Scleraldehyde as a stabilizing agent for collagen scaffold preparation

Schizophyllan is a natural polysaccharide, produced by fungi of the genus Schizophyllum. Periodate oxidation specifically cleaves the vicinal glycols in schizophyllan to form their dialdehyde derivatives. The present study investigates the interaction of scleraldehyde with Type I collagen membrane. The formation of the inter and intra interaction between scleraldehyde and the collagen fibres results in significant increase in viscosity of collagen. Crosslinking efficiency of scleraldehyde was found to increase with concentration of scleraldehyde. Scleraldehyde interacted collagen membrane exhibited an increase in thermal stability by 29 °C at pH 8. The gelling time of collagen fibrils was found to decrease with increase in concentration of scleraldehyde due to shift in nucleation centre. Swelling degree of collagen membrane was also found to decrease with increase in concentration of scleraldehyde. Scleraldehyde treated collagen membrane exhibited 93% resistance to collagenase. The modified collagen membrane exhibited non-toxicity towards the fibroblasts cells. The modified collagen membrane by scleraldehyde finds application as a stabilizing agent in scaffold preparation.     

Effects of temperature and shear on the activity of acid phosphatase in a tubular membrane reactor

The effect of temperature on the activity of acid phosphatase [orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] immobilized as a gel layer on the inner wall of ultrafiltration tubular membranes by both copolymerization/gelation and cogelation has been investigated. Both forms of gel-immobilized enzyme showed fairly good stability, the activation energy of their inactivation being significantly lower than that of the free enzyme and of the heat denaturation of proteins in general. The shear effect on the cogelled enzyme was also studied at different temperatures and Reynolds numbers. The results indicated that the cogelled enzyme is a more convenient form for continuous operation in the tubular membrane reactor (TMR), a reactor configuration particularly suitable for industrial applications.     

类人胶原蛋白-透明质酸血管支架的性能及生物相容性

将类人胶原蛋白与透明质酸按不同比例复合,控制透明质酸的终浓度(W/V)分别为0、0.01%、0.05%、0.1%,用京尼平交联,采用真空冷冻干燥方法构建出血管支架材料。通过扫描电镜、XPS分析、拉力测试、压力爆破实验、细胞毒性实验、血管支架细胞种植实验及小鼠皮下植入等方法对其表面超微结构、表面元素组成、力学性能、细胞毒性等级、细胞相容性、组织相容性进行了研究。结果表明:当透明质酸的含量为0.05%时,类人胶原蛋白-透明质酸支架的孔径比较均匀,孔隙率达94.38%,应力为(1000.8±7.9)kPa,爆破压力为(1058.6±8.2)kPa,细胞毒性实验合格,同时具有良好的细胞相容性、组织相容性及降解性能。     

Bacteriostatic properties of biomatrices against common orthopaedic pathogens

Tissue-engineered grafts for tissue regeneration include either mature or progenitor cells seeded onto biomatrices that provide shape and support for developing tissue. Popular biomaterials used in orthopaedic surgery include collagen type I, hyaluronic acid, hydroxyapatite, and polylactic polyglycolic acid (PLGA). Biomatrices with bacteriostatic properties may be beneficial in promoting tissue-engineered graft survival in patients susceptible to infection. We evaluated the bacteriostatic effects of these biomaterials on the growth of the four most common orthopaedic bacterial pathogens: Staphylococcus aureus, Staphylococcus epidermidis, beta-hemolytic Streptococcus, and Pseudomonas aeruginosa. Hyaluronic acid demonstrated the largest bacteriostatic effect on these pathogens by inhibiting bacterial growth by an average of 76.8% (p = 0.0005). Hydroxyapatite and collagen inhibited growth on average by 49.7% (p = 0.011) and 37.5% (p = 0.102), respectively. PLGA exhibited the least bacteriostasis with an average inhibition of 9.8% (NS) and actually accelerated the growth of beta-hemolytic Streptococcus and P. aeruginosa.     

Substitution of aspartic acid with glutamic acid increases the unfolding transition temperature of a protein

Proteins from thermophiles are more stable than those from mesophiles. Several factors have been suggested as causes for this greater stability, but no general rule has been found. The amino acid composition of thermophile proteins indicates that the content of polar amino acids such as Asn, Gln, Ser, and Thr is lower, and that of charged amino acids such as Arg, Glu, and Lys is higher than in mesophile proteins. Among charged amino acids, however, the content of Asp is even lower in thermophile proteins than in mesophile proteins. To investigate the reasons for the lower occurrence of Asp compared to Glu in thermophile proteins, Glu was substituted with Asp in a hyperthermophile protein, MjTRX, and Asp was substituted with Glu in a mesophile protein, ETRX. Each substitution of Glu with Asp decreased the Tm of MjTRX by about 2 degrees C, while each substitution of Asp with Glu increased the Tm of ETRX by about 1.5 degrees C. The change of Tm destabilizes the MjTRX by 0.55 kcal/mol and stabilizes the ETRX by 0.45 kcal/mol in free energy.     

