An innovative method in venous coronary cast technique / 대한해부학회지

Polyurethane foam (PU foam) is a new material which is being used in producing both macro-anatomical and micro-anatomical specimens. PU foam is simple to use, without need for special equipment. The present study was carried out to evaluate morphology of coronary sinus and its tributaries. During the study, we encountered few problems in carrying out injections. Coronary sinus and its tributaries were difficult to cannulate since the coronary sinus lacks a vascular stem, around which ligature can be tied before injection so that the cannula can be held in place. In contrast, in majority of the organs it is easy to inject since they possess tubular vascular stem to hold the cannula in place. A new device was developed which could be used to cannulate coronary sinus orifice to inject the casting media. The second problem we faced was saponification of adipose tissue. This made corrosion of the soft tissue difficult. Hence in this study, we describe the device we have developed to place in the coronary sinus orifice, and how saponified adipose tissue was taken care during the actual maceration step.

Antiobesity and antihyperlipidemic effect of Ixora coccinea on Triton X-100 induced hyperlipidemia in rats: An approach to evaluate asymmetrical temperature distribution analysis using thermography / 中草药·英文版

Objective: Obesity and hyperlipidemia is the major cause of many pathological diseases with an increase side effects using allopathic drugs. The present study focuses on the effect of Ixora coccinea on Triton X-100 induced hyperlipidemia in rats and associated complications. Methodology: In vitro radical scavenging activity of I. coccinea was assessed using DPPH, FRAP and hydrogen peroxide. In vivo antiobesity and antihyperlipidimic activity of I. coccinea was tested in Triton X-100 induced hyperlipidemic rats and assessed for its biochemical parameters in blood and tissue samples. The relationship between physiological responses and regulation of body temperature was investigated by using animal surface temperature images captured with infrared camera. Results: The results of mineral analysis, antioxidant, total flavonoid and phenolic content represented high amount of mineral and had the potential to scavenge free radicals tested with DPPH, FRAP and hydrogen peroxide radicals with dose dependent activity. The highest activity was observed in aqueous extract, DPPH with 71.5% inhibition, FRAP with 56.8%, H2O2 with 33% activity at 100 µg/mL concentration. Triton X-100 induced hyperlipidemic rats when treated with I. coccinea aqueous extract showed significant activity by regulating the biochemical parameters and maintaining the lipid profile by decreasing TC, LDL-C, VLDL-C, TG and improving HDL-C levels. Similarly, the elevated levels of creatinine, urea, uric acid, AST, ALT, ALP due to induction of hyperlipidemia, were brought back to near normal levels after treatment with I. coccinea. The levels of tissue anti-oxidants enzymes like SOD and CAT were also found to be improved in treated I. coccinea groups. The whole body asymmetrical temperature distribution analysis showed that significant decreases in temperature was observed in obesity induced groups but a gradual increase in temperature (2%–5%) was observed after treatment. Conclusion: Thus, the results indicated that I. coccinea can be a drug of choice to decrease the risk of complications associated with hyperlipidemia and obesity.

Preparation of bioactive denatured acellular dermal matrix from burn mice riched in mice bone marrow mesenchymal stem cells / 中华烧伤杂志

