Melatonin alleviates circadian rhythm disruption exacerbating DSS-induced colitis by inhibiting the distribution of HMGB1 in intestinal tissues.

Circadian rhythm disruption (CRD) is regarded as a risk factor for inflammatory bowel disease (IBD), and it was reported to suppress the level of melatonin, which execute anti-inflammatory effects. High mobility group box 1 protein (HMGB1) is a member of the damage-associated molecular pattern (DAMP) family and has been verified as an IBD-associated inflammatory cytokine that mediates the TLR4-NF-κB pathway. However, no exact mechanism has been illustrated among melatonin, disrupted circadian rhythm and inflammatory bowel disease, as well as regarding the effect of melatonin on HMGB1. In the present study, we aimed to explore the role of relationship with HMGB1. CRD aggravated DSS-induced colitis by worsening colonic inflammation and tissue injury, as well as by enhancing HMGB1 translocation, which could be reversed by ethyl pyruvate, an HMGB1 antagonist. Moreover, melatonin treatment attenuated these disorders and the shuttling of HMGB1 in the intestinal epithelial cells (IECs), the effect of which could be partly reversed by the melatonin antagonist luzindole. The protective role of melatonin may be tightly related to the translocation of HMGB1 in IECs. Accordingly, these results suggested that melatonin may be a new therapeutic beneficial option in IBD patients, especially for those with circadian rhythm disruption.

Topical and effective hemostatic medicines in the battlefield.

Uncontrolled hemorrhage has been considered as one of the most important factors for causing death on the battlefront. If given timely and efficient hemostatic medicines in pre-hospital setting, patients will obtain more time and chance to wait for medical treatment so as to save their lives. However, there is not a certain answer about which kind of hemostatic drugs can achieve efficacious effect to hemostasis in the battle. This review aims to summarize effective hemostatic medicines applied in battlefield from 41 articles. After analyzing and comparing the efficacy and complications of those products, we conclude that Fibrin Sealant Dressing, Celox and Woundstat are prior to other materials to stanch life-threatening extremity hemorrhage on the battlefield based on present research in the related area. Therefore, in the prevalence of some inevitable battlefield throughout the world, especially in the Middle Eastern countries, our findings suggest for the first time that the effective hemostatic device is not only a key point to link pre-hospital and hospital care but also an essential way to increase the survival rate of battlefront in the foreseeable future.

Workload influence on fatigue related psychological and physiological performance changes of aviators.

OBJECTIVE: We evaluated a variety of non-invasive physiological technologies and a series of test approaches for examination of aviator performances under conditions of mental workload in order to provide a standard real-time test for physiological and psychological pilot fatigue assessments. METHODS: Twenty-one male aviators were selected for a simulated flight in a hypobaric cabin with artificial altitude conditions of 2400 meter above sea level. The simulated flight lasted for 1.5 h, and was repeated for two times with an intervening 0.5 h rest period outside the hypobaric cabin. Subjective criteria (a fatigue assessment instrument [FAI]) and objective criteria (a standing-position balance test as well as a critical flicker fusion frequency (CFF) test) were used for fatigue evaluations. RESULTS: No significant change was observed in the FAI scores before and after the simulated flight, indicating that there was no subjective fatigue feeling among the participants. However, significant differences were observed in the standing-position balance and CFF tests among the subjects, suggesting that psychophysiological indexes can reflect mental changes caused by workload to a certain extent. The CFF test was the simplest and clearly indicated the occurrence of workload influences on pilot performances after a simulated flight. CONCLUSIONS: Results showed that the CFF test was the easiest way to detect workload caused mental changes after a simulated flight in a hypobaric cabin and reflected the psychophysiological state of aviators. We suggest that this test might be used as an effective routine method for evaluating the workload influences on mental conditions of aviators.

[Bioremediation of chromium (VI) contaminated site by reduction and microbial stabilization of chromium].

