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Collagen’s long half-life (in skin approximately 10 years) makes this protein highly susceptible to glycation and formation of the advanced glycation end products (AGEs). Accumulation of cross-linking AGEs in the skin collagen has several detrimental effects; thus, the opportunity for non-invasive monitoring of skin glycation is essential, especially for diabetic patients. In this paper, we report using the time-resolved intrinsic fluorescence of collagen as a biomarker of its glycation. Contrary to the traditional fluorescence intensity decay measurement at the arbitrarily selected excitation and detection wavelengths, we conducted systematic wavelength- and time-resolved measurements to achieve time-resolved emission spectra. Changes in the intrinsic fluorescence kinetics, caused by both collagen aggregation and glycation, have been detected.

The procollagen C-propeptides of the fibrillar collagens play key roles in the intracellular assembly of procollagen molecules from their constituent polypeptides chains, and in the extracellular assembly of collagen molecules into fibrils. Here we review recent advances in understanding the molecular mechanisms controlling C-propeptide trimerization which have revealed the importance of inter-chain disulphide bonding and a small number of charged amino acids in the stability and specificity of different types of chain association. We also show how the crystal structure of the complex between the C-propeptide trimer of procollagen III and the active fragment of procollagen C-proteinase enhancer-1 leads to a detailed model for accelerating release of the C-propeptides from procollagen by bone morphogenetic protein-1 and related proteinases. We then discuss the effects of disease-related missense mutations in the C-propeptides in relation to the sites of these mutations in the three-dimensional structure. While in general there is a good correlation between disease severity and structure-based predictions, there are notable exceptions, suggesting new interactions involving the C-propeptides yet to be characterized. Mutations affecting proteolytic release of the C-propeptides from procollagen are discussed in detail. Finally, the roles of recently discovered interaction partners for the C-propeptides are considered during fibril assembly and cross-linking.

The Ehlers–Danlos syndromes (EDS) are a group of rare inherited connective tissue disorders. Vascular EDS (vEDS) is caused by pathogenic variants in COL3A1, most frequently glycine substitutions. We describe the phenotype of the largest series of vEDS patients with glutamic acid to lysine substitutions (Glu>Lys) in COL3A1, which were all previously considered to be variants of unknown significance.

We report the effects of quercetin, a flavonoid present in the human diet, on early stage beta-amyloid (Aβ) aggregation, a seminal event in Alzheimer’s disease. Molecular level changes in Aβ arrangements are monitored by time-resolved emission spectral (TRES) measurements of the fluorescence of Aβ’s single tyrosine intrinsic fluorophore (Tyr). The results suggest that quercetin binds β-amyloid oligomers at early stages of their aggregation, which leads to the formation of modified oligomers and hinders the creation of β-sheet structures, potentially preventing the onset of Alzheimer’s disease.

Background: Reagent‐supported thromboelastometry with the rotation thrombelastography (e.g. ROTEM®) is a whole blood assay that evaluates the visco‐elastic properties during blood clot formation and clot lysis. A hemostatic monitor capable of rapid and accurate detection of clinical coagulopathy within the resuscitation room could improve management of bleeding after trauma. Objectives: The goals of this study were to establish whether ROTEM correlated with standard coagulation parameters to rapidly detect bleeding disorders and whether it can help to guide transfusion. Methods: Ninety trauma patients were included in the study. At admission, standard coagulation assays were performed and ROTEM parameters such as clot formation time (CFT) and clot amplitude (CA) were obtained at 15 min (CA15) with two activated tests (INTEM, EXTEM) and at 10 min (CA10) with a test analyzing specifically the fibrin component of coagulation (FIBTEM). Results: Trauma induced significant modifications of coagulation as assessed by standard assays and ROTEM. A significant correlation was found between prothrombin time (PT) and CA15‐EXTEM (r = 0.66, P < 0.0001), between activated partial thromboplastin time and CFT‐INTEM (r = 0.91, P < 0.0001), between fibrinogen level and CA10‐FIBTEM (r = 0.85, P < 0.0001), and between platelet count and CA15‐INTEM (r = 0.57, P < 0.0001). A cutoff value of CA15‐EXTEM at 32 mm and CA10‐FIBTEM at 5 mm presented a good sensitivity (87% and 91%) and specificity (100% and 85%) to detect a PT > 1.5 of control value and a fibrinogen less than 1 g L−1, respectively. Conclusions: ROTEM is a point‐of‐care device that rapidly detects systemic changes of in vivocoagulation in trauma patients, and it might be a helpful device in guiding transfusion.

