Fracture of tendon collagen

A study of rat-tail tendon fibers, about 200 μ in diameter, by scanning electron microscopy of fractured specimens reveals that these crimped fibers are largely made up of cylindrical units, about 10 μ in diameter. These, in turn, are made up of the well-known collagen fibrils, ca. 0.1 μ in diameter, which have been observed previously by transmission electron microscopy and small-angle x-ray diffraction. Both the 10-μ and 0.1-μ cylindrical units were found to be arranged parallel to the macroscopic fiber axis rather than helically about it, as has been often proposed in the past. Significant improvement in resolution of the fracture surfaces was obtained by dehydration of the tendon. Microscopy of undehydrated tendon fibers undergoing solubilization by dilute aqueous acetic acid confirmed the results obtained by fractography.     

Posttranslational modifications of collagen studied by off-line coupling of HPLC and CE

A typical example of non-enzymatic change of collagen is glycation (the Maillard reaction, formation of advanced glycation end products) resulting from the reaction of sugars with the epsilon-amino group of lysine. Posttranslational non-enzymatic modifications of collagen by sugars were studied. Collagenous tissues were incubated as a test protein separately with both glucose and ribose. The collagen mixture was digested by bacterial collagenase and separated by reversed-phase HPLC (in a Jupiter Proteo 90 A column). The eluate from this HPLC separation was collected as seven fractions and consecutively analysed by CE in a bare fused silica capillary (57/50 cm x 75 mm id) using 100 mM sodium 1-heptanesulfonate in 100 mM phosphate buffer, pH 2.5 (NaH2PO4 adjusted to pH by phosphoric acid). The chromatographic and electromigration behaviour of individual peptides varied considerably. This off-line HPLC-CE coupling made it possible to discover minor changes in the structure of collagen caused by posttranslational modifications. A new HPLC-CE technique for peptide analysis was developed, and applied to the identification of posttranslational modifications in slowly metabolised test proteins.     

The effects of urea and n-propanol on collagen denaturation: using DSC, circular dicroism and viscosity

The effect of urea and n-propanol on circular dichroism (CD) and viscosity of purified type1 collagen solution at various temperatures and differential scanning calorimetry (DSC) of rat-tail tendon (RTT) collagen fibre have been studied. CD reveals a spectrum with a positive peak at around 220 nm and a negative peak at 200 nm characteristics of collagen triple helix. The molar ellipticity decreases as the concentration of urea increases up to particular concentration (collagen solution treated with 265 μM of urea) and after that it increases (collagen solution treated with 500 μM of urea). There is a linear decrease in molar ellipticity as the concentration of n-propanol increases. Denaturation temperature of urea and n-propanol treated with purified collagen solution has been studied using viscosity method. Additives such as urea and n-propanol decrease the thermal stability of collagen triple helix in solution and in RTT collagen fibre. Thermal helix to coil transition of urea and n-propanol treated collagen depends on the degree of hydration and the concentration of these additives. Thermodynamic parameters such as the peak temperature, enthalpy of activation, and energy of activation for collagen-gelatin transition for native, urea and n-propanol treated RTT collagen fibre has been calculated using DSC. The change in the thermodynamic parameters has been observed for native, urea and n-propanol treated RTT collagen fibres. The experimental results show that the change in the water structure, dehydration and desolvation induced by different additives such as urea and n-propanol on RTT may vary with the type of denaturation.     

Sensitive silver staining of protein in sodium dodecyl sulfate-polyacrylamide gels using an azo dye, calconcarboxylic acid, as a silver-ion sensitizer

A highly sensitive silver staining method for detecting proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was developed. It is based on the silver nitrate staining method but also employs an azo dye, calconcarboxylic acid (NN), as a silver-ion sensitizer. It increases silver binding on protein bands or spots by the formation of a silver-dye complex and also increases the reducing power of silver ions to metallic silver by NN itself with formaldehyde. After a 2 h gel fixing step, the protocol including sensitization, silver-ion impregnation, and reduction steps can be completed in 1 h. The sensitivity is superior to that of silver stain with glutardialdehyde as a silver-ion sensitizer. The detection limit of NN-silver stain is 0.05-0.2 ng protein. Considering the high sensitivity without using glutardialdehyde, the NN-silver stain would be useful for routine silver staining of proteins.     

