[Endothelial protective effect of simvastatin on coagulation system in septic rats].

Objective: To investigate the endothelial protective effects of simvastatin on the coagulation system in septic rats. Methods: A total of 54 SD male rats were divided into 3 groups. Six healthy rats were intraperitoneally injected with normal salineas control group. Twenty-four rats in septic group were intraperitoneally injected with normal saline followed by lipopolysaccharide 2.5 mg. Study group had 24 rats intraperitoneally injected with simvastatin followed by lipopolysaccharide. Plasma von Willebrand factor (vWF), thrombomodulin (TM), platelet activating factor (PAF) and antithrombin-Ⅲ (AT-Ⅲ) were tested at 1 h, 3 h, 6 h and 12 h after treatment. Scanning electron microscopy and transmission electron microscopy were used to observe the morphology and apoptosis of rat aorta endothelial cells. Results: Compared with healthy control group, vWF [(68.3±4.8) ng/ml, (59.2±5.1) ng/ml, (74.2±20.1) ng/ml, (53.5±4.0)ng/ml, respectively], TM [(1.4±0.3) ng/ml, (1.6±0.4) ng/ml, (2.8±0.9) ng/ml, (1.4±0.5) ng/ml, respectively], PAF [(29.1±6.5) pg/ml, (28.6±1.5) pg/ml, (28.7±2.7) pg/ml, (18.2±4.1) pg/ml, respectively] and AT-Ⅲ [(262.2±38.1)µg/ml, (233.0±70.4) µg/ml, (218.7±54.7) µg/ml, (162.2±37.2) µg/ml, respectively] were significantly increased in the sepsis group at 1 h, 3 h, 6 h and 12 h (P<0.05). Compared with the sepsis group, the plasma levels of PAF in simvastatin intervention group at 1 h [(15.6±2.5) pg/ml, 3 h(10.4±5.3) pg/ml, 6 h (9.3±1.4) pg/ml, 12 h(11.0±2.7) pg/ml] were significantly decreased, so were the TM level at 6 h (1.6±0.9) ng/ml, and the AT-Ⅲ levels at 1 h[(190.3±29.2) µg/ml],6 h [(104.4±33.6) µg/ml] and 12 h [(73.6±39.0) µg/ml, P<0.05]. Conclusion: In the condition of sepsis, toxins and over-activated inflammatory factors damage the vascular endothelium. A large amount of circulating vWF, TM, PAF, and AT-Ⅲ cause early hypercoagulability. Simvastatin significantly reduces plasma amount of these procoagulants, suggesting it smodification of coagulopathy and vascular protective effectsin a septic rat model.

Effect of gene transfection timing on TGF-ß1 expression in rabbit mandibular distraction gap.

Transforming growth factor-ß1 (TGF-ß1) is a member of the TGF-ß superfamily, and plays an important role in promoting various stages of intramembranous and endochondral bone formation. It is one of the major growth factors that influence new bone formation in the distraction gap during distraction osteogenesis (DO). The major problem of DO is the time required for the treatment. Reports show that gene therapy accelerates osteogenesis, which can significantly benefit patients with DO. However, the optimal timing of gene transfection has not yet been reported. In this study, we used the New Zealand rabbit mandibular DO model for transfecting recombinant plasmid pIRES-hVEGF165-hBMP2 during the latency, distraction, and consolidation periods of DO. The TGF-ß1 levels in the distraction gap were detected at different time-points by immunohistochemistry and analyzed semi-quantitatively with the CMIAS-2001A computerized image analyzer. The TGF-ß1 levels peaked after 7 days and decreased after 14 days of consolidation in each group. In contrast, the TGF-ß1 levels in the transfected distraction period group were significantly higher than those in the other groups. After 28 days of consolidation, TGF-ß1 levels decreased and there was no significant difference among the groups. These results indicated that the genes transfected in the distraction period up-regulated the expression of TGF-ß1 more than in the latency and consolidation periods, which promoted bone formation in the distraction gap through a series of biological effects. Thus, we obtained a remarkable effect on new bone formation, and showed that the distraction period is optimal for gene therapy.

