial cells and the peritubular cell cytoplasm by day one of postnatal development. The differentiation of contractile cells in the wall is accompanied by progressive organization of the pericellular matrix into a continuous basement membrane. Although fibronectin is visible at birth, it is gradually removed from the tubule wall.     

The predominant roles of the sequence periodicity in the self‐assembly of collagen‐mimetic mini‐fibrils

Collagen fibrils represent a unique case of protein folding and self‐association. We have recently successfully developed triple‐helical peptides that can further self‐assemble into collagen‐mimetic mini‐fibrils. The 35 nm axially repeating structure of the mini‐fibrils, which is designated the d‐period, is highly reminiscent of the well‐known 67 nm D‐period of native collagens when examined using TEM and atomic force spectroscopy. We postulate that it is the pseudo‐identical repeating sequence units in the primary structure of the designed peptides that give rise to the d‐period of the quaternary structure of the mini‐fibrils. In this work, we characterize the self‐assembly of two additional designed peptides: peptide Col877 and peptide Col108rr. The triple‐helix domain of Col877 consists of three pseudo‐identical amino acid sequence units arranged in tandem, whereas that of Col108rr consists of three sequence units identical in amino acid composition but different in sequence. Both peptides form stable collagen triple helices, but only triple helices Col877 self‐associate laterally under fibril forming conditions to form mini‐fibrils having the predicted d‐period. The Co108rr triple helices, however, only form nonspecific aggregates having no identifiable structural features. These results further accentuate the critical involvement of the repeating sequence units in the self‐assembly of collagen mini‐fibrils; the actual amino acid sequence of each unit has only secondary effects. Collagen is essential for tissue development and function. This novel approach to creating collagen‐mimetic fibrils can potentially impact fundamental research and have a wide range of biomedical and industrial applications.     

Concentration‐dependent and surface‐assisted self‐assembly properties of a bioactive estrogen receptor α‐derived peptide

We have synthesized a 17‐mer peptide (ERα17p) that is issued from the hinge region of the estrogen receptor α and which activates the proliferation of breast carcinoma cells in steroid‐deprived conditions. In the present paper, we show that at a concentration of ~50 μM, it rapidly forms amyloid‐like fibrils with the assistance of electrostatic interactions and that at higher concentrations, it spontaneously forms a hydrogel. By using biophysical, spectral and rheological techniques, we have explored the structural, biophysical and mechanical characteristics of ERα17p with respect to fibril formation and gelation. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Transglutaminase-2 interaction with heparin: identification of a heparin binding site that regulates cell adhesion to fibronectin-transglutaminase-2 matrix

Heparan sulfate proteoglycans are critical binding partners for extracellular tranglutaminase-2 (TG2), a multifunctional protein involved in tissue remodeling events related to organ fibrosis and cancer progression. We previously showed that TG2 has a strong affinity for heparan sulfate (HS)/heparin and reported that the heparan sulfate proteoglycan syndecan-4 acts as a receptor for TG2 via its HS chains in two ways: by increasing TG2-cell surface trafficking/externalization and by mediating RGD-independent cell adhesion to fibronectin-TG2 matrix during wound healing. Here we have investigated the molecular basis of this interaction. Site-directed mutagenesis revealed that either mutation of basic RRWK (262-265) or KQKRK (598-602) clusters, forming accessible heparin binding sequences on the TG2 three-dimensional structure, led to an almost complete reduction of heparin binding, indicating that both clusters contribute to form a single binding surface. Mutation of residues Arg(19) and Arg(28) also led to a significant reduction in heparin binding, suggesting their involvement. Our findings indicate that the heparin binding sites on TG2 mainly comprise two clusters of basic amino acids, which are distant in the linear sequence but brought into spatial proximity in the folded "closed" protein, forming a high affinity heparin binding site. Molecular modeling showed that the identified site can make contact with a single heparin-derived pentasaccharide. The TG2-heparin binding mutants supported only weak RGD-independent cell adhesion compared with wild type TG2 or mutants with retained heparin binding, and both heparin binding clusters were critical for TG2-mediated cell adhesion. These findings significantly advance our knowledge of how HS/heparin influences the adhesive function of TG2.     

