Artificial Cystine Research Articles

Value of color Doppler twinkling artifact and CT in distinguishing uric acid stones from cystine stones: an experimental study

Objective To quantitatively analyze the value of color Doppler twinkling artifact and CT in distinguishing urinary stone composition of uric acid from that of cystine, and to assess the ability of color Doppler twinkling artifact and CT to diagnose urinary stone composition of uric acid. Methods Artificial stones made of uric acid and cystine were inserted in porcine kidneys respectively. Color Doppler ultrasound and CT scanning were performed in vitro and twinkling artifact videos were recorded. The CT attenuation value, the width and length of twinkling artifact (TAW, TAL) were measured. The color pixels representing twinkling artifact intensity (TAI) were calculated. The differences between artificial cystine and artificial uric acid stones in TAW, TAL, TAI and CT attenuation value were compared. The receiver operating characteristic (ROC) curve was used to assess the ability of TAW, TAL, TAI and CT attenuation value to diagnose urinary stone composition of uric acid. Results The TAW [(4.16±1.54)mm vs (4.97±1.93)mm], TAL[(3.75±1.05)mm vs (5.12±2.51)mm] and TAI [(3 005.50±812.33) vs (5 433.86±2 505.81)] of artificial cystine stones were less than those of artificial uric acid stones while the CT attenuation value of artificial cystine stones [(573.75±110.10)HU vs (364.09±67.28)HU] were greater (P<0.05). The ROC curve was displayed to diagnose urinary stone composition of uric acid, and the area under curve of CT attenuation value was bigger than that of TAI (0.936 vs 0.817, Z=2.308, P=0.021). Combination of color Doppler twinkling artifact and CT value would dramatically increase the diagnostic accuracy to 98%. Conclusions Both color Doppler ultrasound and CT were reliable examinations for distinguishing urinary stone composition of cystine from that of uric acid. Combination of color Doppler ultrasound and CT would be good references to diagnose urinary stone composition of uric acid. Key words: Ultrasonography, Doppler, color; Calculi; Twinkling artifact; Tomography, X-ray computer

Natural and Artificial Cystine Knots for Assembly of Homo- and Heterotrimeric Collagen Models

Native collagens are molecules that are difficult to handle because of their high tendency towards aggregation and denaturation. It was discovered early on that synthetic collagenous peptides are more amenable to conformational characterization and thus can serve as useful models for structural and functional studies. Single-stranded collagenous peptides of high propensity to self-associate into triple-helical trimers were used for this purpose as well as interchain-crosslinked homotrimers assembled on synthetic scaffolds. With the growing knowledge of the biosynthetic pathways of natural collagens and the importance of their interchain disulfide crosslinks, which stabilize the triple-helical structure, native as well as de novo designed cystine knots have gained increasing attention in the assembly of triple-stranded collagen peptides. In addition, natural sequences of collagens were incorporated in order to biophysically characterize their functional epitopes. This review is focused on the methods developed over the years, and future perspectives for the production of collagen-mimicking synthetic and recombinant triple-helical homo- and heterotrimers.

Synthetic heterotrimeric collagen peptides as mimics of cell adhesion sites of the basement membrane.

Collagen type IV forms a network in the basement membrane into which other constituents of the tissue are incorporated. It also provides cell-adhesion sites that are specifically recognized by cell-surface receptors, i.e., the integrins. Different from the ubiquitous sequential RGD adhesion motif found in most of the matrix proteins, in collagen type IV, the responsible binding sites for alpha1beta1 integrin have been identified as Asp461 of the two alpha1 chains and Arg461 of the alpha2 chain. Because of the heterotrimeric character of this collagen, the spatial geometry of the binding epitope depends not only on the triple-helical fold, but decisively even on the stagger of the chains. To investigate the effects of chain registration on the conformational properties and binding affinities of this adhesion epitope, two synthetic heterotrimeric collagen peptides consisting of the identical three chains were assembled by an artificial cystine knot in two different registers, i.e., in the most plausible alpha2alpha1alpha1' and less probable alpha1alpha2alpha1' chain alignment. A detailed conformational characterization of both trimers allowed to correlate their different binding affinities for alpha1beta1 integrin with the degree of local plasticity of the two different triple helices. Optimal local breathing of the rod-shaped collagens is apparently crucial for selective recognition by proteins interacting with these main components of the extracellular matrix.

