Bombyx batryticatus Cocoonase Inhibitor Separation, Purification, and Inhibitory Effect on the Proliferation of SMCC-7721 HeLa-Derived Cells.

The present study was to isolate and purify Bombyx batryticatus cocoonase inhibitor (BBCI) and to evaluate its inhibitory effect on the proliferation of SMCC-7721 cells. BBCI was purified from the crude proteins of Bombyx batryticatus using affinity chromatography with cocoonase as the ligand, its N-terminal amino acid sequence was determined using the Edman degradation method, and its inhibiting activity on SMCC-7721 cell proliferation was detected in vitro using the MTT method and in vivo in tumor-bearing nude mice. The purified BBCI presented as a single band in SDS-PAGE, the molecular weight determined by time-of-flight mass spectrometry was 13,973.63 Da, and its N-terminal amino acid sequence was VRNKRQSNDD. BBCI was a noncompetitive cocoonase inhibitor with an average Michaelis constant of 76.50, and it inhibited cocoonase activity with an inhibition ratio of 1 : 1 (molar). BBCI could inhibit the proliferation of SMCC-7721 cells in vitro with the IC50 being about 260.52 µg/ml within 36 h of treatment and inhibit the SMCC-7721 tumor growth in nude mice by subcutaneous injection of BBCI around the tumor, where the tumor inhibitory effect was dose dependent. BBCI did not significantly influence the spleen coefficient of the mice. In conclusion, to the best of our knowledge, the present study is the first to report that BBCI, which was purified from Bombyx batryticatus, was a serine proteinase inhibitor with antitumor activity.

[Research on medical speciality of traditional Chinese medicines using dot-immunoblotting method based on polyclonal antibody prepared from traditional Chinese medicines with hot/cold nature].

OBJECTIVE: To research on the substantial foundation of the medical speciality of Chinese traditional medicines from immunogenicity. METHOD: Control antigen with hot nature was prepared from the mixture of the aqueous extracts of three Chinese traditional medicines with three typical hot nature of Alpinia officinarum, Cinnamomum cassia and Curculigo orchioides, while that with cold nature prepared with Rheum palmatum, Anemarrhena asphodeloides, Coptis chinensis, and polyclonal antibody was prepared by immunizing rabbit with control antigen. Dot blotting was performed between the polyclonal antibody of control antigen and the aqueous extracts of nine Chinese traditional medicines on a piece of PVDF membrane, and the blotting signals were analyzed by the software of Quantity One. RESULT: Blotting signals with hot control antigen of nine Chinese traditional medicines in descending were Zingiber officinale, Aconitum carmichaeli, Eucommia ulmoides, Fraxinus rhynchophylla, Lonicera japonica, Anemarrhena asphodeloides, Coptis chinensis, Rheum palmatum and Phellodendron chinense, which degree of similarity to control antigen in peak value were 57.33%, 43.56 %, 34.16%, 30.2%, 28.81%, 26.53%, 21.68%, 17.62% and 14.85%, respectively. Blotting signals with cold control antigen were Rheum palmatum, Anemarrhena asphodeloides, Coptis chinensis, Phellodendron chinense, Zingiber officinale, Lonicera japonica, Fraxinus rhynchophylla, Eucommia ulmoides and Aconitum carmichaeli in descending, of which degree of similarity to cold control antigen in peak value were 55.22%, 54.23%, 46.72%, 34.08%, 30.3%, 24.48%, 24.33%, 20.35% and 15.17%, respectively. Results of cluster analysis with Wistar's method showed that nine medicines were classified into two groups, one group included Phellodendron chinense, Anemarrhena asphodeloides, Coptis chinensis, Rheum palmatum, another was Zingiber officinale, Aconitum carmichaeli, Eucommia ulmoides, Fraxinus rhynchophylla, Lonicera japonica. CONCLUSION: Blotting signals of nine medicines with control antigen regularly varied with the alteration of medicine nature. The more similarity degree of the tested medicine to control antigen was smaller, the more distance of the tested medicine to control antigen was further. Dot immunoblotting was a practical and effective new method in researching the substantial foundation of the medical speciality of Chinese traditional medicines.

[Investigation and management of wound on head and neck from the disaster area in Wenchuan earthquake].

