Antihypertensive activity of chitin derivatives

To develop angiotensin I converting enzyme (ACE) inhibitory chitin derivatives based on the properties of ACE inhibitors, chitins with different degree of deacetylation were chemically modified by grafting 2-chloroethylamino hydrochloride onto chitin at the C-6 position. Three kinds of chitin derivatives were prepared and designated as aminoethyl-chitin (AEC) with 10% degree of deacetylation, aminoethyl-chitin with 50% degree of deacetylation (AEC50), and aminoethyl-chitin with 90% degree of deacetylation (AEC90). IC50 values of three chitin derivatives on ACE were 0.064 microM (AEC), 0.038 microM (AEC50), and 0.103 microM (AEC90). The results of Dixon plots revealed that AEC50 was a competitive inhibitor, and the inhibition constant (Ki) value was 0.021 microM. In addition, the antihypertensive effect of AEC50 on spontaneously hypertensive rats (SHR) was evaluated, and the result showed that it effectively decreased systolic blood pressure (SBP) in a dose-dependent manner.     

壳寡糖对大肠杆菌抑菌活性研究

分析壳寡糖对大肠杆菌抑菌效果的影响因素.采用摇瓶法和ELISA板法对不同浓度的壳寡糖进行抑菌试验;比较不同pH、不同脱乙酰度的壳寡糖对大肠杆菌抑菌效果的差异;比较不同聚合度的单一聚合度壳寡糖抑菌效果的差异.壳寡糖浓度大于5 mg/mL时抑菌效果与同浓度苯甲酸钠相近;pH为4时,0.156 mg/mL的壳寡糖溶液抑菌活性即能超过90%;pH为7时,5 mg/mL的壳寡糖才能达到90%抑菌活性.脱乙酰度为95%时,5 mg/mL的壳寡糖溶液抑菌活性能超过97%;脱乙酰度为45%时,40 mg/mL的壳寡糖溶液抑菌活性仅有56%;聚合度大于4的单一聚合度壳寡糖40 mg/mL时抑菌活性能达到99%.结果表明:提高壳寡糖溶液浓度、降低pH、提高脱乙酰度,能提高壳寡糖的抑菌活性,单一聚合度壳寡糖聚合度越高,对大肠杆菌的抑制作用越强.此外,采用ELISA板的方法进行实验,即节省试药又方便快捷.     

Chitosan oligosaccharides suppress production of nitric oxide in lipopolysaccharide-induced N9 murine microglial cells in vitro

Chitosan oligosaccharides (COS) have been reported to exert many biological activities, such as antioxidant, antitumor and anti-inflammatory effects. In the present study, we examined the effect of COS on nitric oxide (NO) production in LPS induced N9 microglial cells. Pretreatment with COS (50?~?200?μg/ml) could markedly inhibit NO production by suppressing inducible nitric oxide synthase (iNOS) expression in activated microglial cells. Signal transduction studies showed that COS remarkably inhibited LPS-induced phosphorylation of p38 MAPK and ERK1/2. COS pretreatment could also inhibit the activation of both nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). In conclusion, our results suggest that COS could suppress the production of NO in LPS-induced N9 microglial cells, mediated by p38 MAPK and ERK1/2 pathways.     

Heat-induced increases in endothelial NO synthase expression and activity and endothelial NO release

Endothelial nitric oxide (NO) synthase (eNOS) is regulated by heat shock protein 90 (HSP90), a heat-inducible protein; however, the effect of heat shock on eNOS expression and eNO release is unknown. Bovine aortic endothelial cells were incubated for 1 h at 37 degrees C, 42 degrees C, or 45 degrees C and cell lysates were evaluated with the use of Western blotting. We observed a 2.1 +/- 0.1-fold increase in eNOS protein content, but no change in HSP90 content, HSP70 content, or HSP90/eNOS association, 24 h after heat shock at 42 degrees C. We also observed a 7.7 +/- 1.5-fold increase in HSP70 protein content, but did not observe a change in eNOS or HSP90 24 h after heat shock at 45 degrees C. eNOS activity and maximal bradykinin-stimulated NO release was significantly increased 24 h after heat shock at 42 degrees C. Heat shock in rats (core temperature: 42 degrees C, 15 min) resulted in a significant increase in aortic eNOS, HSP90, and HSP70 protein content. The aorta from heat-shocked rats exhibited a decreased maximal contractile response to phenylephrine, which was abolished by preincubation with NG-nitro-l-arginine. We conclude that prior heat shock is a physical stimulus of increased eNOS expression and is associated with an increase in eNOS activity, agonist-stimulated NO release, and a decreased vasoconstrictor response.     