The Role of Cystine Knots in Collagen Folding and Stability. Part 1. Conformational Properties of (Pro‐Hyp‐Gly)5 and (Pro‐(4S)‐FPro‐Gly)5 Model Trimers with an Artificial Cystine Knot.

ChemInformVolume 34, Issue 42 Natural Products The Role of Cystine Knots in Collagen Folding and Stability. Part 1. Conformational Properties of (Pro-Hyp-Gly)5 and (Pro-(4S)-FPro-Gly)5 Model Trimers with an Artificial Cystine Knot. Dirk Barth, Dirk Barth MPI Biochem., D-82152 Martinsried, GermanySearch for more papers by this authorHans-Juergen Musiol, Hans-Juergen Musiol MPI Biochem., D-82152 Martinsried, GermanySearch for more papers by this authorMarkus Schuett, Markus Schuett MPI Biochem., D-82152 Martinsried, GermanySearch for more papers by this authorStella Fiori, Stella Fiori MPI Biochem., D-82152 Martinsried, GermanySearch for more papers by this authorAlexander G. Milbradt, Alexander G. Milbradt MPI Biochem., D-82152 Martinsried, GermanySearch for more papers by this authorChristian Renner, Christian Renner MPI Biochem., D-82152 Martinsried, GermanySearch for more papers by this authorLuis Moroder, Luis Moroder MPI Biochem., D-82152 Martinsried, GermanySearch for more papers by this author Dirk Barth, Dirk Barth MPI Biochem., D-82152 Martinsried, GermanySearch for more papers by this authorHans-Juergen Musiol, Hans-Juergen Musiol MPI Biochem., D-82152 Martinsried, GermanySearch for more papers by this authorMarkus Schuett, Markus Schuett MPI Biochem., D-82152 Martinsried, GermanySearch for more papers by this authorStella Fiori, Stella Fiori MPI Biochem., D-82152 Martinsried, GermanySearch for more papers by this authorAlexander G. Milbradt, Alexander G. Milbradt MPI Biochem., D-82152 Martinsried, GermanySearch for more papers by this authorChristian Renner, Christian Renner MPI Biochem., D-82152 Martinsried, GermanySearch for more papers by this authorLuis Moroder, Luis Moroder MPI Biochem., D-82152 Martinsried, GermanySearch for more papers by this author First published: 23 September 2003 https://doi.org/10.1002/chin.200342200AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume34, Issue42October 21, 2003 RelatedInformation

The role of cystine knots in collagen folding and stability, part II. Conformational properties of (Pro-Hyp-Gly)n model trimers with N- and C-terminal collagen type III cystine knots.

In mature collagen type III the homotrimer is C-terminally cross-linked by an interchain cystine knot consisting of three disulfide bridges of unknown connectivity. This cystine knot with two adjacent cysteine residues on each of the three alpha chains has recently been used for the synthesis and expression of model homotrimers. To investigate the origin of correct interchain cysteine pairings, (Pro-Hyp-Gly)(n) peptides of increasing triplet number and containing the biscysteinyl sequence C- and N-terminally were synthesised. The possibilities were that this origin may be thermodynamically coupled to the formation of the collagen triple helix as happens in the oxidative folding of proteins, or it could represent a post-folding event. Only with five triplets, which is known to represent the minimum number for self-association of collagenous peptides into a triple helix, air-oxidation produces the homotrimer in good yields (70 %), the rest being intrachain oxidised monomers. Increasing the number of triplets has no effect on yield suggesting the formation of kinetically trapped intermediates, which are not reshuffled by the glutathione redox buffer. N-terminal incorporation of the cystine knot is significantly less efficient in the homotrimerisation step and also in terms of triple-helix stabilisation. Compared to an artificial C-terminal cystine knot consisting of two interchain disulfide bridges, the collagen type III cystine knot produces collagenous homotrimers of remarkably high thermostability, although the concentration-independent refolding rates are not affected by the type of disulfide bridging. Since the natural cystine knot allows ready access to homotrimeric collagenous peptides of significantly enhanced triple-helix thermostability it may well represent a promising approach for the preparation of collagen-like innovative biomaterials. Conversely, the more laborious regioselectively formed artificial cystine knot still represents the only synthetic strategy for heterotrimeric collagenous peptides.