OBJECTIVE: To investigate and analyze the wounded's state of ear, nose, throat, neck and head injury in Wenchuan earthquake. METHODS: The 206 wounded cases, who was treated in No. 452 Hospital of People's Liberation Army, were investigated specially with emphasis on injury cause, severity and treatment. RESULTS: The injured 165 cases among the 206 were in hospital, while the cases who related to the injury of ear, nose and throat were 37 cases (22.4%). Among the inpatients, the trauma of otorhinolaryngology and head and neck included: ear injuries totally 13 cases (including hemotympanum 2 cases), extraneous matter 4 cases, haemorrhagic 4 cases, nasalis and the fracture of nasal bone and nasal sinuses 7 cases (including cerebrospinal rhinorrhea 1 case), zygomatic abscess 1 case, fracture of mandible 4 cases, lip injuries 2 cases and hoarse 2 cases. The inpatients were wounded mostly because of falling and stepping. All the inpatients recovered well after properly management by ENT doctors. CONCLUSIONS: Maxillofacial injury of the wounded those were medical evacuation in the earthquake area, was ignored more readily comparing to the injury of other spots, so specialist should examine early and treat properly the people as soon as possible.

Longitudinal residual strain and stress-strain relationship in rat small intestine.

BACKGROUND: To obtain a more detailed description of the stress-free state of the intestinal wall, longitudinal residual strain measurements are needed. Furthermore, data on longitudinal stress-strain relations in visceral organs are scarce. The present study aims to investigate the longitudinal residual strain and the longitudinal stress-strain relationship in the rat small intestine. METHODS: The longitudinal zero-stress state was obtained by cutting tissue strips parallel to the longitudinal axis of the intestine. The longitudinal residual stress was characterized by a bending angle (unit: degrees per unit length and positive when bending outwards). Residual strain was computed from the change in dimensions between the zero-stress state and the no-load state. Longitudinal stresses and strains were computed from stretch experiments in the distal ileum at luminal pressures ranging from 0-4 cmH2O. RESULTS: Large morphometric variations were found between the duodenum and ileum with the largest wall thickness and wall area in the duodenum and the largest inner circumference and luminal area in the distal ileum (p < 0.001). The bending angle did not differ between the duodenum and ileum (p > 0.5). The longitudinal residual strain was tensile at the serosal surface and compressive at the mucosal surface. Hence, the neutral axis was approximately in the mid-wall. The longitudinal residual strain and the bending angle was not uniform around the intestinal circumference and had the highest values on the mesenteric sides (p < 0.001). The stress-strain curves fitted well to the mono-exponential function with determination coefficients above 0.96. The alpha constant increased with the pressure, indicating the intestinal wall became stiffer in longitudinal direction when pressurized. CONCLUSION: Large longitudinal residual strains reside in the small intestine and showed circumferential variation. This indicates that the tissue is not uniform and cannot be treated as a homogenous material. The longitudinal stiffness of the intestinal wall increased with luminal pressure. Longitudinal residual strains must be taken into account in studies of gastrointestinal biomechanical properties.

[Specific inhibition of Raf-1 gene expression in HNE1 by RNA interference].

OBJECTIVE: To silence the expression of Raf-1 gene in HNE1 cells using vector-based RNA interference (RNAi) technique. METHODS: The vector containing the human U6 promoter was used for targeted gene silencing when a dsDNA oligonucleotide encoding an appropriate shRNA was ligated into the vector, and 67nt oligonucleotide fragment was inserted into the downstream of the U6 promoter. Plasmids containing different Raf-1 target sequences [ (1) pshuttle-Raf-1-a( 225), (2) pshattle-Raf-1-b ( 358) and (3) pshuttle-Raf-1-c(474)], were transfected into HNE1 cells. Expression of Raf-1 mRNA was assayed by RT-PCR. Apoptosis were determined by cytometry. RESULTS: Vector-based RNAi had advantages over antisense RNA because it could be delivered to the target cell more efficiently, and effect could last longer. Raf-1 expression could be inhibited by plasmid-expressed shRNA. Three different targeting sequences were selected from Raf-1 gene, and the inhibitory effect of pSIREN shuttle-Raf-1-b (358) was biggest. CONCLUSION: Raf-1 expression in HNE1 cells can be inhibited significantly using plasmid-based RNAi.