Reactive oxygen species scavenging activity of aminoderivatized chitosan with different degree of deacetylation

Chitosans with different degree of deacetylation were prepared from crab shell chitin in the presence of alkali. Aminoderivatized chitosan derivatives were prepared in addition of amino functional groups at a hydroxyl site in the chitosan backbone. Six kinds of aminoderivatized chitosan such as aminoethyl-chitosan (AEC90), dimethylaminoethyl-chitosan (DMAEC90), and diethylaminoethyl-chitosan (DEAEC90), which were prepared from 90% deacetylated chitosan, and AEC50, DMAEC50 and DEAEC50, which were prepared from 50% deacetylated chitosan, were prepared and their reactive oxygen species (ROS) scavenging activities were investigated against hydroxyl radical, superoxide anion radical and hydrogen peroxide. The electron spin resonance (ESR) spectrum revealed that AEC90 showed the highest scavenging effects against hydroxyl and superoxide anion radical, the effects were 91.67% and 65.34% at 0.25 and 5 mg/mL, respectively. For hydrogen peroxide scavenging effect, DEAEC90 exhibited the strongest activity. These results suggest that the scavenging effect depends on their degree of deacetylation and substituted group.     

Inhibition of proinflammatory cytokine-induced invasiveness of HT-29 cells by chitosan oligosaccharide

The effect of chitosan oligosaccharide (COS, 1 kDa 相似文献   

Chitin/chitosan transformation by thermo-mechano-chemical treatment including characterization by enzymatic depolymerization

Chitosan, the deacetylated derivative of chitin, was until recently produced by hydrolysis in 50% (w/v) NaOH. Application of thermo-mechano-chemical technology to chitin deacetylation was evaluated as an alternative method of chitosan production. This process consists of a cascade reactor unit operating under reduced alkaline conditions of 10% (w/v) NaOH. Prior mercerization of chitin at 4 degrees C for 24 h was required for high deacetylation yields. Sudden decompression of the aqueous alkaline suspension of mercerized chitin resulted in near complete deacetylation of chitin. Reactor residence time was 90 s at 230 degrees C prior to decompression. The chitosan produced was characterized by elemental analysis, (13)C-NMR and enzymatic depolymerization. Enzymatic determination of the degree of acetylation of chitin/chitosan mixtures was also investigated. Relative chitinase and/or chitosanase digestibilities were shown to be strongly dependent on chitin deacetylation. Based on enzymatic digestibilities, the alkaline aqueous high shear process does not appear to produce significant secondary products. Correlation of chitosanase digestibility with percentage of deacetylation provides a simple biological assay to study chitosan composition.     

Thermal inactivation of foot-and-mouth disease viruses in suspension

The heat resistance of foot-and-mouth disease virus (FMDV) strains isolated from outbreaks in Thailand was investigated in phosphate-buffered saline (PBS) at 50, 60, 70, 80, 90, and 100 degrees C. The first-order kinetic model fitted most of the observed linear inactivation curves. The ranges of decimal-reduction time (D value) of FMDV strains at 50, 60, 70, 80, 90, and 100 degrees C were 732 to 1,275 s, 16.37 to 42.00 s, 6.06 to 10.87 s, 2.84 to 5.99 s, 1.65 to 3.18 s, and 1.90 to 2.94 s, respectively. The heat resistances of FMDV strains at lower temperature (50 degrees C) were not serotype specific. The effective inactivating temperature is approximately 60 degrees C. Heat resistances of FMDV strains at 90 and 100 degrees C were not statistically different (P > 0.05), while the FMDV serotype O (OPN) appeared to be the most heat resistant at 60 to 80 degrees C. The other observed inactivation curves were linear with shoulder or tailing (biphasic curves). The shoulder effect was mostly observed at 90 and 100 degrees C, while the tailing effect was mostly observed at 50 to 80 degrees C. The adjusted D values in the case of shoulder and tailing effects did not affect the overall estimated heat resistance of these FMDV strains, so even unadjusted D values of deviant inactivation curves were legitimate. The z values of FMDV serotypes O, A, and Asia 1 were 21.78 to 23.26, 20.75 to 22.79, and 19.87 degrees C, respectively. The z values of FMDV strains studied were not statistically significantly different (P > 0.05). The results of this study indicated that the heat resistance in PBS of FMDV strains from Thailand was much less than had been reported for foreign epidemic FMDV strains.     