Changes in thermal and electrical properties of bone as a result of 1 MGy-dose gamma-irradiation

Determination of temperature dependencies of electric conductivity and thermal properties by differential scanning calorimetry (DSC) allow to analyse the processes of charge and heat transport in the bone being a complex collagen-hydroxyapatite (HAP)-water system. Modification of the bone structure by high doses of gamma-radiation changes the electrical and thermal properties of the bone. Electrical conductivity (sigma) of the bone decreases with consecutive heating runs. The decrease in sigma observed for irradiated samples was explained by the scission of the main chain of collagen macromolecule. Irradiation decreased the hydration level in the bone, its denaturation temperature and increased both enthalpy and entropy of the denaturation process.     

Adaptive Changes in Cardiac Fibroblast Morphology and Collagen Organization as a Result of Mechanical Environment

There is a growing body of work in the literature that demonstrates the significant differences between 2D versus 3D environments in cell morphologies, spatial organization, cell-ECM interactions, and cell signaling. The 3D environments are generally considered more realistic tissue models both because they offer cells a surrounding environment rather than just a planar surface with which to interact, and because they provide the potential for more diverse mechanical environments. Many studies have examined cellular-mediated contraction of 3D matrices; however, because the 3D environment is much more complex and the scale more difficult to study, little is known regarding how mechanical environment, cell and collagen architecture, and collagen remodeling are linked. In the current work, we examine the spatial arrangement of neonatal cardiac fibroblasts and the associated collagen organization in constrained and unconstrained collagen gels over a 24 h period. Collagen gels that are constrained by their physical attachment to a mold and similar gels, which have been detached (unconstrained) from the mold and subsequently contract, offer two simple mechanical models by which the mechanisms of tissue homeostasis and wound repair might be examined. Our observations suggest the presence of two mechanical regimes in the unconstrained gels: an outer ring where cells orient circumferentially and local collagen aligns with the elongated cells; and a central region where unaligned stellate/bipolar cells are radially surrounded by collagen, similar to that seen throughout constrained gels. The evolving organization of cell alignment and surrounding collagen organization suggests that cellular response may be due to the cellular perception of the apparent stiffness of local physical environment.     

Hyaluronic acid of Streptococcus sp. as a potent elicitor for induction of systemic resistance against plant diseases

The potential of hyaluronic acid (HA) in inducing systemic resistance to cucumber, tomato and pepper was tested in planta. In the study, HA was found to be a potent agent for suppressing disease caused by Cucumber Mosaic Virus (CMV) (in pepper), Pseudomonas syringae pv. tomato (tomato speck disease), Xanthomonas axonopodis pv. vesicatoria (tomato spot disease), Pseudomonas syringae pv. lachrymans (cucumber angular leaf spot), and Colletotrichum orbiculare (cucumber anthracnose). Disease control was obtained with spraying, injection and drenching of plants with HA. HA did not exhibit direct antimicrobial action against the pathogens tested. Studies carried out in transgenic tobacco indicated that defense genes PR 1a and PDF 1.2 were activated upon treatment with HA, demonstrating salicylic acid (SA) and jasmonic acid (JA) pathways getting activated during defense. Further work is warranted to evaluate the use of HA-mediated disease suppression in crop plants.     

Synthesis of a C-linked hyaluronic acid disaccharide mimetic

The synthesis of a C-disaccharide that is designed as a mimetic for the repeating unit disaccharide of hyaluronic acid is described. The target compound was obtained via the SmI2-promoted coupling reaction of the sulfone, 2-acetamido-4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-1,2-dideoxy-1-pyridinylsulfonyl-beta-D-glucopyranose (6), and the aldehyde, p-methoxyphenyl 2,3-di-O-benzyl-4-deoxy-4-C-formyl-6-O-p-methoxybenzyl-beta-D-glucopyranoside (14).     

Alterations in growth temperature range and fatty acid composition of Thermus as a result of plasmid elimination

Elimination of plasmids from Thermus flavus, T. thermophilus and three wild Thermus strains caused alterations in growth temperature range, pigmentation and membrane fatty acids without affecting viability. Following plasmid elimination all Thermus strains lost their ability to grow above 70°C. In addition, the minimum growth temperature was lowered by 5–10°C. Fatty acids were reduced by an average of approximately 35%. In addition, the contribution of iso- and anteisobranched fatty acids were altered in four of the five strains. The iso C_15:0/iso C_17:0 ratio approached 1.0 in all strains, whereas the anteiso C_15:0/anteiso C_17:0 was reduced to 0.2. The iso C_16:0/normal-C_16:0 ratio increased in all strains due to an increase in iso C_16:0 in four strains and a reduction in normal-C_16:0 relative to iso C_16:0 in one strain. However, it was evident that the plasmid-free strains were able to compensate for these alterations in membrane fluidity to a certain extent by reducing the average chain length of isobranched acids. Altered fatty acid metabolism at the level of precursors may have influenced membrane composition and consequently growth temperature range.     