Studies of the local conformational properties of the cell-adhesion domain of collagen type IV in synthetic heterotrimeric peptides.

Collagen type IV is a specialized form of collagen that is found only in basement membranes. It is involved in integrin-mediated cell-adhesion processes, and the responsible binding sites for the alpha1beta1 integrin cell receptor have been identified as Asp461 of the two alpha1 chains and Arg461 of the alpha2 chain. In the most plausible stagger of native collagen type IV the alpha2 chain is the tailing one. This has recently been confirmed by the differentiated binding affinities of synthetic heterotrimeric collagen peptides in which the chains were staggered in this native register as well as in the less plausible alpha1alpha2alpha1' register with an artificial cystine knot. In the present work, two heterotrimeric collagen peptides with chain registers identical to the previous ones were synthesized for fluorescence resonance energy transfer and emission anisotropy measurements, exploiting the native Phe464 in the alpha2 chain as donor and an Ile467Tyr mutation in the alpha1' chain as acceptor fluorophore. This fluorophore pair allowed extraction of more detailed information on the conformational properties of the cell-adhesion epitope incorporated into the central part of the trimeric collagen model peptides. A comparison of the experimentally derived values of the interfluorophore distance and of the orientation factor kappa(2) with the values extracted from the molecular model of the trimer in the native stagger confirmed a triple-helical structure of the adhesion-site portion at low temperature. The thermal unfolding of this central domain was specifically monitored by emission anisotropy, allowing unambiguous assignment of the three structural domains of the trimeric collagen molecules detected by microcalorimetry, with the integrin binding site as the portion of weakest triple-helical stability flanked by two more stable triple-helical regions. The results are consistent with the picture of a conformational microheterogeneity as the responsible property for selective recognition of collagens by interacting proteins.

Conformation-dependent side reactions in interstrand-disulfide bridging of trimeric collagenous peptides by regioselective cysteine chemistry.

Conversion of single-chain or disulfide-bridged dimeric collagenous peptides into Cys(Npys) derivatives as activated species for subsequent regioselective thiol/disulfide exchange reactions leads to side products whose origin and nature was determined by HPLC and ESI-MS. In both cases the high tendency of the educts to self-associate into triple-helical homotrimers, as assessed by their dichroic properties in the reaction media, is responsible for the failure of this well established cysteine chemistry. Only by optimizing the synthetic strategy or by exploiting a kinetic control of the reaction, could these conformation-dependent limitations be more or less efficiently bypassed for the regioselective assembly of heterotrimeric collagen model peptides crosslinked with artificial cystine knots.