Inhibition of heat shock protein synthesis and protein glycosylation by stepdown heating

Mammalian cells exhibit increased sensitivity to hyperthermic temperatures of 38-43 degrees C after an acute high-temperature heat shock; this phenomenon is known as the stepdown heating (SDH) effect. We characterized the SDH effect on (1) the synthesis of major heat shock proteins, HSP110, 90, 72/70, 60 (35S-amino acids label), (2) on heat-induced protein glycosylation (3H-D-mannose label), and (3) on thermotolerance expression, using cell survival as an endpoint. Partitioning of label between soluble and insoluble cell fractions was separately examined. Synthesis of high molecular weight HSPs (HSP110, 90, and 72/70) was increased both by acute (10 min, 45 degrees C) and chronic (1-6 h, 41.5 degrees C) hyperthermia, primarily in the soluble cytosol fraction. SDH (10 min, 45 degrees C + 1 to 6 h, 41.5 degrees C) completely inhibited labeling of HSP110, partially inhibited HSP90 labeling, and had virtually no effect on HSP72/70 synthesis, when compared with chronic hyperthermia alone. At the cell survival level, SDH increased sevenfold the rate of cell killing at 41.5 degrees C, but reduced the expression of thermotolerance by only a factor of two. This suggests that SDH sensitization did not result from changes in HSP72/70 synthesis, nor solely from inhibition of thermotolerance. 35S-labeled HSP60 and HSP50 were found primarily in the cellular pellet fraction after both acute and chronic hyperthermia. SDH completely inhibited 35S-labeling of both HSP60 and HSP50. Labeling of GP50 with 3H-D-mannose was also completely inhibited by SDH. Moreover, SDH progressively reduced N-acetylgalactosaminyl-transferase activity. The data demonstrate that heat sensitization by SDH is accompanied by complex and selectively inhibitory patterns of HSP synthesis and protein glycosylation. Profound inhibition of HSP110, HSP60, and HSP50/GP50 labeling suggests that these may be associated with mechanisms of SDH sensitization.     

Sulfated chitooligosaccharides as prolyl endopeptidase inhibitor

Prolyl endopeptidase (PEP, EC 3.4.21.26) is a proline-specific endopeptidase with a serine-type mechanism, which digests small peptide-like hormones, neuroactive peptides, and various cellular factors. PEP has been involved in neurodegenerative disorders, therefore, the discovery of PEP inhibitors can revert memory loss caused by amnesic compounds. In this study, we prepared hetero-chitooligosaccharides (COSs) with different molecular sizes using ultrafiltration (UF) membrane reactor system from hetero-chitosan with different degrees of deacetylation (DD; 90%, 75% and 50% deacetylation), and synthesized sulfated COSs (SCOSs). PEP inhibitory activities of SCOSs were evaluated and the results showed that 50% deacetylated SCOSs (50-SCOSs) exhibited higher inhibitory activities than those of 90% and 75% deacetylated SCOSs (90-SCOSs and 75-SCOSs). Among the 50-SCOSs (50-SCOS I, 5000–10,000 Da; 50-SCOS II, 1000–5000 Da; 50-SCOS III, below 1000 Da), 50-SCOS II possessed the highest inhibitory activity and IC₅₀ value was 0.38 mg/ml. Kinetics studies with 50-SCOS II indicated a competitive enzyme inhibition with a K_i value of 0.78 mg/ml. It was concluded that the 50-SCOS II may be useful for PEP inhibitor and for developing a new type PEP inhibitor from carbohydrate based materials.     