Dexmedetomidine vs remifentanil intravenous anaesthesia and spontaneous ventilation for airway foreign body removal in children.

BACKGROUND: To compare the safety and efficacy of dexmedetomidine/propofol (DP)-total i.v. anaesthesia (TIVA) vs remifentanil/propofol (RP)-TIVA, both with spontaneous breathing, during airway foreign body (FB) removal in children. METHODS: Seventy-seven children undergoing rigid bronchoscopy for FB removal were randomly allocated to receive either RP-TIVA and spontaneous ventilation (Group RP, n=38) or DP-TIVA and spontaneous ventilation (Group DP, n=39). Heart rate, arterial pressure, pulse oxygen saturation (Sp(O2)), respiratory rate, end-tidal CO2 (E'(CO2)), and induction time were recorded. Adverse events, the intervention for these events, and postoperative care duration were also assessed. RESULTS: The mean induction times were comparable between the two groups (Group RP 12.2 min vs Group DP 13.1 min, P>0.05). At the end of the procedure, the mean (E'(CO2)) was higher in Group RP (Group RP 6.8 kPa vs Group DP 5.8 kPa, P<0.001), and respiratory rate was lower in Group RP (Group RP 20.4 vs Group DP 35.8, P<0.001). Additionally, the perioperative haemodynamic profile was more stable in Group DP than that in Group RP. The incidence rate of breath-holding and intervention were comparable between the two groups. In the post-anaesthesia care unit (PACU), no hypoxaemia was observed, and emergence time increased in Group DP (Group DP 65.1 min vs Group RP 23.8 min, P<0.0001). The incidence of cough in PACU was higher in Group RP (Group RP 55.3% vs Group DP 10.3%, P<0.0001). CONCLUSIONS: Compared with RP-TIVA, DP-TIVA provided more stable respiratory and haemodynamic profiles, but required a longer recovery time. Clinical trial registration China Clinical Research Information Service, ChiCTR-TRC-13003018.

Description of durancin TW-49M, a novel enterocin B-homologous bacteriocin in carrot-isolated Enterococcus durans QU 49.

AIMS: To characterize the novel bacteriocin produced by Enterococcus durans. METHODS AND RESULTS: Enterococcus durans QU 49 was isolated from carrot and expressed bactericidal activity over 20-43 degrees C. Bacteriocins were purified to homogeneity using the three-step purification method, one of which, termed durancin TW-49M, was an enterocin B-homologous peptide with most identical residues occurring in the N-terminus. Durancin TW-49M was more tolerant in acidic than in alkali. DNA sequencing analysis revealed durancin TW-49M was translated as a prepeptide of the double-glycine type. Durancin TW-49M and enterocin B expressed similar antimicrobial spectra, in which no significant variation due to the diversity in their C-termini was observed. CONCLUSIONS: Durancin TW-49M, a novel nonpediocin-like class II bacteriocin, was characterized to the amino acid and genetic levels. The diverse C-terminal parts of durancin TW-49M and enterocin B were hardly to be suggested as the place determining the target cell specificity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first and comprehensive study of a novel bacteriocin produced by Ent. durans. The high homology at the N-terminal halves between durancin TW-49M and enterocin B makes them suitable to study the structure-function relationship of bacteriocins and their immunity proteins.

Toxic Effects in Siganus oramin by dietary exposure to 4-tert-octylphenol.

The integrated toxicities of 4-tert-octylphenol (t-OP) on Siganus oramin were investigated by dietary administration at doses of 5, 25 and 125 mg/kg body weight over 28 days. Significant increase was observed in the activity of hepatic glutathione S-transferase at 125 mg/kg on both day 14 and 28 in males, and at all doses on day 28 in females, and in hepatosomatic index at 25 mg/kg on day 14 in both sexes. Plasma levels of testosterone and cortisol decreased significantly at all doses on day 28. Histopathologic changes in liver, spleen, intestine and testis deteriorated with increasing doses and duration. The results suggest that S. oramin is sensitive to t-OP, and the above endpoints may be potential biomarkers for evaluating toxicities of environmental pollutants such as t-OP.