Background: The complex geometry of the right ventricle (RV) and poor endocardial definition make quantitative assessment of RV function difficult. Doppler tissue imaging may be helpful in quantifying RV function through measurement of tricuspid annular velocity (TAV). This prospective study assessed the feasibility of using color Doppler tissue imaging to measure TAV using a novel transgastric RV inflow view in patients undergoing cardiac surgery. Methods: We used the transgastric RV inflow view and measured the TAV using Doppler tissue imaging and quantitative analysis software. We also measured left ventricular fractional area of contraction and hemodynamic variables. We compared values before and after cardiopulmonary bypass in patients undergoing coronary artery bypass graft. Results: TAV could be measured in 19 of 23 patients (83%) undergoing coronary artery bypass graft. There was a significant decrease postbypass in TAV: isovolumic acceleration was 1.71 +/- 0.59 versus 1.32 +/- 0.66 m/s^2, isovolumic velocity was 4.34 +/- 1.19 versus 3.13 +/- 1.35 cm/s, and systolic annular descent velocity was 5.15 +/- 1.15 versus 3.77 +/- 1.18 cm/s. There was a significant change in heart rate and cardiac index without any change in stroke volume index. There was no change in left ventricular function (fractional area of contraction: 54 +/- 10 vs 52 +/- 10%). Conclusion: Determination of TAV using the transgastric RV inflow view is feasible and may provide quantitative information on systolic RV function in patients undergoing coronary artery bypass graft. We found a decrease in systolic TAV after cardiopulmonary bypass without a significant change in stroke volume index. .

Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that binds to the C-propeptide of procollagen I and can enhance the activities of procollagen C-proteinases up to 20-fold. To determine the molecular mechanism of PCPE activity, the interactions of the recombinant protein with the procollagen molecule as well as with its isolated C-propeptide domain were studied using surface plasmon resonance (BIAcore) technology. Binding required the presence of divalent metal cations such as calcium and manganese. By ligand blotting, calcium was found to bind to the C-propeptide domains of procollagens I and III but not to PCPE. By chemical cross-linking, the stoichiometry of the PCPE/C-propeptide interaction was found to be 1:1 in accordance with enzyme kinetic data. The use of a monoclonal antibody directed against the N-terminal region of the C-propeptide suggested that this region is probably not involved in binding to PCPE. Association and dissociation kinetics of the C-propeptide domains of procollagens I and III on immobilized PCPE were rapid. Extrapolation to saturation equilibrium yielded apparent equilibrium dissociation constants in the range 150–400 nm. In contrast, the association/dissociation kinetics of intact procollagen molecules on immobilized PCPE were relatively slow, corresponding to a dissociation constant of 1 nm. Finally, pN-collagen (i.e.procollagen devoid of the C-terminal propeptide domain) was also found to bind to immobilized PCPE, suggesting that PCPE binds to sites on either side of the procollagen cleavage site, thereby facilitating the action of procollagen C-proteinases.

Recently several cDNAs have been described encoding lysyl oxidase-like proteins. Their deduced amino acid sequences are characterized by a strong similarity in the C-terminal region, corresponding to the lysyl oxidase family catalytic domain, and by marked differences in the N-terminal regions. Different biological functions have been described for lysyl oxidases in addition to their traditionally assumed cross-linking role. To answer the question of whether these different functions are carried out by different lysyl oxidases, purified and active forms of these enzymes are required. At present only the classical form of lysyl oxidase has been purified and characterized. The purpose of this study was to isolate and characterize the lysyl oxidase-like protein. In view of the strong sequence homology with the C-terminal domain of other lysyl oxidases, we chose to purify the protein from bovine aorta using antibodies specific to the N-terminal domain of the proenzyme. We have isolated a 56-kDa protein identified by amino acid sequencing as the bovine lysyl oxidase-like precursor, which is cleaved at the Arg-Arg-Arg sequence at positions 89–91 by a furin-like activity, as revealed after deblocking of the N-terminal residue. The immunopurified protein was largely inactive, but further processing in vitroby bone morphogenetic protein-1 led to an enzyme that was active on elastin and collagen substrates.