壳寡糖诱导植物防御反应中一氧化氮信号的研究

壳寡糖可以增强植物对病虫害的防御能力,为了深入研究壳寡糖的作用机理,首次运用荧光酶标仪及一氧化氮(Nitric oxide,NO)荧光探针Diaminofluorescein diacetate (DAF-2DA)对壳寡糖诱导的NO信号进行研究。研究发现,不同浓度的壳寡糖均可诱导烟草悬浮细胞产生NO;NO的清除剂Carboxy-PTIO potassium salt(cPTIO)和一氧化氮合酶(Nitric oxide synthase,NOS)抑制剂N^ω-nitro-L-arginine methyl Ester(L-NAME)可以明显抑制NO的产生;硝酸还原酶(Nitrate reductase, NR)的抑制剂叠氮化钠和钨酸钠对NO的产生无影响;Ca²⁺流相关抑制剂氯化镧和钌红均可抑制NO的产生。NO和Ca²⁺流的相关抑制剂可明显抑制壳寡糖诱导的抗性相关基因的表达。结果显示:壳寡糖主要通过NOS酶催化合成NO,且NO参与调节壳寡糖诱导的抗性相关基因的表达,在此过程中,Ca²⁺可以调节NO的合成。     

Nitric oxide production and its functional link with OIPK in tobacco defense response elicited by chitooligosaccharide

Chitooligosaccharide (COS) or oligochitosan has been shown to induce tobacco defense responses which are connected with nitric oxide (NO) and OIPK (oligochitosan-induced Ser/Thr protein kinase). The aim of this study was to reveal the relationship between NO production and OIPK pathway in the defense response of tobacco elicited by COS. NO generation was investigated by epidermal strip bioassay and fluorophore microscope using fluorophore diaminofluorescein diacetate (DAF-2DA). Tobacco epidermal cells treated with COS resulted in production of NO, which was first present in chloroplast, then in nucleus, finally in the whole cell; this NO production was sensitive to NO scavenger cPTIO and the mammalian NO synthase (NOS) inhibitor l-NAME, suggesting that NOS-like enzyme maybe involved in NO generation in tobacco epidermal cells. However, NOS and nitrate reductase (NR, EC 1.6.6.1) inhibitors reduced NO content in tobacco leaves by using NO Assay Kit, suggesting both NOS and NR were involved in NO production in tobacco leaves. Using a pharmacological approach and western blotting, we provide evidence that NO acts upstream of OIPK expression. NO scavenger, NOS inhibitor partly blocked the activation of OIPK and the activities of several defense-related enzymes induced by COS; treatment with NO donor sodium nitroprusside (SNP) induced the activation of OIPK and enhanced the defense systems. The results suggest that COS is able to induce NO generation, which results in up-regulation the activities of some defense-related enzymes through an OIPK-dependent or independent pathway.     

Influence of knee angle and individual flexibility on the flexion-relaxation response of the low back musculature.

In many occupational settings (e.g. agriculture and construction) workers are asked to maintain static flexed postures of the low back for extended periods of time. Recent research indicates that the resulting strain in the viscoelastic, ligamentous tissues may have a deleterious effect on the stability of the spine and the normal reflex response of spinal tissues. The purpose of this study was to evaluate the previously described flexion-relaxation response in terms of the interactive effect of trunk flexion angle (30 degrees, 50 degrees, 70 degrees, 90 degrees ), knee flexion angle (0 degrees (straight knees), 20 degrees, 40 degrees ) and individual flexibiliteky (low, medium, and high). These conditions were tested under two levels of loading: no load (just supporting the weight of the torso) and trunk extension moment equal to 50% of the subject's posture-specific maximum voluntary trunk extension capacity. Surface electromyographic (EMG) data were collected from the multifidus, the longissimus, the iliocostalis, the vastus medialis, the rectus femoris, the vastus lateralis, the biceps femoris, and the gastrocnemius-soleus group from a sample of eight male participants as they performed isometric weight holding tasks in the postures defined by the combinations of trunk angle and knee angle. The results of this study showed that knee angle did have a significant effect on the lumbar extensor muscle activity but only consistently at the 90 degrees trunk angle. Participant flexibility showed a consistent trend of decreasing lumbar extensor muscle activity with decreased flexibility across all trunk angle values. Most interesting was the interactive response of flexibility and knee angle, wherein the flexibility of the participant influenced the trunk angles at which the knee flexion angle affected the flexion-relaxation response. Highly flexible subjects showed an effect of knee angle on the flexion-relaxation response only at the 90 degrees trunk angle; subjects in the medium flexibility category showed a similar response in both the 70 degrees and 90 degrees trunk angles; subject in the low flexibility group showed no knee angle effect on the flexion-relaxation response. Overall the results confirm previous results with regard to the contribution of the passive tissues to the overall trunk extension moment but also show that the tension in the bi-articular biceps femoris, which was influenced by knee flexion angle and flexibility, affects the ratio of active extensor moment contributions of the lumbar extensor musculature to passive extensor moment contributions from the muscular and ligamentous tissues. The results of this study provide empirical data describing this complicated, interactive response.     