Objective@#To investigate the preparation of bioactive denatured acellular dermal matrix (DADM) from burn mice riched in mice bone marrow mesenchymal stem cells.@*Methods@#Twelve BALB/c mice were collected and 20% total body surface area scalds (hereinafter referred to as burns) with deep partial thickness were inflicted on the back skin of each mouse. After removing epidermis, the burned skin were collected and divided into Triton X-100 group and elhylene diamine tetraacetic acid (EDTA) group according to the random number table, with 15 samples in each group. Samples in Triton X-100 group and EDTA group were respectively placed in mixture of 2.5 g/L Triton X-100 and 2.5 g/L trypsin solution and mixture of 0.2 g/L EDTA and 2.5 g/L trypsin solution for sustained vibration and elution for 24 hours to make mice DADM. The general appearance of DADM was observed. The structure and arrangement of collagen fibers of DADM were observed by scanning electron microscope and tissue structure of DADM were observed by fluorescence microscope. Bone marrow mesenchymal stem cells (BMSCs) from mice were transplanted in mice DADM in the two groups with concentration of 2×105 cells per well to prepare bioactive mice DADM. After cultured for 3 days, tissue structure of bioactive mice DADM was observed by hematoxylin and eosin staining, distribution and number of BMSCs of bioactive mice DADM were observed by immunofluorescence staining. Proliferation of BMSCs of bioactive mice DADM after cultured for 2 h, 1 d, 3 d, and 5 d was detected by cell count kit-8. Data were processed with analysis of variance for repeated measurement and t test.@*Results@#(1) Mice DADM in the two groups were white in appearance with certain tenacity and elasticity. Mice DADM in the two groups maintained good three-dimensional porous network structure. Collagen fibers of mice DADM in EDTA group were with good continuity, and collagen fibers of mice DADM in Triton X-100 group were fractured in varying degrees. Mice DADM in the two groups were decellularized completely, and the collagen fibers were loose and arranged disorderly. The continuity of tissue structure of mice DADM in EDTA group was better than that of mice DADM in Triton X-100 group. (2) After cultured for 3 days, the BMSCs in bioactive mice DADM in the two groups were evenly distributed. The number of bioactive BMSCs in mice DADM in EDTA group was 37±7, which was significantly more than that of mice DADM in Triton X-100 group (25±8, t=0.128, P<0.05). The proliferation of bioactive BMSCs in mice DADM in Triton X-100 group and EDTA group was similar at 2 hours and on day 1 after cultured (t=1.292, 0.656, P>0.05). On 3, 5 days after cultured, the proliferation of bioactive BMSCs in mice DADM in EDTA group was significantly higher than that of mice DADM in Triton X-100 group (t=2.309, 14.128, P<0.05 or P<0.01).@*Conclusions@#Mice DADM prepared by decellularization of EDTA has better three-dimensional porous network structure and good continuity of collagen fiber. The BMSCs in bioactive DADM from burn mice prepared by transplanting BMSCs are evenly distributed with large quantity and strong proliferative capacity, which has the potential to be good autologous dermal substitute.

Serodiagnosis of human neurocysticercosis using antigenic components of Taenia solium metacestodes derived from the unbound fraction from jacalin affinity chromatography

The aim of the present study was to analyse Taenia solium metacestode antigens that were derived from the unbound fraction of jacalin affinity chromatography and subsequent tert-octylphenoxy poly (oxyethylene) ethanol Triton X-114 (TX-114) partitioning in the diagnosis of human neurocysticercosis (NCC). Immunoassays were designed to detect T. solium-specific IgG antibodies by ELISA and immunoblot. Serum samples were collected from 132 individuals who were categorised as follows: 40 had NCC, 62 presented Taenia spp or other parasitic diseases and 30 were healthy individuals. The jacalin-unbound (J unbound ) fraction presented higher sensitivity and specificity rates than the jacalin-bound fraction and only this fraction was subjected to subsequent TX-114 partitioning, resulting in detergent (DJ unbound ) and aqueous (AJ unbound ) fractions. The ELISA sensitivity and specificity were 85% and 84.8% for J unbound , 92.5% and 93.5% for DJ unbound and 82.5% and 82.6% for AJ unbound . By immunoblot, the DJ unbound fraction showed 100% sensitivity and specificity and only serum samples from patients with NCC recognised the 50-70 kDa T. solium-specific components. We conclude that the DJ unbound fraction can serve as a useful tool for the differential immunodiagnosis of NCC by immunoblot.

Interaction of Losartan potassium with the micelles of Triton X 100.