On the relationship of amino acid composition to silver staining of proteins in electrophoresis gels: II. Peptide sequence analysis.

The quantification of proteins in silver-stained electrophoresis gels has been limited by the differences in "stainability" of different proteins. Despite efforts by many researchers, the precise basis of the reaction between silver reagents and polypeptides is still unclear, and, depending on the formulation, may even differ. We have tested the hypothesis that differences in stainability among proteins can be attributed to differences in di- or tripeptide composition. The results indicate that some order of protein structure other than short peptides accounts for the staining differences observed.     

Synthesis,characterization, and application of monodisperse gelatin-stabilized silver nanospheres in reduction of aromatic nitro compounds

Monodisperse colloidal silver nanospheres were synthesized by the reaction of silver nitrate, hydroxylammonium hydrosulphate (NH₂OH)₂ · H₂SO₄ and sodium hydroxide in the presence of gelatin as stabilizer. Colloidal nanospheres were characterized by UV-vis absorption spectroscopy, transmission electron microscopy, X-ray diffraction and dynamic light scattering. X-ray diffraction data confirmed that the silver nanospheres were crystalline with face-centered-cubic structure. Transmission electron microscopy analysis revealed the formation of homogeneously distributed silver nanoparticles of spherical morphology and size of the nanoparticles was in the range of 0.7–5.2 nm. Silver nanospheres were stable for more than two months when stored at ambient temperature. Size and size distribution were studied by varying pH, reaction temperature, silver ion concentration in feed solution, concentration of reducing agent and concentration of the stabilizing agent. Catalytic activity of silver nanospheres was tested for the reduction reaction of nitro compounds in sodium borohydride solution. Monodisperse silver nanospheres showed excellent catalytic activity towards the reduction of aromatic nitro compounds. The reduction rate of aromatic nitro compounds had been observed to follow the sequence 4-nitrophenol > 2-nitrophenol > 3-nitrophenol.     

三角形银纳米片的合成及其影响因素

报道了一种在不需光照的条件下制备三角形银纳米片的新方法.在表面活性剂BRIJ35存在下,将硝酸银用硼氢化钠和柠檬酸钠还原,在不同温度范围内反应形成各种尺度的三角形银纳米片,探讨了硼氢化钠浓度、BRIJ35浓度和表面活性剂的种类和溶液pH值等因素对银纳米颗粒形貌和尺寸的影响.     

Fast horizontal electrophoresis. II. Development of fast automated staining procedures using PhastSystem

The development of equipment for fast automated staining is described. It is possible to handle staining procedures with up to 20 steps and nine different solutions. To increase the reaction rate in the reaction chamber, the gels are rotated and high temperatures are used. The temperature in the reaction chamber is controlled between room temperature and 50 degrees C. Increased temperature, above 20 degrees C, generally results in faster staining and destaining. However, some reactions proceed better at a low temperature, including fixation of proteins with TCA, and the development step in silver staining, where increased temperatures cause a high background stain. Silver staining using acidic silver nitrate solution is preferred, due to easy preparation and good storage stability of the reagents. This method also causes little precipitation of silver on the walls of the reaction chamber. Silver staining is accomplished within one hour. Staining with PhastGel Blue is accomplished within 30 min.     

Spectrophotometric assay of superoxide anion formed in Maillard reaction based on highly water-soluble tetrazolium salt.