Time-dependent viscoelastic properties along rat small intestine.

AIM: To measure the time-dependent (viscoelastic) behavior in the change of the small intestinal opening angle and to test how well the behavior could be described by the Kelvin model for a standard linear solid. METHODS: Segments from the duodenum, jejunum, and ileum were harvested from 10 female Wistar rats and the luminal diameter, wall thickness, and opening angle over time (theta(t)) were measured from rings cut from these segments. RESULTS: Morphometric variations were found along the small intestine with an increase in luminal area and a decrease in wall thickness from the duodenum to the ileum. The opening angle obtained after 60 min was highest in the duodenum (220.8+/-12.9 degrees) and decreased along the length of the intestine to 143.9+/-8.9 degrees in the jejunum and 151.4+/-9.4 degrees in the ileum. The change of opening angle as a function of time, fitted well to the Kelvin model using the equation theta(t)/theta(o) = [-eta exp (-lambda t)] after the ring was cut. The computed creep rate lambda did not differ between the segments. Compared to constant calculated from pig aorta and coronary artery, it showed that alpha agreed well (within 5%), eta was three times larger than that for vascular tissue, and lambda ranged +/-40% from the value of the pig coronary artery and was a third of the value of pig aorta. CONCLUSION: The change of opening angle over time for all the small intestine segments fits well to the standard linear spring-dashpot model. This viscoelastic constant of the rat small intestine is fairly homogenous along its length. The data obtained from this study add to a base set of biomechanical data on the small intestine and provide a reference state for comparison to other tissues, diseased intestinal tissue or intestinal tissue exposed to drugs or chemicals.

Morphology and stress-strain properties along the small intestine in the rat.

The stress-strain relationship is determined by the inherent mechanical properties of the intestinal wall, the geometric configurations, the loading conditions and the zero-stress state of the segment. The purpose of this project was to provide morphometric and biomechanical data for rat duodenum, jejunum and ileum. The circumferential strains were referenced to the zero-stress state. Large morphometric variations were found along the small intestine with an increase in the outer circumferential length and luminal area and a decrease in wall thickness in distal direction. The serosal residual strain was tensile and decreased in distal direction (P < 0.05). The mucosal residual strain was compressive and the absolute value decreased in distal direction (P < 0.001). The stress-strain experiments showed that the duodenum was stiffest. All segments were stiffest in longitudinal direction (P < 0.05). In conclusion, axial variation in morphometric and biomechanical properties was found in the small intestine. The zero-stress state must be considered in future biomechanical studies in the gastrointestinal tract.

Morphometric and biomechanical intestinal remodeling induced by fasting in rats.

The function of the small intestine is mechanical to a large degree. To understand the function it is necessary to know how the mechanical stresses and strains can be computed. Nutrition plays an important role in the maintenance of normal gut structure and function. The small intestine undergoes functional changes when food is withheld. To explore the morphological and biomechanical remodeling during starvation, intestinal segments from the fed and fasted rat duodenum, jejunum, and ileum were investigated. After seven days of fasting the animals lost 22% of the body weight and the intestinal mass per length decreased by nearly 40% in the duodenum. Fasting decreased the plasma levels of glucose, insulin, triglyceride, and cholesterol whereas the level of free fatty acids increased (P < 0.001). Fasting decreased the outer circumferential length, wall thickness, wall area, inner circumferential length, and luminal area at the three locations (P < 0.001). Histological examination showed that the mucosal and the submucosal thickness decreased during fasting (P < 0.001), whereas the muscle layers were unchanged. The residual strain on the mucosal surface was compressive. The serosal residual strain was tensile and increased with the highest values after four days of fasting in the duodenum and jejunum (P < 0.001). Fasting shifted the stress-strain curves to the right in both circumferential and longitudinal directions at the three locations (P < 0.04). In conclusion pronounced biomechanical and structural remodeling occurred in the small intestine during fasting for up to one week. Since the contractile properties depend on the passive properties (according to the well-known Hill's model), it can be predicted that the smooth muscle contractile function will also change.