Chaperone-dependent regulation of endothelial nitric-oxide synthase intracellular trafficking by the co-chaperone/ubiquitin ligase CHIP

Endothelial nitric-oxide synthase (eNOS), the enzyme responsible for production of endothelial NO, is under tight and complex regulation. Proper cellular localization of eNOS is critical for optimal coupling of extracellular stimulation with NO production. In addition, the molecular chaperone Hsp90 interacts with eNOS and positively regulates eNOS activity. Hsp90 is modulated by physical interaction with its co-chaperones. CHIP (carboxyl terminus of Hsp70-interacting protein) is such a co-chaperone that remodels the Hsp90 heterocomplex and causes protein degradation of some Hsp90 substrates through the ubiquitin-protein isopeptide ligase activity of CHIP. Here we show that CHIP incorporated into the eNOS.Hsp90 complex and specifically decreased soluble eNOS levels in transiently transfected COS cells. Surprisingly, in contrast to the effects of the Hsp90 inhibitor geldanamycin, which induces eNOS ubiquitylation and its subsequent protein degradation, CHIP did not target eNOS for ubiquitylation and proteasome-dependent degradation. Instead, CHIP partitioned soluble eNOS into an insoluble and inactive cellular compartment, presumably through its co-chaperone activity. This effect seems to be due to displacement of eNOS from the Golgi apparatus, which is otherwise required for trafficking of eNOS to the plasmalemma and subsequent activation. Consistent with observations from overexpression studies, eNOS localization to the membrane and activity were increased in mouse lung endothelial cells lacking CHIP. Taken together, these results demonstrate a novel co-chaperone-dependent mechanism through which eNOS trafficking is regulated and suggest a potentially generalized role for CHIP in protein trafficking through the Golgi compartment.     

Synergistic effects between aminoethyl-chitosans and β-lactams against methicillin-resistant Staphylococcus aureus (MRSA)

Two kinds of aminoethyl-chitosans (AEC), AEC90 and AEC50, which had degrees of deacetylation of 90% and 50%, respectively, were prepared and their synergistic effects in combination with β-lactams including ampicillin, penicillin, and oxacillin against two standard methicillin-resistant Staphylococcus aureus (MRSA) strains and twelve clinical isolated MRSA strains were investigated. When AECs and β-lactams were combined, synergistic effects were observed with fractional inhibitory concentration (FIC) indices of 0.252–0.508, and the MICs of β-lactams in the presence of AECs were dramatically reduced.     

Chitooligosaccharides induce apoptosis in human myeloid leukemia HL-60 cells

In this study we propose a novel anticancer agent using hetero-chitooligosaccharide (hetero-COS). To examine the possibility of the hetero-COS as a anticancer agent, we prepared nine kinds of hetero-COS with relatively higher molecular weights (90, 75 and 50-COS I, 5-10kDa), medium molecular weights (90, 75 and 50-COS II, 1-5kDa), and lower molecular weights (90, 75 and 50-III, below 1kDa), and their anticancer properties were investigated on HL-60 cells using flow cytometry and morphological analysis. The results obtained indicate that 90-COS III, which is relatively higher degree of deacetylation and lower molecular weights, showed the highest anticancer activity, and the data showed the anticancer property of the hetero-COSs depended on their degree of deacetylation values and molecular weight.     