Procollagen C-propeptide domains direct chain association during intracellular assembly of procollagen molecules. In addition, they control collagen solubility during extracellular proteolytic processing and fibril formation and interact with cell surface receptors and extracellular matrix components involved in feedback inhibition, mineralization, cell growth arrest, and chemotaxis. At present, three-dimensional structural information for the C-propeptides, which would help to understand the underlying molecular mechanisms, is lacking. Here we have carried out a biophysical study of the recombinant C-propeptide trimer from human procollagen III using laser light scattering, analytical ultracentrifugation, and small angle x-ray scattering. The results show that the trimer is an elongated molecule, which by modeling of the x-ray scattering data appears to be cruciform in shape with three large lobes and one minor lobe. We speculate that each of the major lobes corresponds to one of the three component polypeptide chains, which come together in a junction region to connect to the rest of the procollagen molecule.

Human unhydroxylated homotrimeric triple-helical collagen I produced in transgenic plants was used as an experimental model to provide insights into the role of hydroxyproline in molecular folding and fibril formation. By using chemically cross-linked molecules, we show here that the absence of hydroxyproline residues does not prevent correct folding of the recombinant collagen although it markedly slows down the propagation rate compared with bovine fully hydroxylated homotrimeric collagen I. Relatively slow cis-trans-isomerization in the absence of hydroxyproline likely represents the rate-limiting factor in the propagation of the unhydroxylated collagen helix. Because of the lack of hydroxylation, recombinant collagen molecules showed increased flexibility as well as a reduced melting temperature compared with native homotrimers and heterotrimers, whereas the distribution of charged amino acids was unchanged. However, unlike with bovine collagen I, the recombinant collagen did not self-assemble into banded fibrils in physiological ionic strength buffer at 20 °C. Striated fibrils were only obtained with low ionic strength buffer. We propose that, under physiological ionic strength conditions, the hydroxyl groups in the native molecule retain water more efficiently thus favoring correct fibril formation. The importance of hydroxyproline in collagen self-assembly suggested by others from the crystal structures of collagen model peptides is thus confirmed experimentally on the entire collagen molecule.

Although the collagen V heterotrimer is known to be involved in the control of fibril assembly, the role of the homotrimer in fibrillar organization has not yet been examined. Here, the production of substantial amounts of recombinant collagen V homotrimer has allowed a detailed study of its role in homotypic and heterotypic fibril formation. After removal of terminal regions by pepsin digestion, both the collagen V heterotrimer and homotrimer formed thin homotypic fibrils, thus showing that diameter limitation is at least in part an intrinsic property of the collagen V triple helix. When mixed with collagen I, however, various complementary approaches indicated that the collagen V heterotrimer and homotrimer exerted different effects in heterotypic fibril formation. Unlike the heterotrimer, which was buried in the fibril interior, the homotrimer was localized as thin filamentous structures at the surface of wide collagen I fibrils and did not regulate fibril assembly. Its localization at the fibril surface suggests that the homotrimer can act as a molecular linker between collagen fibrils or macromolecules in the extracellular matrix or both. Thus, depending on their respective distribution in tissues, the different collagen V isoforms might fulfill specific biological functions.

L’American Heart Association en collaboration avec l’International Liaison Committee On Resuscitation vient de proposer une actualisation des recommandations pour la réanimation cardio-pulmonaire. C’est l’occasion de faire le point sur les travaux récents et les principaux progrès dans le traitement de l’arrêt cardiaque, concernant en particulier le massage cardiaque, la ventilation, la défibrillation ou le traitement pharmacologique. Pour terminer, nous résumons ce qui paraît essentiel et novateur dans les nouveaux concepts de la réanimation cardio-pulmonaire.

The precise molecular mechanisms that determine the three-dimensional architectures of tissues remain largely unknown. Within tissues rich in extracellular matrix, collagen fibrils are frequently arranged in a tissue-specific manner, as in certain liquid crystals. For example, the continuous twist between fibrils in compact bone osteons resembles a cholesteric mesophase, while in tendon, the regular, planar undulation, or “crimp”, is akin to a precholesteric mesophase. Such analogies suggest that liquid crystalline organisation plays a role in the determination of tissue form, but it is hard to see how insoluble fibrils could spontaneously and specifically rearrange in this way. Collagen molecules, in dilute acid solution, are known to form nematic, precholesteric and cholesteric phases, but the relevance to physiological assembly mechanisms is unclear. In vivo, fibrillar collagens are synthesised in soluble precursor form, procollagens, with terminal propeptide extensions. Here, we show, by polarized light microscopy of highly concentrated (5–30 mg/ml) viscous drops, that procollagen molecules in physiological buffer conditions can also develop long-range nematic and precholesteric liquid crystalline ordering extending over 100 μm2domains, while remaining in true solution. These observations suggest the novel concept that supra-fibrillar tissue architecture is determined by the ability of soluble precursor molecules to form liquid crystalline arrays, prior to fibril assembly.