ACOUSTIC AND MECHANICAL PROPERTIES OF ARTIFICIAL STONES IN COMPARISON TO NATURAL KIDNEY STONES

Standardized and reproducible artificial kidney stone models are important for performing comparative studies of different lithotripsy modalities. The acoustic and mechanical properties of renal calculi dictate the manner by which stones interact with the mechanical stresses produced by shock wave lithotripsy (SWL) or intracorporeal lithotripsy modalities. We have developed a novel artificial kidney stone model that is made of natural substances found in real kidney stones. These stone models appear to be much closer in physical properties to natural kidney stones than previously used stone models. The acoustic and mechanical properties of six groups of artificial stone models were compared to corresponding natural stones of similar compositions. Moreover, three groups of artificial stone models made of plaster-of-Paris were compared to their natural counterparts. In terms of acoustic properties, stone density was measured using a pycnometer based on Archimedes' principle, whereas longitudinal and transverse (or shear) wave propagation speeds were measured using an ultrasound pulse transmission technique. These values were used to calculate wave impedance and dynamic mechanical properties (bulk modulus, Young's modulus, and shear modulus) of the stones. The microhardness of the stones was measured and the effect of composition on stone fragility was evaluated. Artificial stones, when compared to natural stones of similar composition, showed similar trends in longitudinal and transverse wave speeds, wave impedance, and dynamic elastic moduli. However, values for the artificial stones were uniformly low compared to those of natural stones, suggesting that these artificial stones may be more amenable to shock wave fragmentation. The results of SWL on stone fragmentation of artificial and natural stones also revealed similar trends with the exception of artificial cystine stones which were found to be the most resistant to shock wave fragmentation. The results indicate that the physical properties of artificial stones made of natural stone materials are comparable to renal calculi of the same chemical composition. The data suggests that these stone phantoms are suitable for performing standardized and reproducible in vitro investigations, especially with regards to fragility of kidney stones of different chemical compositions during SWL.

Heterotrimeric collagen peptides as fluorogenic collagenase substrates: synthesis, conformational properties, and enzymatic digestion.

The collagenase cleavage site of collagen type I, i.e., the sequence portions 772-784 (P(4)-P(9)') and 772-785 (P(4)-P(10)') of the two alpha1-chains and the sequence portion 772-784 (P(4)-P(9)') of the alpha2-chain, were assembled in an alpha1alpha2alpha1' register by C-terminal cross-linking of these peptides with an artificial cystine knot. The triple-helical conformation of the construct was stabilized by N-terminal extensions with (Gly-Pro-Hyp)(5) repeats. The gaps in the sequence alignment were filled up, and the alpha1-chain was dansylated and the alpha1'-chain was acylated with a tryptophan residue to place in spatial proximity the two chromophores for an efficient fluorescence resonance energy transfer. Although the incorporation of the two N-terminal chromophores leads to partial destabilization of the overall triple-helical fold, the heterotrimer behaved as a collagen-like substrate of the matrix metalloproteinases MMP-1 and MMP-13. Cleavage of the fluorogenic heterotrimer leads to a 6-fold increase in fluorescence intensity, thus making it a useful fluorogenic substrate for interstitial collagenases. With this folded heterotrimeric collagen molecule it was shown that fluorescence resonance energy transfer, as applied so far only for the design of linear fluorogenic enzyme substrates, can also be exploited in conformation dependency.

Improving cystine stone therapy: an in vitro study of dissolution

Objectives. To perform an in vitro study analyzing the possibilities in therapeutic strategies for an effective treatment of cystine stones. Methods. Artificial stones made of cystine [Bon(n)-Stones] with a ball-shaped size of 0.9 cm in diameter were used. Chemolysis of artificial cystine stones with different solvents (artificial urine, physiologic sodium chloride solution, acetylcysteine, tris-(hydroxymethylene)-aminomethane [THAM], and combinations of these) was investigated. An experimental arrangement with computer-assisted online measurement of data simulating the physiologic conditions in the upper urinary tract at varying pH values was used. Results. All solutions showed a statistical improvement in the solubility of cystine stones compared with artificial urine and physiologic sodium chloride solution. The combination of THAM (pH 10) and 2% acetylcysteine was most effective (13.91 ± 1.73 mg/hr) and demonstrated a 41-fold higher ability to dissolve cystine calculi compared with artificial urine. Conclusions. Our data indicate that local chemolysis with special solutions is an effective treatment modality in cystine stone therapy.