Synthesis of proteoglycans and hyaluronic acid by long-term cultures of testicular cells from immature and pubertal rats

Long-term cultures of somatic testicular cells derived from immature and pubertal rats were used to study the synthesis of proteoglycans (PG) and hyaluronic acid (HA). Labelled PG and HA in the culture medium, membrane-associated and intracellular pools were characterized by gel filtration, ion exchange chromatography and selected enzymatic and chemical treatments. Somatic cells synthesize a PG containing both heparan and chondroitin/dermatan sulfate (CS/DS) chains and a PG containing only CS/DS chains. No major qualitative changes in the type of PG were observed in cells derived from immature and pubertal animals. However, significant age-dependent differences in the cell distribution pattern of PG and HA were determined. This may have implications in the regulation of spermatogenesis.     

凝胶层析在发酵液中分离提纯透明质酸中的应用

采用葡聚糖凝胶G-100,以0.1 mol/L氯化钠溶液为洗脱剂,在优化的凝胶层析条件(层析柱高度30 cm、进样量2 ml、洗脱流速1 ml/4 min)下,对透明质酸发酵液进行分离纯化,得到了高纯度的透明质酸.HA提取率达到79.85%,蛋白质去除率为84.93%.     

Changes in the blood-testis barrier as a result of streptococcal sensitization

Permeability of the histo-hematic barrier in the guinea pig and mouse testes immunized with streptococci has been studied radiometrically and luminescent microscopically with application of labelled bovine serum albumin, human gamma-globulin and blue dye preparations. The permeability of the hemato-testicular barrier increases on the 7th--30th day after immunization. Test-preparations penetrate from the vessels of the intercanalicular connective tissue layers through the membrane of the convoluted tubules. The testicular autoantigen is revealed in blood. The disturbance in permeability of the hemato-testicular barrier precedes or coincides in time with the developing destruction of the testicular spermatogenic elements.     

Changes in tissue protein levels as a result of endurance exercise

We previously reported that endurance training increases amino acid catabolism. In this study, the effects of an acute endurance exercise bout on tissue protein levels and urea excretion have been investigated. Exhaustive exercising of trained rats resulted in an increase in ammonia excretion but there was no significant change in urea excretion. Protein levels of muscle and liver were significantly decreased by an exhaustive bout of exercise. In muscle, both the soluble and myofibrillar protein fractions were decreased in exhausted rats. These results demonstrate that during exercise there is a net loss of protein in muscle and liver.     

Electrochemical properties of heme-protein in lauric acid films and its application as a biosensor

In this research, the enhancement of electron-transfer activity of hemoglobin (Hb) in lauric acid film was investigated for the first time. This type of composite film was made on a glassy carbon electrode by a casting method. Cyclic voltammetric result of the modified electrode displays a well-defined redox peak, which was attributed to the direct electrochemical response of Hb. Our results illustrate that Hb exchange electrons directly with electrode and exhibits the characteristics of peroxidase. When we use this modified electrode as a biosensor, it gives excellent performance in the electrocatalytic reduction of hydrogen peroxide (H₂O₂). The parameters such as pH and applied potential of the biosensor influencing in H₂O₂ detection were optimized carefully. Through the optimal conditions, the proposed biosensor shows the linear range for H₂O₂ determination was from 1×10⁻⁵ to 1.25×10⁻⁴ mol L⁻¹ with a detection limit of 1×10⁻⁷ mol L⁻¹. The biosensor retained more than 90% of the initial response after 14 d.     

Thermal inactivation of D-amino acid oxidase from Trigonopsis variabilis occurs via three parallel paths of irreversible denaturation

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a long-known flavoenzyme whose most important biocatalytic application is currently the industrial production of 7-amino-cephalosporanic acid (7-ACA) from cephalosporin C. Lacking mechanistic foundation, rational stabilization of TvDAO for improved process performance remains a problem. We report on results of thermal denaturation studies at 50 degrees C in which two purified TvDAO forms were compared: the native enzyme, and a site-specifically oxidized protein variant that had the side chain of cysteine108 converted into a sulfinic acid and lost 75% of original specific activity. Although inactivation time courses for both enzymes are fairly well described by simple single-exponential decays, the underlying denaturation mechanisms are shown by experiments and modeling to be complex. One main path leading to inactivation is FAD release, a process whose net rate is determined by the reverse association rate constant (k), which is 25-fold lower in the oxidized form of TvDAO. Cofactor dissociation is kinetically coupled to aggregation and can be blocked completely by the addition of free FAD. Aggregation is markedly attenuated in the less stable Cys108-SO(2)H-containing enzyme, suggesting that it is a step accompanying but not causing the inactivation. A second parallel path, characterized by a k-value of 0.26/h that is not dependent on protein concentration and identical for both enzymes, likely reflects thermal unfolding reactions. A third, however, slow process is the conversion of the native enzyme into the oxidized form (k < 0.03/h). The results fully explain the different stabilities of native and oxidized TvDAO and provide an inactivation mechanism-based tool for the stabilization of the soluble oxidase.