Effect of chitooligosaccharides on calcium bioavailability and bone strength in ovariectomized rats

Chitosan polymer with deacetylation degree of 93% was hydrolyzed with an endo-type chitosanase (35,000 U/g protein) with substrate to enzyme ratio of 1 to 1.5 for 18 h in a batch reactor, and then the resultant hydrolysates were fractionated into four different molecular weights using an ultrafiltration (UF) membrane reactor system. An in vitro study elucidated that four kinds of chitooligosaccharides (COSs) could efficiently inhibit the formation of insoluble calcium salts in the neutral pH. In vivo effects of COSs on Ca bioavailability were further studied in the osteoporosis modeling rats induced by ovariectomy and concurrent low calcium intake. During the experimental period corresponding to the menopause with the osteoporosis disease, calcium retention was increased and bone turnover was decreased by COS IV supplementation in the ovariectomized (OVX) rats. After the low Ca diet, COS IV diet including both normal level of calcium and vitamin D significantly decreased calcium loss in feces and increased calcium retention compared to the control diet. The levels of femoral total calcium, bone mineral density (BMD), and femoral strength were also significantly increased by the COS IV diet in a similar level to those of CPP diet group. In the present study, the results proved the beneficial effects of low molecular chitooligosaccharide (COS IV) in preventing negative mineral balance.     

Effect of temperature and humidity on development, survival and oviposition in laboratory populations of Eriworm, Philosamia ricini (Boisduval) (Lepidoptera Saturniidae)

Experiments were conducted on laboratory populations of Eriworm, Philosamia ricini Boisduval to study the effect of temperature and humidity on development, growth, survival and oviposition. Experiments were performed at four different temperatures (22 +/- 2 degrees C, 26 +/- 2 degrees C, 28 +/- 2 degrees C and 34 +/- 1 degrees C) each with three different humidities (50%, 70% and 90% relative humidity). Shortest developmental period (42 days) was observed in 28 +/- 2 degrees C and 70% RH. Longest developmental period was (63.6 days) observed in 22 +/- 2 degrees C at 50% RH. Highest larval weight (9.1 g) was found in 28 +/- 2 degrees C at 70% RH. Best pupal weight was observed in 26 +/- 2 degrees C at 70% RH. Weight of silk material was found to be maximum in 26 +/- 2 degrees C at 70% RH. Survival was best in 28 +/- 2 degrees C and 26 +/- 2 degrees C at 70% RH. Oviposition was found to be highest in 28 +/- 2 degrees C at 70% RH.     

Molecular dynamics and dissipative particle dynamics simulations for prediction of miscibility in polyethylene terephthalate/polylactide blends

The miscibility of polyethylene terephthalate (PET)/polylactide (PLA) blends is studied through atomistic molecular dynamics (MD) and mesoscale dissipative particle dynamics (DPD) simulation. Five PET/PLA blends (with the weight ratio at 90/10, 70/30, 50/50, 30/70 and 10/90) as well as pure PET and PLA are examined. The solubility parameter values obtained by using the MD simulation are in good agreement with the reference data. The Flory–Huggins parameters, χ, which are computed for different blends and determined from the cohesive energy densities, with the radial distribution functions g(r) of the inter-molecular atoms, suggest that PET is completely miscible with PLA over the entire composition range. This is further proved by the mesoscopic morphologies of PET/PLA blends. All the simulation results are qualitatively consistent with the experimental results, and demonstrate that the modelling strategies in this study may serve as a powerful tool for predicting miscibility and mesoscopic morphology of polymer blends.     

Induction of the NO synthesis in lactobacilli under stress conditions

An increase in the nitric oxide (NO) biosynthesis in Lactobacillus plantarum 8P-A3 cells takes place under strong stress influence, which leads to a considerable decrease in the microbial cell viability: heating at 70 degrees C and 80 degrees C, prolonged cultivation, toxic effect of hexylresorcinol. The factors, which do not lead to cell death, such as heating at 60 degrees C, 50 microg/ml homoserine lactone, Bacillus intermedius 7P ribonuclease (binase) in concentrations up to 300 microg/ml, do not induce NO synthesis. The activation of the NO biosynthesis in response to stress treatment evidences to universality of key-mechanisms of stress response in cells differing in the level of their organization as well as to important role of nitric oxide in them.