Fibrils of embryonic cartilage are heterotypic alloys formed by collagens II, IX, and XI and have a uniform diameter of ∼20 nm. The molecular basis of this lateral growth control is poorly understood. Collagen II subjected to fibril formation in vitroproduced short and tapered tactoids with strong D-periodic banding. The maximal width of these tactoids varied over a broad range. By contrast, authentic mixtures of collagens II, IX, and XI yielded long and weakly banded fibrils, which, strikingly, had a uniform width of about 20 nm. The same was true for mixtures of collagens II and XI lacking collagen IX as long as the molar excess of collagen II was less than 8-fold. At higher ratios, the proteins assembled into tactoids coexisting with cartilage-like fibrils. Therefore, diameter control is an inherent property of appropriate mixtures of collagens II and XI. Collagen IX is not essential for this feature but strongly increases the efficiency of fibril formation. Therefore, this protein may be an important stabilizing factor of cartilage fibrils.

We have used one- (OPE) and two-photon (TPE) excitation with time-correlated single-photon counting techniques to determine time-resolved fluorescence intensity and anisotropy decays of the wild-type Green Fluorescent Protein (GFP) and two red-shifted mutants, S65T-GFP and RSGFP. WT-GFP and S65T-GFP exhibited a predominant ∼3ns monoexponential fluorescence decay, whereas for RSGFP the main lifetimes were ∼1.1ns (main component) and ∼3.3ns. The anisotropy decay of WT-GFP and S65T-GFP was also monoexponential (global rotational correlation time of 16±1ns). The ∼1.1ns lifetime of RSGFP was associated with a faster rotational depolarization, evaluated as an additional ∼13ns component. This feature we attribute tentatively to a greater rotational freedom of the anionic chromophore. With OPE, the initial anisotropy was close to the theoretical limit of 0.4; with TPE it was higher, approaching the TPE theoretical limit of 0.57 for the colinear case. The measured power dependence of the fluorescence signals provided direct evidence for TPE. The general independence of fluorescence decay times, rotation correlation times, and steady-state emission spectra on the excitation mode indicates that the fluorescence originated from the same distinct excited singlet states (A*, I*, B*). However, we observed a relative enhancement of blue fluorescence peaked at ∼440nm for TPE compared to OPE, indicating different relative excitation efficiencies. We infer that the two lifetimes of RSGFP represent the deactivation of two substates of the deprotonated intermediate (I*), distinguished by their origin (i.e., from A* or B*) and by nonradiative decay rates reflecting different internal environments of the excited-state chromophore.

Bromide, iodide and tetraphenylborate quaternary salts of four new highly fluorescent dyes have been produced by the reaction of the 6-methoxyquinoline heterocyclic nitrogen base with 3-bromo-1-propanol, methyl iodide and methyl bromide with one of the dyes having its counter-ion exchanged for the tetraphenylborate ion. Three of the dyes, unlike the base, are readily water soluble, the tetraphenylborate salt only slightly water soluble and all are fluorescent over a broad pH range. The dyes have been characterised in terms of their water solubility, their steady-state fluorescence spectra and their bi-exponential fluorescence lifetimes, which are all found to be in the range ≈ 10–30 ns. We have found that the cationic dyes readily bind to negatively charged colloidal silica particles and because of the suitably long fluorescence lifetimes we have been able to determine the particle size of Du Pont's SM30 colloidal silica using time-resolved fluorescence anisotropy. The average particle diameter, determined using three of the dyes, was 6.8 ± 0.6 nm, which compares well with Du Pont's mean value of 7 nm.