A novel highly water-soluble tetrazolium salt, WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt), can be reduced to water-soluble formazan with a superoxide anion. Here, the WST-1 assay was applied to detect the superoxide anion generated during the Maillard reaction. A sample solution containing carbonyl compounds such as glyceraldehyde and glycolaldehyde (5 mM) and Na-acetyl-L-lysine (10 mM) was incubated for 2 days at 37 degrees C. The detection of a superoxide anion generated in the sample was performed by the WST-1 assay, and the result was compared with the cytochrome c assay. The reduction of WST-1 was almost perfectly (86-96%) inhibited by the addition of the superoxide dismutase (SOD). On the contrary, the reduction of cytochrome c was slightly (20-25%) inhibited by the addition of SOD. A similar result was observed for the addition of 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron). These results mean that the specificity of WST-1 to the superoxide anion was superior to that of cytochrome c. It was also possible to continuously monitor superoxide anion generation during the Maillard reaction by the coexistence of WST-1 in the reaction solution.     

Synthesis of 1-[(nitroxyalkoxy)methoxy]-1-triazene 2-oxides,new hybrid nitrogen monoxide donors

1-[(Nitroxyalkoxy)methoxy]-3,3-dialkyl-1-triazene 2-oxides were obtained by the reaction of 3,3-dialkyl-1-hydroxy-1-triazene 2-oxide sodium salts with β- or γ-nitroxyalcohol chloromethyl ethers or by the reaction of 3,3-dialkyl-1-(bromoalkoxymethoxy)-1-triazene 2-oxides with silver nitrate. These compounds can be of interest as new hybrid NO donors in living organisms.     

Capillary electrophoresis and circular dichroism study of trichosanthin and its mutants

A new chromogenic reagent, 2-(2-quinolylazo)-5-diethylaminoaniline (QADEAA) was synthesized. A highly sensitive, selective and rapid method for the determination of silver based on the rapid reaction of silver (I) with QADEAA has been developed. In the presence of sodium citrate-sodium hydroxide buffer solution (pH 6.5) and sodium dodecyl sulfonate (SDS) medium, QADEAA reacts with silver to form a violet complex of molar ratio 1:2 (silver to QADEAA). The molar absorptivity of the complex is 1.39x10(5) l mol(-1) cm(-1) at 580 nm. Beer's law is obeyed in the range of 0.01-0.6 mug ml(-1). The relative standard deviation for 11 replicate samples of 0.2 mug ml(-1) is 1.67%. This method can be applied to the determination of silver in water with satisfactory results.     

Improved separation of alpha chains of collagen type I, type III, and type V by noninterrupted electrophoresis using thioglycolic acid as a negatively charged reducer

Improved separation of alpha chains of collagen type I (alpha 1 [I]2 alpha 2[I]), type III(alpha 1[III]3), and type V (alpha 1[V]alpha 3[V])was achieved by noninterrupted sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a negatively charged reducer, thioglycolic acid. The thioglycolic acid, added to the running buffer of the cathodic reservoir, in the middle of electrophoresis quickly migrated in the gel anode, reducing interchain disulfide linkages in collagen type III and dissociating it into its alpha chain monomer, alpha 1[III], without an interruption of electrophoresis. The alpha chain, alpha 1[III], migrated more slowly than the alpha 1 [I] and alpha 2[I] chains of collagen type I, resulting in an excellent separation of alpha 1[III] from alpha 1[I]. The mobility of alpha 1[III] could be controlled by varying the time of thioglycolic acid addition to the running buffer. This enabled us not only to separate alpha 1[III] from alpha 1[I] and alpha 1[V], but also to precisely quantitate these alpha chains, even at low protein loading of mixed samples.     

Intensification of peroxidase-diaminobenzidine staining using gold-sulfide-silver: a rapid and highly sensitive method for visualization in immunoblotting.

A highly sensitive and rapid visualization method for protein detection by immunoblotting is described. Proteins blotted onto a Durapore membrane were visualized by the following procedure: after conventional peroxidase-based staining with 3,3'-diaminobenzidine (DAB), the produced DAB precipitates were intensified by treating with (i) gold trichloride (acid), (ii) sodium sulfide, and (iii) a developer containing silver nitrate. This postintensification method was employed for the detection of the genetic polymorphism of human proteins, such as deoxyribonuclease I in urine, and group specific component, transferrin and alpha 1-antitrypsin in serum after polyacrylamide gel-isoelectric focusing, followed by immunoblotting. This postintensification technique was found to be simple, giving up to 16- to 64-fold amplification of the conventional peroxidase-DAB staining.     