Generic inhibition of amyloidogenic proteins by two naphthoquinone-tryptophan hybrid molecules

Amyloid formation is associated with several human diseases including Alzheimer's disease (AD), Parkinson's disease, Type 2 Diabetes, and so forth, no disease modifying therapeutics are available for them. Because of the structural similarities between the amyloid species characterizing these diseases, (despite the lack of amino acid homology) it is believed that there might be a common mechanism of toxicity for these conditions. Thus, inhibition of amyloid formation could be a promising disease-modifying therapeutic strategy for them. Aromatic residues have been identified as crucial in formation and stabilization of amyloid structures. This finding was corroborated by high-resolution structural studies, theoretical analysis, and molecular dynamics simulations. Amongst the aromatic entities, tryptophan was found to possess the most amyloidogenic potential. We therefore postulate that targeting aromatic recognition interfaces by tryptophan could be a useful approach for inhibiting the formation of amyloids. Quinones are known as inhibitors of cellular metabolic pathways, to have anti- cancer, anti-viral and anti-bacterial properties and were shown to inhibit aggregation of several amyloidogenic proteins in vitro. We have previously described two quinone-tryptophan hybrids which are capable of inhibiting amyloid-beta, the protein associated with AD pathology, both in vitro and in vivo. Here we tested their generic properties and their ability to inhibit other amyloidogenic proteins including α-synuclein, islet amyloid polypeptide, lysozyme, calcitonin, and insulin. Both compounds showed efficient inhibition of all five proteins examined both by ThT fluorescence analysis and by electron microscope imaging. If verified in vivo, these small molecules could serve as leads for developing generic anti-amyloid drugs.     

Proper calibration of ultrasonic power enabled the quantitative analysis of the ultrasonication-induced amyloid formation process

To elucidate the mechanisms of ultrasonication on the amyloid fibril formation, we quantitatively determined the ultrasonic power using both calorimetry and potassium iodide (KI) oxidation, and under the properly calibrated ultrasonic power, we investigated the ultasonication-induced amyloid formation process of the mouse prion protein (mPrP(23-231)). These methods revealed that the ultrasonic power in our system ranged from 0.3 to 2.7 W but entirely dependent on the positions of the ultrasonic stage. Intriguingly, the nucleation time of the amyloid fibrils was found to be shortened almost proportionally to the ultrasonic power, indicating that the probability of the occurrence of nucleus formation increases proportionally to the ultrasonic power. Moreover, mPrP(23-231) formed two types of aggregates: rigid fibrils and short fibrils with disordered aggregates, depending on the ultrasonic power. The nucleation of rigid fibrils required an ultrasonic power larger than 1.5 W. While at the strong ultrasonic power larger than 2.6 W, amyloid fibrils were formed early, but simultaneously fine fragmentation of fibrils occurred. Thus, an ultrasonic power of approximately 2.0 W would be suitable for the formation of rigid mPrP(23-231) fibrils under the conditions utilized (ultrasonication applied for 30 s every 9 min). As ultrasonication has been widely used to amplify the scrapie form of the prion protein, or other amyloids in vitro, the calorimetry and KI oxidation methods proposed here might help determining the adequate ultrasonic powers necessary to amplify them efficiently.     