This study is an investigation of the physicochemical interaction of Losartan potassium (LST K), an angiotensin-II receptor (type AT1) antagonist, with micelles of triton X, a nonionic surfactant. The effect of micelles on the spectral properties of LSTK was monitored on at pH 7.4 and at room temperature. The spectrum of LST K showed gradual and progressive bathochromic and hypochromic shift in presence of increasing concentrations of triton X 100. The binding constant Kb of LST K to triton X 100 micelles was calculated using the differential absorbance at λ = 225 nm& was found to be 4.13 ± 0.35 ×105 mol-1 L. By using pseudo-phase model, the partition coefficient between the bulk water and Triton X 100 micelles, Kx, was calculated from both differential absorbance Δ A225, Kx = 2.26 ±0.12 x105 mol-1 L. The binding of LST K to Triton X 100 micelles implied a shift in drug acidity constant (Δ pKa = 0.8).

Experimental study of preparing acellular human tendon scaffold by chemical extraction methods / 中华显微外科杂志

Objective To explore a method of preparing the acellular tendons of human with chemical approaches.Methods From April 2011 to June 2012,several treatments were performed on human tendons using 1.00％ tri(n-butyl) Phosphate (TnBP) combinded with Triton X-100 at the concentration of 0,0.25％,0.50％,1.00％ respectively.Specimens were examined using histological observation,scanning electron microscopy,biomechanical testing and hydroxyproline quantitation.The results were then compared with fresh tendons of control group,so as to value the characteristics of decellularized human tendon scaffold.Results Acellular human tendons had glossy surface,intact aponeurotic membrane and satisfactory flexibility.A small number of disrupted cells remained in the 1.00％ TnBP + 0％ Triton X-100 treated tissue,while other three experimental groups successfully eliminate all cells.Intact and regular collagen architecture was retained in 1.00％ TnBP +0.25％ Triton X-100 treated tissue.1.00％ TnBP + 0.50％ Triton X-100 and 1.00％ TnBP + 1.00％ Triton X-100 treated tissue were nearly identical to 1.00％ TnBP +0.25％ Triton X-100 treated tissue,but the interval of collagen was slightly wider than the control group,the maximum load (385.22 ± 80.32N,398.22 ± 127.20N),ultimate tensile strength(46.69 ± 16.30Mpa,46.20 ±5.52Mpa) and hydroxyproline content(0.282663 ± 0.0110109 μg/mg,0.279355 ± 0.0102129 μg/mg) were statistically lower (P ＜ 0.05) compared with those of the control group,maximum load(533.28 ± 135.77N),ultimate tensile strength (65.56 ± 14.40Mpa) and hydroxyproline content (0.292882 ± 0.0100988 μg/mg) respectively.Conclusion The decellularization treatment with 1.00％ TnBP + 0.25％ Triton X-100 could be optimized for preparing acellular human tendons.

Remoção de endotoxina presente em meio fermentado contendo biomoléculas utilizando sistemas micelares de duas fases aquosas / Endotoxin removal from fermentation broth containing biomolecules using two-phase micellar system