Chromium (VI) contaminated soil samples were collected from a chemical plant in Suzhou. Firstly, the reduced soil was prepared by adding reagent (Stone-sulfure reagent) into polluted soil to transfer most chromium (VI) into chromium (III), then a nutrient solution was introduced into the reduced soil, and the stabilized soil was obtained after 60 days culturing. The chromium (VI) content of the three kinds of soil was analyzed. The results showed that the chromium (VI) content in toxicity characteristic leaching liquid (TCLL) dropped by 96. 8% (from 8.26 mg · L(-1) to 0.26 mg · L(-1)), and the total chromium content dropped by 95.7% (from 14.66 mg · L(-1) to 0.63 mg · L(-1)) after bioremediation in 5% nutrient solution. Additionally, the durability of chromium stabilization was tested by potassium permanganate oxidation and sterilization of microbe-treated soil. After oxidation, the chromium (VI) content in TCLL of the reduced soil was increased from 8.26 mg · L(-1) to 14.68 mg · L(-1). However, the content after bioremediation was decreased to 2.68 mg · L(-1). The results of sterilization demonstrated that the death of microbe had no significant effect on the stabilization of chromium. Consequently, the research in this paper demonstrated the feasibility of bioremediation of chromium (VI) polluted soil through reduction followed by stabilization/soilidification, and provided a technique with low cost but high efficiency.

Effect of Hypotensive Resuscitation with a Novel Combination of Fluids in a Rabbit Model of Uncontrolled Hemorrhagic Shock.

OBJECTIVE: The aim of this study was to compare the effects of hypotensive and normotensive resuscitation with a novel combination of fluids via lactate Ringer's solution (LRS), 6% hydroxyethyl starch 130/0.4 solution (HES), and 7.5% hypertonic saline solution (HSS) at early stage of uncontrolled hemorrhagic shock (UHS) before hemostasis. METHODS: New Zealand white rabbits (n = 32) underwent UHS by transecting the splenic parenchyma, followed by blood withdrawal via the femoral artery to target mean arterial pressure (MAP) of 40-45 mmHg. Animals were distributed randomly into 4 groups (n = 8): in group Sham, sham operation was performed; in group HS, UHS was untreated; in group HS-HR, UHS was treated by hypotensive resuscitation with HSS and LRS+HES (ratio of 2∶1) to MAP of 50-55 mmHg; in group HS-NR, UHS was treated by normotensive resuscitation with HSS and LRS+HES (ratio of 2∶1) to MAP of 75-80 mmHg. Outcomes of hemodynamics, inflammatory and oxidative response, and other metabolic variables were measured and the histopathological studies of heart, lung and kidney were performed at the end of resusucitation. RESULTS: Hypotensive resuscitation with the novel combination of fluids for UHS rabbits decreased blood loss, maintained better stabilization of hemodynamics, and resulted in relatively higher hematocrit and platelet count, superior outcomes of blood gas, and lower plasma lactate concentration. Besides, hypotensive resuscitation attenuated the inflammatory and oxidative response significantly in UHS rabbits. CONCLUSION: Hypotensive resuscitation with the novel combination of fluids via HSS and LRS+HES (ratio of 2∶1) has an effective treatment at early stage of UHS before hemostasis.

Structural importance of the acyl group in substrate specificity of purified bovine lysophospholipase D.

The structural importance of the acyl group in lysophosphatidylcholine (LPC) as substrate of purified bovine lysophospholipase D (lysoPLD) was investigated. Among LPCs with saturated acyl chains, the K(m) value decreased according to the length of the acyl chain (C12-C16) up to the palmitoyl group, while the V(m) value showed no remarkable change. But, the extension of the acyl size to C18, as observed with 1-stearoyl LPC (K(m), 8.5 mM), rather resulted in a remarkable increase in the K(m) value. Meanwhile, the introduction of one double bond in the C18 saturated acyl chain led to a remarkable reduction in the K(m) value, as observed with 1-oleoyl LPC (K(m), 0.48 mM). Furthermore, 1-linoleoyl LPC (K(m), 56 microM) with two double bonds exhibited a smaller K(m) value than 1-oleoyl LPC, suggesting that the unsaturation degree might be important in augmenting the binding affinity of LPCs. A similar phenomenon was also observed with 1-arachidonoly LPC (K(m), 79 microM) or 1-docosahexaenoyl LPC (K(m), 36 microM). Overall, the order of catalytic efficiency (V(m)/K(m) value) of those LPCs seemed to be affected by the K(m) value rather than the V(m) value, which differed by at most threefold among LPC derivatives. Next, the introduction of a hydroperoxide group into 1-linoleoyl-LPC or 1-arachidonoyl LPC led to a further reduction in K(m) values (1-hydroperoxylinoleoyl LPC, 26 microM; 1-hydroperoxyarachidonoyl LPC, 33 microM), accompanied by a further increase in the V(m)/K(m) values. Additionally, phosphatidylcholines (PCs) with an oxidized acyl chain at sn-2 position were found to be efficient as 1-palmitoyl LPC as substrates of lysoPLD. Taken together, the catalytic efficiency of LPCs or oxidized PCs as substrates of lysoPLD seems to be determined by the property of the acyl chain, length of the acyl chain, unsaturation degree and oxidation status.