The collagens produced by chick embryo chondrocytes cultured in alginate beads were investigated both biochemically and ultrastructurally. The cartilage phenotype is maintained for at least 14 days, as indicated by the production of the cartilage-specific collagens II, IX, and XI and the absence of collagen I. There were differences in the distributions of collagens among the three different compartments analyzed (cells and their associated matrix, further-removed matrix (released by alginate solubilization), and culture medium), with large amounts of collagen IX (mainly in proteoglycan form) in the culture medium. Inhibition of lysyl oxidase activity by β-aminopropionitrile led to an overall decrease in collagen production. In contrast to the biochemical observations, collagen ultrastructure in the extracellular matrix of alginate cultures was not in the form of the expected 64-nm banded fibrils, but rather in the form of segment-long-spacing-like crystallites. This abnormal structure is likely to be a result of alginate disrupting normal assembly. We conclude that, in this system, the native fibrillar structure of the collagenous matrix is not essential for the maintenance of the differentiated phenotype of chondrocytes.

Procollagen C-proteinase enhancer (PCPE) is a 55 kDa glycoprotein that increases the activity of procollagen C-proteinase (PCP)/bone morphogenetic protein-1 (BMP-1) during C-terminal processing of fibrillar collagen precursors. Here we show that the 36 kDa, active fragment of PCPE enhances the activity of both the short (mouse) and long (chick) forms of PCP/BMP-1. The activity of PCPE is not associated with the formation of sedimentable procollagen aggregates. In addition, PCPE (36 kDa) has no effect in vitro on N-termnial procollagen processing by highly purified procollagen N-proteinase. Finally, when the amount of PCP is adjusted so that the rate of C-terminal processing remains constant, PCPE (36 kDa) has no effect on the assembly of collagen or pN-collagen in vitro following C-terminal processing of the corresponding precursors.

Tyrosine-rich acidic matrix protein (TRAMP; 22 kDa extracellular matrix protein; dermatopontin) is a protein that co-purifies with lysyl oxidase and with dermatan sulphate proteoglycans, with possible functions in cell—matrix interactions and matrix assembly. Using a rabbit polyclonal antiserum raised against porcine TRAMP, which cross-reacts with both the human and murine forms of the protein, we show by immunoblotting that TRAMP has a widespread tissue distribution, including skin, skeletal muscle, heart, lung, kidney, cartilage and bone. In cultures of human skin fibroblasts, TRAMP incorporates both [ 35S]sulphate and [ 3H]tyrosine and is secreted into the medium, as shown by immunoprecipitation. Amino acid analysis of immunoprecipitated TRAMP demonstrates that many of the tyrosine residues in TRAMP are sulphated.

Life tables were calculated for 205 South Australians with cystic fibrosis. An improvement in survival was noted between 1948 and 1982. Ninety‐three per cent of patients who were diagnosed as having cystic fibrosis after 1973 were alive at 14 years of age, compared with 40% of those who were diagnosed between 1948 and 1973. A Cystic Fibrosis Clinic was established in 1973 and much of this improvement is attributed to the care provided by this centre. Deaths from meconium ileus fell from 58% of infants with this complication between 1948 and 1973 to only 8% between 1973 and 1983, in spite of the increasing incidence of patients who were chronically colonized with Pseudomonas aeruginosa(currently 68% of patients). These figures are similar to those from Victoria and from other cystic fibrosis centres in North America. The improvement in survival means that adults now comprise a quarter of the patients with cystic fibrosis in South Australia, and that adult institutions need to be aware of these patients and their needs.

A cross-sectional analytic survey of 92 patients permitted to attempt vaginal delivery after previous lower segment cesarean section (“trial of scar”) is reported. Variables which may predict mode of delivery were assessed. Fifty patients (54.3%) were delivered vaginally; 42 patients (45.7%) had repeat cesarean sections in labor. There were three cases of scar dehiscence (3.2%). There was no maternal or fetal mortality. When the cervix was less than 3cm dilated at initial examination in labor, 10 of 37 patients (27%) were delivered vaginally, compared to 38 of 55 patients (69%) who were delivered vaginally when the cervix was greater than 3cm dilated: Assessment of cervical dilatation on admission in labor proved to be the most significant prognostic factor at the onset of labor, with regard to successful vaginal delivery in a patient with a lower segment cesarean section scar.

DENIS H. LYNN. 2008. The Ciliated Protozoa: Characterization, Classification, and Guide to the Literature, 3rd ed. Springer. ISBN: 978‐1‐4020‐8238‐2. 606 p. $249