Foi investigada a utilização de Sistema Micelar de Duas Fases Aquosas (SMDFA) para remoção de lipolissacarídeos (LPS) de preparações contendo proteínas recombinantes de interesse farmacêutico, como a proteína verde fluorescente (GFPuv). Os SMDFA são constituídos por soluções de tensoativos contendo micelas e oferecem ambientes hidrofóbico e hidrofílico, que possibilitam seletividade na partição de biomoléculas de acordo com sua hidrofobicidade, permitindo a remoção de LPS contaminante. Neste trabalho, foi realizada a implementação do método para a quantificação de LPS em amostras contaminadas e a obtenção de LPS e GFPuv puros a partir de cultivo de E. coli recombinante. Além disso, foi estudada a influência do Triton X-114 na metodologia de quantificação de LPS, e a adição de MgSO4, CaCl2, KI e (NH4)2SO4 na partição de GFPuv e LPS puros em SMDFA. E ainda, realizou-se um planejamento experimental (22) para avaliar os maiores KGFPuv e porcentoRECGFPuv. O homogeneizado celular de E. coli foi testado nas melhores condições obtidas com o planejamento experimental. E finalmente, o processo por cromatografia de afinidade por íons metálicos (IMAC) foi empregado para investigar a adsorção de LPS em matriz IDA-Ca2+. Conforme os resultados obtidos, o TX-114 causou elevada interferência no método cinético cromogênico, em função da similaridade desta molécula com os LPS. Os LPS apresentaram partição preferencial para a fase concentrada em micelas, com altos valores de remoção, por centoREMLPS>98,0 por cento. Ao contrário, a GFPuv foi recuperada preferencialmente na fase diluída, na qual existe maior volume disponível, resultando em valores de KGFPuv>1. A adição de sais ocasionou diminuição nos valores KGFPuv, provavelmente por causa da carga negativa que GFPuv adquiriu nas condições avaliadas. Os resultados do planejamento experimental mostraram que a melhor condição de partição obtida foi na região do ponto central, 4,0 por cento (p/p) a 60,0°C, com KGFPuv>10. O processo por IMAC apresentou as maiores...

The Aqueous Two-Phase Micellar System (ATPMS) was investigated for endotoxin (LPS) removal from preparations containing recombinant proteins of pharmaceutical interest, such as the green fluorescent protein (GFPuv). These systems usually consist of micellar surfactants solutions and offer both hydrophobic and hydrophilic environments, providing selectivity to the biomolecules partitioning according to its hydrophobicity. In this work, the implementation of the method for LPS quantification in contaminated samples was accomplished, as well as the obtaining of pure LPS and GFPuv from recombinant E. coli. Furthermore, the influence of Triton X-114 in the methodology for LPS quantification was studied, as the addition of MgSO4, CaCl2, KI, and (NH4)2SO4 into the partition of pure GFPuv and LPS in ATPMS. In addition, a statistical design (22) was carried out to evaluate the highest KGFPuv and percentRECGFPuv. The E. coli cell lysate was tested under optimum conditions obtained with the statistical design. And, finally, the process by ionmetal affinity chromatography (IMAC) was used to investigate the adsorption of LPS in IDA-Ca2+ matrix. The results showed that the TX-114 caused high interference in the kinetic chromogenic method, according to the similarity of this molecule to LPS. The LPS showed preferential partitioning to the micellerich phase, with high values of removal, percentREMLPS>98.0 percent. In the other hand, the GFPuv was preferentially recovered in the micelle-poor phase, in which there is greater volume available resulting in values of KGFPuv>1. The addition of salts caused a reduction in the values KGFPuv, probably because of the negative charge that the GFPuv acquired at the conditions evaluated. The results of the statistical design showed that the best partitioning condition obtained was in the central point region, 4.0 percent (wt/wt) at 60.0°C, with KGFPuv>10. The process by IMAC showed the highest adsorption of LPS-IDA-Ca+2 capacities at the conditions of lower pH...

Retainment of membrane binding capacity of non-palmitoylated Gs alpha mutants expressed in COS-1 cells

Heterotrimeric guanine nucleotide binding regulatory proteins (G proteins) transduce extracellular signals into intracellular signals by coupling receptors and effectors. Because most of the G protein-coupled receptors are integral proteins, the G proteins need to have a membrane binding capacity to receive signals from the receptors. The alpha subunit of G protein binds tightly to the cytoplasmic face of the plasma membrane without any membrane spanning domain. Fatty acylation of G alpha with myristic acid or palmitic acid, in addition to the beta gamma subunits, plays an important role in anchoring the G alpha subunit. The reversible and dynamic palmitoylation of the alpha subunit of stimulatory G protein (Gs alpha) has been suggested as essential for its membrane attachment. However, in our previous experiments, Gs alpha deleted in the amino terminus containing palmitoylation site, retained its binding capacity when expressed in COS cells. Thus, to evaluate the role of palmitoylation in Gs alpha membrane binding, we constructed and expressed non-palmitoylated mutants of Gs alpha and analyzed their subcellular distributions in COS-1 cells. We found that non-palmitoylated mutants of Gs alpha, C3S- and G2A/C3S Gs alpha, retained their membrane binding capacities in COS-1 cells, demonstrating that palmitoylation is not essential for membrane binding of Gs alpha in COS-1 cells. We also found that the palmitoylation did not change significantly the distribution of Gs alpha in Triton X-114 partition. These results suggest that the palmitoylation of Gs alpha may produce different effects on membrane binding depending on cell types.