Inhibition of lysophospholipase D activity by unsaturated lysophosphatidic acids or seed extracts containing 1-linoleoyl and 1-oleoyl lysophosphatidic acid.

Lysophospholipase D (lysoPLD), generating lipid mediator lysophosphatidic acid (LPA) from lysophosphatidyclcholine (LPC), is known to be inhibited by lysophosphatidic acids. Meanwhile, some plant lipids are known to contain lysophospholipids as minor components. Therefore, it is interesting to test whether edible seed samples, rich in phospholipids, may contain lysophospholipids, which express a strong inhibition of lysoPLD activity. First, the structural importance of fatty acyl group in LPAs was examined by determining the inhibitory effect of various LPAs on bovine lysoPLD activity. The most potent in the inhibition of lysoPLD activity was linoleoyl-LPA ( K i, 0.21 microM), followed by arachidonoyl-LPA ( K i, 0.55 microM), oleoyl-LPA ( K i, 1.2 microM), and palmitoyl-LPA ( K i, 1.4 microM), based on the fluoresecent assay. The same order of inhibitory potency among LPA analogs with different acyl chains was also found in the spectrophotometric assay. Subsequently, the extracts of 12 edible seeds were screened for the inhibition of lysoPLD activity using both spectrophotometric and fluorescent assays. Among seed extracts tested, the extract from soybean seed, sesame seed, or sunflower seed (30 mg seed weight/mL) was found to exhibit a potent inhibition (>80%) of lysoPLD activity. In further study employing ESI-MS/MS analysis, major LPA components in seed extracts were identified to be 1-linoleoyl LPA, 1-oleoyl LPA, and 1-palmitoyl LPA with 1-linoleoyl LPA being more predominant. Thus, the potent inhibition of lysoPLD activity by seed extracts might be ascribed to the presence of LPA with linoleoyl group rather than other acyl chains.

Reductive depolymerization of bovine thyroglobulin multimers via enzymatic reduction of protein disulfide and glutathionylated mixed disulfide linkages.

The nascent thyroglobulin (Tg) multimer molecule, which is generated during the initial fate of Tg in ER, undergoes the rapid reductive depolymerization. In an attempt to determine the depolymerization process, various types of Tg multimers, which were generated from deoxycholate-treated/reduced Tg, partially unfolded Tg or partially unfolded/reduced Tg, were subjected to various GSH (reduced glutathione) reducing systems using protein disulfide isomerase (PDI), glutathione reductase (GR), glutaredoxin or thioredoxin reductase. The Tg multimers generated from deoxycholate-treated/reduced Tg were depolymerized readily by the PDI/GSH system, which is consistent with the reductase activity of PDI. The PDI/GSH-induced depolymerization of the Tg multimers, which were generated from either partially unfolded Tg or partially unfolded/reduced Tg, required the simultaneous inclusion of glutathione reductase, which is capable of reducing glutathionylated mixed disulfide (PSSG). This suggests that PSSG was generated during the Tg multimerization stage or its depolymerization stage. In particular, the thioredoxin/thioredoxin reductase system or glutaredoxin system was also effective in depolymerizing the Tg multimers generated from the unfolded Tg. Overall, under the net GSH condition, the depolymerization of Tg multimers might be mediated by PDI, which is assisted by other reductive enzymes, and the mechanism for depolymerizing the Tg multimers differs according to the type of Tg multimer containing different degrees and types of disulfide linkages.

Inactivation of protein disulfide isomerase by alkylators including alpha,beta-unsaturated aldehydes at low physiological pHs.