The intrinsic factor (IF)-cobalamin receptor binding site is located in the amino-terminal portion of IF.

Intrinsic factor has two binding sites, one each for cobalamin and for the ileal receptor recognizing the intrinsic factor-cobalamin complex. To obtain initial functional mapping of these domains, cDNAs encoding intact rat and human intrinsic factor or fragments thereof were expressed transiently in COS-1 cells or in an in vitro transcription/translation system. Deletion of as little as 12% of the amino acids from the carboxyl terminus resulted in loss of cobalamin binding activity. On the other hand, the receptor binding region of intrinsic factor appears localized to a restricted region in the amino-terminal portion of the protein. Only those transcription/translation fragments of rat or human intrinsic factor tested that contained amino acid residues 25 to 62 (out of 399) showed calcium-dependent binding to isolated kidney brush borders, the shortest sequence corresponding with 20 consecutive amino acids. In contrast, a 232-amino acid carboxyl-terminal fragment of rat intrinsic factor and 243- and 338-amino acid carboxyl-terminal fragments of human intrinsic factor showed no receptor binding activity.

The minor components of the rat asialoglycoprotein receptor are apically located in neonatal enterocytes.

Because specific uptake of the asialoglycoprotein haptocorrin has been reported in suckling distal intestine, evidence of the asialoglycoprotein receptor in rat ileum was sought. On Western blot, two different polyclonal antisera against purified rat holoreceptor recognized only proteins of the size of the minor receptor components (51 and 55 kilodaltons) in both suckling and adult rat intestine. Messenger RNA encoding the minor component (RHL-2/3) was detected in total RNA extracted from rat ileum. Calcium-specific binding of porcine or rat haptocorrin-[57Co]cobalamin complexes was detected in the brush border membranes of distal suckling rat and porcine small intestine. This binding was almost completely blocked by another asialoglycoprotein, asialofetuin. The pH optimum for binding was 6-9, with a Ka of 0.25 nmol/L and a capacity of 4.6 fmol/mg protein. These properties, with the exception of the low capacity, are all consistent with those of the intact receptor on hepatocytes. The intestinal receptor was localized not to the basolateral membrane, as in the liver, but to the apical brush border, as suggested by the binding data. Furthermore, immunoperoxidase stains of paraffin-embedded tissue detected strong binding to the brush border and apical phagolysosome of mid and distal suckling rat intestine. These data show that, contrary to expectations, the minor components of the asialoglycoprotein receptor are functional and expressed in the apical membrane of the suckling intestine. The abundance of the protein by Western blot suggests possible roles for it in the neonatal gut other than haptocorrin binding.

Estimation of surface-bound heparin activity: a comparison of methods.

We compared two assays for estimating the amount of active heparin bound to a catheter surface: 1) a kinetic assay based on the inactivation of thrombin by antithrombin III, and 2) thrombin uptake. Both assays were used to estimate the amount of heparin activity on a series of catheters coated with no heparin, covalently bound heparin, and ionically bound heparin. The kinetic assay produced estimates of surface-bound heparin activity and showed that some binding methods resulted in destruction of most of the heparin's biologic activity. In contrast, the thrombin uptake assay did not correlate with the amount of heparin activity on the catheter surface. Substantial thrombin uptake was found on surfaces coated with no heparin or inactive heparin, while low thrombin uptake was found on surfaces with high levels of heparin activity in the kinetic assay. We conclude that: 1) a kinetic assay based on the heparin accelerated inactivation of thrombin by antithrombin III can be used to estimate the amount of active heparin bound to a catheter surface, and 2) thrombin uptake studies do not correlate with heparin activity and do not predict which heparin binding method will result in the highest concentration of active heparin on the catheter surface.

The mode of anchoring and precursor forms of sucrase-isomaltase and maltase-glucoamylase in chicken intestinal brush-border membrane. Phylogenetic implications.