Darwinian transformation of a 'scarcely nutritious fluid' into milk

In an early challenge to an aspect of Darwin's theory of natural selection, Jackson Mivart contended that milk could not have evolved 'from a scarcely nutritious fluid from an accidentally hypertrophied cutaneous gland'. The evolutionary change from a gland secretion to milk involves an increase in calcium and protein concentrations by up to 100- and 1000-fold, respectively. Even so, the challenge, we suggest, is not just a problem of scale. An increase in the concentrations of calcium and phosphate brings an increased risk of calcification of the secretory gland because calcium phosphate is highly insoluble. In addition, two of the four constituent milk casein proteins (κ and α(S2)) aggregate to produce toxic amyloid fibrils. It is proposed that both problems were solved through the cosecretion of ancestral β- and κ-caseins to form a stable amorphous aggregate of both proteins with sequestered amorphous calcium phosphate, that is, a primordial casein micelle. Evolutionarily, a gradual increase in the concentration of casein micelles could therefore produce progressively more nutritious fluids for the neonate without endangering the reproductive potential of the mother.     

CD4+ lymphocytes require platelets for adhesion to immobilized fibronectin in flow: role of beta(1) (CD29)-, beta(2) (CD18)-related integrins and non-integrin receptors

The role of platelets in T-lymphocytes adhesion is not clear yet. Herpesvirus saimiri (HVS)-infected CD4(+) T-lymphocytes were placed into polystyrene plates pre-coated with fibronectin. The adherent T-cells were enumerated by image analysis. Under static condition, 38+/-10cells/mm(2) adhered and addition of gel-filtered platelets (GFP) and PMA enhanced cell adhesion 4.3- and 4.1-fold. Using PMA plus GFP 11.9-fold enhancement in cell adhesion was achieved. In contrast, under flow (200s(-1)), neither basal adhesion nor following separate addition of PMA or GFP was observed, whereas combined addition of PMA and GFP induced noticeable adhesion (34cells/mm(2)). The adhesion was inhibited by blockade of alpha(5)-integrin (CD49e, 87%), beta(2)-integrin (CD18, 78%), CD40L (60%), PSGL-1 (CD162, 60%), and CD40L plus PSGL-1 (83%). Thus, activated platelets promote activated T-cell adhesion to fibronectin under flow via integrins (alpha(5)beta(1), and alpha(L)beta(2)), CD40-CD40L and P-selectin-PSGL-1 mediated interactions.     

Regulated expression of FLRT genes implies a functional role in the regulation of FGF signalling during mouse development

Within the mammalian genome, there are many multimember gene families that encode membrane proteins with extracellular leucine rich repeats which are thought to act as cell adhesion or signalling molecules. We previously showed that the members of the NLRR gene family are expressed in a developmentally restricted manner in the mouse with NLRR-1 being expressed in the developing myotome. The FLRT gene family shows a similar genomic layout and predicted protein secondary structure to the NLRRs so we analysed expression of the three FLRT genes during mouse development. FLRTs are glycosylated membrane proteins expressed at the cell surface which localise in a homophilic manner to cell-cell contacts expressing the focal adhesion marker vinculin. Each member of the FLRT family has a distinct, highly regulated expression pattern, as was seen for the NLRR family. FLRT3 has a provocative expression pattern during somite development being expressed in regions of the somite where muscle precursor cells migrate from the dermomyotome and move into the myotome, and later in myotomal precursors destined to migrate towards their final destination, for example, those that form the ventral body wall. FLRT3 is also expressed at the midbrain/hindbrain boundary and in the apical ectodermal ridge, regions where FGF signalling is known to be important, suggesting that the role for FLRT3 in FGF signalling identified in Xenopus is conserved in mammals. FLRT1 is expressed at brain compartmental boundaries and FLRT2 is expressed in a subset of the sclerotome, adjacent to the region that forms the syndetome, suggesting that interaction with FGF signalling may be a general property of FLRT proteins. We confirmed this by showing that all FLRTs can interact with FGFR1 and FLRTs can be induced by the activation of FGF signalling by FGF-2. We conclude that FLRT proteins act as regulators of FGF signalling, being induced by the signal and then able to interact with the signalling receptor, in many tissues during mouse embryogenesis. This process may, in part, be dependent on homophilic intercellular interactions between FLRT molecules.