Protein disulfide isomerase (PDI) is known to contain the thioredoxin box motif with a low pKa cysteine residue. To investigate the reactivity of PDI with thiol modifiers at low physiological pHs, either the reduced (PDIred) or oxidized form (PDIoxid) of PDI was exposed to various alkylating ragents. When PDI was incubated with iodoacetamide at pH 6.3 for 30 min at 38 degrees C, a remarkable inactivation (>90%) of PDIred was caused by iodoacetamide (IC50=8 microM). However, PDIoxid was only slightly inactivated (approximately 18%) by iodoacetamide. Similarly, PDIred was significantly inactivated by N-ethylmaleimide (NEM), but PDIoxid was not. When the inactivation by these alkylators was analyzed by pseudo-first order kinetics, NEM (k3=1.75x10(-2) s(-1); K(i)=124 microM) was observed to be more potent than iodoacetamide (k3=9.1x10(-3) s(-1); K(i)=311 microM). Interestingly, the inactivation of PDIred by iodoacetamide was greater at pH 6.3 than pH 7.0, in contrast to a similar inactivation potency of NEM at both pHs. Moreover, the maximal inactivation of PDIred or PDIoxid by iodoacetamide was mainly observed around pH 6.0. In addition, PDIred was found to be inactivated by acrolein (IC50=10 microM) at pH 6.3, and this inactivation was also greater at pH 6.3 than at pH 7. Based on these results, we suggest that PDIred is susceptible to inactivation by alkylators including endogenous alpha,beta-unsaturated aldehydes at low physiological pHs.

Multimerization of bovine thyroglobulin, partially unfolded or partially unfolded/reduced; involvement of protein disulfide isomerase and glutathionylated disulfide linkage.

Fate of the nascent thyrolglobulin (Tg) molecule is characterized by multimerization. To establish the formation of Tg multimers, the partially unfolded/reduced Tg or deoxycholate-treated/ reduced Tg was subjected to protein disulfide isomerase (PDI)-mediated multimerization. Oxidized glutathione/PDI-mediated formation of multimeric Tg forms, requiring at least an equivalent molar ratio of PDI/Tg monomer, decreased with increasing concentration of reduced glutathione (GSH), suggesting the oxidizing role of PDI. Additional support was obtained when PDI alone, at a PDI/Tg molar ratio of 0.3, expressed a rapid multimerization. Independently, the exposure of partially unfolded Tg to GSH resulted in Tg multimerization, enhanced by PDI, according to thiol-disulfide exchange. Though to a lower extent, a similar result was observed with the dimerization of deoxycholate-pretreated Tg monomer. Consequently, it is implied that intermolecular disulfide linkage may be facilitated at a limited region of unfolded Tg. In an attempt to examine the multimerization site, the cysteine residue-rich fragments of the Tg were subjected to GSH-induced multimerization; a 50 kDa fragment, containing three vicinal dithiols, was multimerized, while an N-terminal domain was not. Present results suggest that the oxidase as well as isomerase function of PDI may be involved in the multimerization of partially unfolded Tg or deoxycholate-treated Tg.

Protective action of CLA against oxidative inactivation of paraoxonase 1, an antioxidant enzyme.

The effect of CLA on paraoxonase 1 (PON1), one of the antioxidant proteins associated with HDL, was investigated for its protective action against oxidative inactivation as well as its stabilization activity. When cis-9 (c9),trans-11 (t11)-CLA and t10,c12-CLA were examined for their protective activity against ascorbate/Cu(2+)-induced inactivation of PON1 in the presence of Ca2+, two CLA isomers exhibited a remarkable protection (Emax, 71-74%) in a concentration-dependent manner (50% effective concentration, 3-4 microM), characterized by a saturation pattern. Such a protective action was also reproduced with oleic acid, but not linoleic acid. Rather, linoleic acid antagonized the protective action of CLA isomers in a noncompetitive fashion. Additionally, the two CLA isomers also protected PON1 from oxidative inactivation by H2O2 or cumene hydroperoxide. The concentration-dependent protective action of CLA against various oxidative inactivation systems suggests that the protective action of CLA isomers may be mediated through their selective binding to a specific binding site in a PON1 molecule. Separately, the inactivation of PON1 by p-hydroxymercuribenzoate (PHMB), a modifier of the cysteine residue, was also prevented by CLA isomers, suggesting the possible existence of the cysteine residue in the binding site of CLA. The c9,t11-CLA isomer seems to be somewhat more effective than t10,c12-CLA in protecting against the inactivation of PON1 by either peroxides or PHMB, in contrast to the similar efficacy of these two CLA isomers in preventing ascorbate/Cu(2+)-induced inactivation of PON1. Separately, CLA isomers successfully stabilized PON1, but not linoleic acid. These data suggest that the two CLA isomers may play a beneficial role in protecting PON1 from oxidative inactivation as well as in its stabilization.