Design and synthesis of new fluorescent probe for rapid and highly sensitive detection of proteins via electrophoretic gel stain

A new fluorescent molecular probe, 2,2′‐(1E,1′E)‐2,2′‐(4‐(dicyanomethylene)‐4H‐pyrane‐2,6‐diyl)bis(ethene‐2,1‐diyl)bis(sodium benzenesulfonate) salt ( 1 ), possessing the cyanopyranyl moieties and two benzene sulfonic acid groups was designed and synthesized to detect proteins in solution and for high‐throughput SDS‐PAGE. Compound 1 exhibited no fluorescence in the absence of proteins; however, it exhibited strong fluorescence on the addition of bovine serum albumin as a result of intramolecular charge transfer. Compared with the conventional protocols for in‐gel protein staining, such as SYPRO Ruby and silver staining, 1 achieves higher sensitivity, even though it offers a simplified, higher throughput protocol. In fact, the total time required for protein staining was 60–90 min under optimum conditions much shorter than that required by the less‐sensitive silver staining or SYPRO Ruby staining protocols. Moreover, 1 was successfully applied to protein identification by mass spectrometry via in‐gel tryptic digestion, Western blotting, and native PAGE together with protein staining by 1 , which is a modified protocol of blue native PAGE (BN‐PAGE). Thus, 1 may facilitate high‐sensitivity protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields.     

The effect of fructose derived carbon shells on the plasmon resonance and stability of silver nanoparticles

Carbon nanoparticles between 10 and 50 nm in diameter and carbon shells of various thickness around silver nanoparticles were synthesized by the hydrothermal reaction of fructose. The effect of the carbon shells on the plasmon resonance of the silver nanoparticles and their stability in sodium chloride solutions was investigated. The shell thickness can be adjusted to have insignificant damping of the plasmon resonance and provide stabilization of the particles in solutions with high ionic strength. Hydrazine–carbonyl cross-linking reactions were performed to link fluorescent dye molecules to carbonyl groups on the carbon shell surface.     

Modified silver staining in 2DE improves protein detection even at extremely low sample concentration

The typical concentration of protein loaded varies from 0.13 to 1.40 μg/μL for a classical silver staining method in 2DE gel. Here, we present a simple modified classical silver staining method by modifying the silver impregnation and development reaction steps. This modified method detects the protein spots at extremely low loaded concentrations, ranging from 0.0048 to 0.0480 μg/μL. We recommend this modified silver staining as an excellent method for the limited biological samples used for silver‐stained 2DE analysis. Altogether, the protocol takes close to two days from first dimension separation to second dimension separation, followed by silver staining, scanning, and analysis.     

Crystallization of silver stearate from sodium stearate dispersions

Silver carboxylates, the common silver source used for photothermographic imaging materials, are normally obtained from the reaction between sodium soap (e.g., sodium stearate) and silver nitrate. They form platelet-like crystals with a lamellar structure in water at room temperature. Light microscopy investigations reveal that the formation of silver stearate (AgSt) crystals follows a diffusion-controlled mechanism. The reaction between the sodium soap and silver nitrate preferentially occurs in solution rather than on the soap fiber solid interface. Cryogenic transmission electron microscopy, together with an on-the-grid reaction technique, provides a useful tool to directly image silver stearate microstructures at the initial stages of AgSt precipitation. The AgSt reaction product first forms particles about 5 nm in size, which is similar to the d-spacing of final AgSt crystals. Those particles aggregate to produce larger and loosely packed embryonic crystals, the precursors to the ultimate silver stearate crystals.     