Chicken intestinal sucrase-isomaltase and maltase-glucoamylase have been isolated in their intact form by detergent solubilization and characterized as to their subunit composition and mode of anchoring in the brush-border membrane. Both are heterodimeric enzyme complexes composed of two subunits each of approximately 140 and 130 kDa. Contrary to the mammalian sucrase-isomaltase, chicken isomaltase was identified as the smaller of the two subunits. As was shown by hydrophobic labeling, only one of the two subunits in each heterodimer is anchored in the bilayer, the smaller 130 kDa isomaltase subunit of the sucrase-isomaltase complex, and the larger 140 kDa subunit of the maltase-glucoamylase complex. Both preparations contain a high-molecular weight polypeptide of approximately 250 kDa which in the case of sucrase-isomaltase could be identified by peptide mapping as a single-chain precursor not (yet) proteolytically processed to the final heterodimer. These first data on the mode of membrane anchoring of non-mammalian glycosidases indicate that they are synthesized, inserted into the membrane, and processed in ways similar to the mammalian enzymes. The fundamental unity between avian and mammalian sucrase-isomaltases suggests that the partial gene duplication of an ancestral isomaltase gene and the subsequent mutation of one of the active sites resulting in pro-sucrase-isomaltase has occurred prior to the separation of mammals from reptiles, i.e. more than 300 million years ago.

ESCA studies of surface chemical composition of segmented polyurethanes.

Surface chemical analysis of two commercially available polyurethanes, i.e., Avcothane and Biomer was carried out by electron spectroscopy for chemical analysis (ESCA). The depth which is subject to analysis is in the range of 50-100 A. The variables studied in this study are the difference in exposure to air or to the mold substrate during the solvent casting process. Model compounds such as a pure polydimethylsiloxane, polyether soft segment and hard segment copolymer were used to identify and assign various ESCA peaks. The air facing surface of Avcothane which is the blood contacting surface is found to be covered mostly with polydimethylsiloxane polymer, with a small amount of polyether soft segment mixed with silicone. Therefore, the hard segment of the polyurethanes is hidden beneath the blood contact surface in Avcothane. In Biomer films, the air facing surface contains a greater concentration of polyether soft segment than the substrate surface. These results are consistent with our previous results obtained by Fourier transform IR internal reflection and Auger electron spectroscopy.

Application of Auger electron spectroscopy for surface chemical analysis of Avcothane.

The effect of the exposure to air or to the substrate during the solvent casting process on the surface chemical composition of Avcothane, a blood compatible biomaterial, was studied by employing Auger electron spectroscopy. The surface layer of 10-15A thickness was analyzed without sputtering, but for the studies probing a deeper layer, argon ion sputtering at a low enough voltage to prevent artifacts was utilized. It is found that the air facing surface which is the blood contact surface contains a greater amount of silicone polymer and a much lower amount of the urethane hard segment in the first 10-15A-deep layer than in the comparably thick layer of the substrate surface. However, the depth-composition profile obtained by sputtering indicate that, probably at a deeper depth, the chemical compositions in terms of silicone polymer and hard segment is comparable both in the air side and the substrate side.

Surface chemical analysis of Avcothane and Biomer by Fourier transform IR internal reflection spectroscopy.

During the solvent casting process, one side of the polymer film is exposed to air while the other side is in contact with a substrate, used as a mold. We have studied the effect of this difference in exposure during casting on the chemical composition of two types of segmented polyurethane, Biomer and Avcothane, by using Fourier transform IR internal reflection spectroscopy. Also, a depth-composition profile was obtained by placing a thin barrier film between the reflection plate and the polymer film. In Avcothane, the air side, which is the blood-contact side, contains a greater amount of the soft segment than the substrate side, and this is more pronounced in the layer closer to the surface. The anisotropy in composition is more drastic when the silicone content is compared. In a layer about 1.5 mu thick, one can detect a greater amount of silicone in the substrate side than in the air side. However, when one averages the concentration in a layer of about 0.8 microns the trend in reversed; i.e., the greater amount of silicone is now present in the air side than in the substrate side. In Biomer films, the anisotropy in chemical composition is less pronounced. Only a modest increase in the relative content of the soft segment/hard segment is observed in the air side when a depth-composition profile is obtained.