Identification of alkylation-sensitive target chaperone proteins and their reactivity with natural products containing Michael acceptor.

Molecular chaperones have a crucial role in the folding of nascent polypeptides in endoplasmic reticulum. Some of them are known to be sensitive to the modification by electrophilic metabolites of organic pro-toxicants. In order to identify chaperone proteins sensitive to alkyators, ER extract was subjected to alkylation by 4-acetamido-4'-maleimidyl-stilbene-2,2'-disulfonate (AMS), and subsequent SDS-PAGE analyses. Protein spots, with molecular mass of 160, 100, 57 and 36 kDa, were found to be sensitive to AMS alkylation, and one abundant chaperon protein was identified to be protein disulfide isomerase (PDI) in comparison with the purified PDI. To see the reactivity of PDI with cysteine alkylators, the reduced form (PDIred) of PDI was incubated with various alkylators containing Michael acceptor structure for 30 min at 38 degrees C at pH 6.3, and the remaining activity was determined by the insulin reduction assay. Iodoacetamide or N-ethylmaleimide at 0.1 mM remarkably inactivated PDIred with N-ethylmaleimide being more potent than iodoacetamide. A partial inactivation of PDIoxid was expressed by iodoacetamide, but not N-ethylmaleimide (NEM) at pH 6.3. Of Michael acceptor compounds tested, 1,4-benzoquinone (IC50, 15 microM) was the most potent, followed by 4-hydroxy-2-nonenal and 1,4-naphthoquinone. In contrast, 1,2-naphthoquinone, devoid of a remarkable inactivation action, was effective to cause the oxidative conversion of PDIred to PDIoxid. Thus, the action of Michael acceptor compounds differed greatly depending on their structure. Based on these, it is proposed that PDI, one of chaperone proteins in ER, could be susceptible to endogenous or xenobiotic Michael acceptor compounds in vivo system.

Role of protein disulfide isomerase in molecular fate of thyroglobulin and its regulation by endogenous oxidants and reductants.

The molecular fate of thyroglobulin (Tg) is controlled by oligomerization, a means of storing Tg at high concentrations, and deoligomerization. The oligomerization of bovine Tg are intermolecular reactions that occur through oxidative processes, such as disulfide and dityrosine formation, as well as isopeptide formation; disulfide formation is primarily responsible for Tg oligomerization. Here, the protein disulfide isomerase (PDI) and/or peroxidase-induced oligomerization of unfolded thyroglobulins, which were prepared by treating bovine Tg with heat, urea or thiol/urea, was investigated using SDS-PAGE analyses. In addition, the enzymatic oligomerization was compared with non-enzymatic oligomerization. The thermally-induced oilgomerization of Tg, dependent on glutathione redox state, was affected by the ionic strength or the presence of a surfactant. Meanwhile, PDI-catalyzed oligomerization, time and pH-dependent, was the most remarkable with unfolded/reduced Tg, which was prepared from a treatment with urea/DTT, while the thermally-unfolded Tg was less sensitive. Similarly, the oligomerization of unfolded/reduced Tg was also mediated by peroxidase. However, PDI showed no remarkable effect on the peroxidase-mediated oligomerization of either the unfolded or unfolded/reduced Tg. Additionally, the reductive deoligomerization of oligomeric Tg was exerted by PDI in an excessively reducing state. Based on these results, it is proposed that PDI catalyzes the oligomerization of Tg through the disulfide linkage and its deoligomerization in the molecular fate, and this process may require a specific molecular form of Tg, optimally unfolded/reduced, in a proper redox state.