Tyrosine nitration site specificity identified by LC/MS in nitrite-modified collagen type IV

Non-enzymatic nitrite induced collagen cross-linking results in changes reminiscent of age-related damage and parallels the well-known model system, non-enzymatic glycation. We have recently observed that nitrite modification of basement membrane proteins can induce deleterious effects on overlying retinal pigment epithelial cells in studies relevant to age-related macular degeneration. The present work was undertaken in order to confirm 3-nitro-tyrosine (3-NT) as a product of the reaction and to identify the site specificity of nitration in collagen IV, a major component of basement membranes. Human collagen type IV was modified via incubation with 200 mM NaNO(2) (pH=7.38) for one week at 37(o)C. The modified protein was prepared in 2 different ways, including acid hydrolysis and trypsin digestion for site specificity determination. The samples were analyzed by LC/MS using a C(12) RP column. Site specificity was determined from tandem MS/MS data utilizing TurboSEQUEST software and the Swiss-Prot sequence database. 3-NT was detected in protein digests and acid hydrolysates of nitrite modified collagen IV. Positive identification with standard 3-NT was confirmed by identical R(t), lambda(max)=279 nm and 355 nm, and m/z=227. Analyses of tryptic digests identified four sites of tyrosine nitration, alpha1(IV)Y348, alpha1(IV)Y534, alpha2(IV)Y327, and alpha2(IV)Y1081. These sites are located in the triple-helical region of the protein and provide clues regarding potential sites for nitrite modification in collagen type IV.     

Factors involved in the nuclearity of silver thiosemicarbazone clusters: cocrystallization of two different sized tetranuclear silver(I) clusters derived from a phosphinothiosemicarbazone ligand

The reaction of silver acetate with a phosphinothiosemicarbazone ligand, HLPPh, yielded the tetranuclear silver compound [Ag 4(LPPh) 4].2MeOH ( 1), which after recrystallization results in the cocrystallization of two different silver clusters, [Ag 4(LPPh) 4] (a)[Ag 4(LPPh) 4] (b).8MeOH ( 2). The factors involved in the assembly of tetranuclear compounds derived from thiosemicarbazone ligands and the structural differences between the two clusters are analyzed herein. Additionally, HLPPh and complex 1 exhibit photoluminescence in solution at room temperature.     

Inhibition of blood coagulation factor XIIIa-mediated cross-linking between fibronectin and collagen by polyamines

Soluble fibronectin is found in body fluids and media of cultured adherent cells. Insoluble fibronectin is found in tissue stroma and in extracellular matrices of cultured cells. Fibronectin is a substrate for factor XIIIa (plasma transglutaminase) and can be cross-linked to collagen and to the alpha chain of fibrin. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to investigate the possibility that factor XIIIa-mediated cross-linking is influenced by polyamines. Spermidine inhibited cross-linking between fibronectin and type I collagen, isolated alpha 1 (I) collagen chains, or iodinated cyanogen bromide fragment 7 of alpha 1 (I) chains (125I-alpha 1 (I)-CB7). Half-maximal inhibition of cross-linking between 125I-alpha (I)-CB7 and fibronectin was observed when 0.1 mM spermine or spermidine was present. Spermidine, 0.7 mM, partially inhibited cross-linking between fibronectin and the alpha chain of fibrin but failed to inhibit cross-linking between the fibrin monomers of a fibrin clot. Spermidine also failed to inhibit cross-linking between fibronectin molecules when aggregation of fibronectin was induced with dithiothreitol. In contrast, 0.7 mM monodanyslcadaverine inhibited fibronectin-collagen, fibronectin-fibrin, fibronectin-fibronectin, and fibrin-fibrin cross-linking. Spermidine or spermine, 0.7 mM, enhanced the cross-linking between molecules of partially amidinated fibronectin, suggesting that N1,8-(di-gamma-glutamyl)-polyamine cross-linkages were formed. Spermidine and spermine failed to enhance cross-linking between monomers of amidinated fibrin. These results indicate that physiologic concentrations of polyamines specifically disturb transglutaminase-catalyzed cross-linking between fibronectin and collagen.