Triton X-100 induces apoptosis in human hepatoma cell lines

The detergent Triton X-100 was used to establish a model for apoptosis in hepatoma cell lines. The electrophoresis of DNA extracted from 0.01% Triton X-100 treated hepatoma cell lines showed DNA ladder formation, a hallmark of apoptosis. The DNA fragmentation appeared within less than 60 min of the Triton X-100 treatment. Chromatin condensation and apoptotic bodies were observed by hematoxylin and eosin (H & E) stain, and fragmented nucleosome was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) test. Apoptosis was semi-quantitated by measuring the lactate dehydrogenase (LDH) level for cytotoxity. It was found that apoptosis had been induced in more than 90% of the cells treated with Triton X-100 for 150 min. These data show that Triton X-100 efficiently induces the apoptotic cell death in hepatoma cell lines.

Comparison of 2 decellularizing methods to prepare rat acelluar allograft spinal cord scaffold / 第三军医大学学报

Objective To compare the effect of 2 decellularizing methods,sodium deoxycholate plus Triton X-100 or plus DNase and Rnase,in the preparation of acelluar allograft spinal cord scaffold in order to provide an ideal natural spinal cord scaffold to bridge the nerve gap.Methods Spinal cord was removed from health rats,and then decellularized by the method of freeze thawing(immersing in 3% sodium deoxycholate followed by the mixture of 1?103 U/ml DNase and RNase),or by chemical extraction(immersing in 1% Triton X-100 and then 1% sodium deoxycholate).HE staining,myelin staining and scanning electron microscopy(SEM) were employed to evaluate the spinal cord scaffold after the 2 methods of decellularization.Results Both cells and myelin were completely decellularized with the 2 methods.In cross section,network of the extracellular matrix was presented without axon,sheath and cells nucleus being seen in the scaffold.Typical network of empty tubes were viewed in longitudinal sections.Conclusion An ideal spinal cord scaffold can be produced with these 2 decellularizing methods in tissue engineering.The scaffold made by the 2 methods have similar three dimensional structure with normal spinal cord,so can be used as a graft to bridge the nerve gap directly or as a scaffold to implant the seeding cells in spinal cord tissue engineering.

Spectroscopic studies on the denaturation of papain solubilized and Triton X-100-solubilized glucoamylase from rabbit small intestine.

Intestinal brush border proteins consist of an enzymatically active hydrophilic moiety attached to a hydrophobic tail. Papain dissociates the hydrophilic part by cleaving off the hydrophobic tail, whereas the detergentTriton X-100 solubilizes the whole molecule. Denaturation by 8 Μ urea or 4 Μ guanidinium chloride does not alter the structure of the papain-solubilized enzyme. An appreciable alteration of the structure of detergent-solubilized enzyme was observed on denaturation. The difference spectra of Triton X-100 (1%)—solubilized enzyme and its urea denatured form shifts and intensifies, with increase in the concentration of the denaturant with an isobestic point at 252 nm. A new band at 280 nm also appears at 4 Μ urea concentration. Papain-solubilized glucoamylase has an ∝ -helical conformation in solution unlike the detergentsolubilized fraction. An elongated structure for the papain solubilized enzyme is inferred from the urea denaturation studies and from molecular weight determinations.